CXIP4 Antibody

What is CXCR4 and why is it a significant research target?

CXCR4 (C-X-C motif chemokine receptor 4) is a G-protein coupled receptor belonging to the G-protein coupled receptor 1 family. It serves as a receptor for the chemokine CXCL12/SDF-1 (stromal cell-derived factor-1), transducing signals by increasing intracellular calcium ion levels and enhancing MAPK1/MAPK3 activation. The canonical human CXCR4 protein consists of 352 amino acid residues with a molecular mass of 39.7 kDa and is primarily localized in lysosomes and the cell membrane . CXCR4 is especially significant because it is expressed in various human cancers, particularly hematologic malignancies, where this receptor and its ligand SDF-1 play crucial roles in cancer progression . Additionally, CXCR4 functions as a coreceptor for HIV-1 entry into target cells, making it important for understanding HIV pathogenesis and developing therapeutic strategies .

What are the primary applications of CXCR4 antibodies in research?

CXCR4 antibodies are extensively utilized across multiple research applications. Flow cytometry represents the most widely used application, allowing researchers to identify and quantify CXCR4-expressing cells. Western blotting and immunohistochemistry (both for frozen and paraffin-embedded sections) are also common applications . Additional methodologies include immunocytochemistry, immunoprecipitation, fluorescence assays, ELISA detection, and microscopy. Over 1900 scientific citations describe the use of CXCR4 antibodies in research, demonstrating their widespread utility . CXCR4 antibodies are particularly valuable for investigating receptor expression patterns, signaling pathways, and for developing therapeutic strategies targeting CXCR4 in cancer and HIV research.

How do different conformational states of CXCR4 impact antibody binding?

Research has revealed that CXCR4 exists in multiple conformational subpopulations that exist in equilibrium on the cell surface. These conformational states are not cell-type specific as previously reported . Different anti-CXCR4 monoclonal antibodies can recognize overlapping epitopes but exhibit varying levels of maximal binding to CXCR4-expressing cells due to these conformational differences. The second extracellular loop (ECL2) appears to be the immunodominant region of CXCR4, with many antibodies targeting residues 179-193 . The ability of antibodies to recognize different CXCR4 conformations correlates with their capacity to inhibit functions such as HIV-1 entry, suggesting functional significance of these conformational states. Small molecule inhibitors like AMD3100 can block binding of antibodies targeting ECL2 but not those targeting the N-terminus, indicating that they disrupt specific conformational domains of the receptor .

How should researchers optimize flow cytometry protocols for CXCR4 detection?

For optimal flow cytometry results with CXCR4 antibodies, researchers should implement several critical methodological considerations. First, select antibodies with demonstrated flow cytometry performance and appropriate fluorophore conjugation that complements your experimental panel design. For intracellular staining, proper fixation with a dedicated Flow Cytometry Fixation Buffer is essential, followed by permeabilization using Flow Cytometry Permeabilization/Wash Buffer I to facilitate antibody access to intracellular antigens . Always include appropriate isotype controls (such as IC0041P) to establish background fluorescence and determine specific binding . When handling conjugated antibodies, protect them from light and avoid freezing to maintain optimal performance . For multicolor panels, carefully consider spectral overlap and implement proper compensation. Be aware that CXCR4 exists in different conformational states that may affect antibody binding, potentially requiring multiple antibody clones targeting distinct epitopes to fully characterize CXCR4 expression .

What experimental approaches can assess CXCR4 antibody blocking efficacy?

To evaluate the blocking efficacy of CXCR4 antibodies against the CXCR4/SDF-1 axis, multiple complementary approaches should be employed. SDF-1 competition assays can measure the ability of antibodies to compete with the natural ligand for receptor binding. Signaling pathway analysis should monitor phosphorylation status of downstream effectors including Akt, Erk1/2, p38, and GSK3β, as these are reduced upon effective CXCR4 blockade . Functional assays such as transwell migration assays and cell proliferation assays provide direct evidence of biological activity inhibition . For antibodies intended for therapeutic development, cytotoxicity assessments including antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assays are crucial, as these represent important mechanisms for targeting neoplastic cells . In vivo efficacy should be evaluated using appropriate xenograft models with various tumor cell lines, similar to the approach described for the humanized antibody hz515H7 . For antibodies targeting CXCR4 as an HIV-1 coreceptor, assessing the ability to block entry of various HIV-1 isolates provides valuable functional data .

What controls are essential when working with CXCR4 antibodies?

Robust experimental design with CXCR4 antibodies requires careful implementation of multiple control types. Isotype controls matching the antibody class, species, and conjugation are essential for establishing background and non-specific binding levels, particularly in flow cytometry applications . Positive control samples should include cell lines or primary cells known to express high levels of CXCR4, such as human immature dendritic cells, platelets, or PBMC monocytes . Negative controls should incorporate cells lacking CXCR4 expression or where CXCR4 has been knocked down/out through genetic manipulation. For functional assays, include blocking controls such as the small molecule CXCR4 inhibitor AMD3100 and competing ligands (SDF-1/CXCL12) . When performing intracellular staining, include controls to assess the effect of fixation and permeabilization on epitope recognition. Given the documented conformational variability of CXCR4, utilizing multiple antibody clones targeting different epitopes can help confirm results and provide a more complete picture of CXCR4 biology .

How do antibody epitope differences affect inhibition of HIV-1 entry?

Anti-CXCR4 monoclonal antibodies exhibit differential capacities to inhibit HIV-1 entry based on their epitope specificity and binding characteristics. Epitope mapping studies reveal that most anti-CXCR4 monoclonal antibodies recognize overlapping epitopes in the second extracellular loop (ECL2), particularly residues 179-193, while some (like 4G10) bind to the N-terminus . Despite recognizing similar epitopes, these antibodies can show marked differences in their ability to block HIV-1 entry. For instance, monoclonal antibodies 701, 718, and 717 demonstrated the most potent inhibition, correlating with their high maximal binding levels to CXCR4-expressing cells . MAb 12G5 showed variable potency against different HIV-1 isolates, strongly inhibiting entry mediated by envelope glycoproteins of NL4-3, DH123, and NDK but only moderately inhibiting entry of HXB2, 84ZR085, and 90CF402 . Interestingly, MAb 716 exhibited intermediate maximal binding but no entry inhibition, and even enhanced entry of isolate 90CF402 by approximately 50% . Additionally, antibodies generally showed greater efficacy against T-cell line adapted (TCLA) isolates compared to primary X4 strains, suggesting strain-specific factors also influence inhibition efficacy .

What are the unique features of engineered CXCR4 antibodies with elongated CDRs?

Innovative antibody engineering approaches have leveraged the unique structure of bovine antibody BLV1H12, which possesses an ultralong heavy chain complementarity determining region 3 (CDRH3), to create novel CXCR4-targeting antibodies. By substituting this extended CDRH3 with modified CXCR4-binding peptides that adopt a β-hairpin conformation, researchers have generated antibodies specifically targeting the ligand binding pocket of the CXCR4 receptor . These engineered antibodies demonstrate several advantageous properties: they selectively bind to CXCR4-expressing cells with binding affinities in the low nanomolar range, effectively inhibit SDF-1-dependent signal transduction, and block cell migration in transwell assays . The engineering approach can be extended beyond CDRH3, as demonstrated by a CDRH2-peptide fusion that binds CXCR4 with an impressive Kd of 0.9 nM . This strategy represents a significant advancement in antibody engineering that could substantially expand antibody functionality by enabling precise targeting of receptor binding pockets, which is typically challenging with conventional antibody development methods .

What dual mechanisms make therapeutic anti-CXCR4 antibodies like hz515H7 unique?

The humanized IgG1 monoclonal antibody hz515H7 exemplifies the potential of therapeutic anti-CXCR4 antibodies through its dual mechanisms of action. The first mechanism involves direct blocking functions: hz515H7 efficiently competes with SDF-1 for CXCR4 binding, induces conformational changes in CXCR4 homodimers, inhibits both CXCR4 receptor–mediated G-protein activation and β-arrestin-2 recruitment, and reduces phosphorylation of downstream effectors such as Akt, Erk1/2, p38, and GSK3β . These effects culminate in inhibition of cancer cell migration and proliferation. The second mechanism involves immune effector functions: hz515H7 induces both antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against neoplastic cells while preserving normal blood cells . In mouse xenograft models, hz515H7 demonstrated antitumor activities against multiple hematologic tumor cell lines, with its Fc-mediated effector functions proving essential in this context . The antibody can also bind to primary tumor cells from acute myeloid leukemia and multiple myeloma patients, suggesting broad clinical applicability . This combination of signaling blockade and immune effector functions makes hz515H7 potentially superior to antagonists that rely solely on receptor blocking.

How can researchers address cross-reactivity concerns with CXCR4 antibodies?

Addressing cross-reactivity is fundamental for ensuring reliable CXCR4 antibody experiments. Researchers should first validate antibody specificity using CXCR4 knockout or knockdown cells as negative controls. When working across species, antibody reactivity must be validated for each species of interest, as different anti-CXCR4 antibodies show varying reactivity profiles with human, mouse, rabbit, and rat samples . Pre-absorption tests can be performed by incubating antibodies with recombinant CXCR4 protein or epitope-specific peptides prior to application. Cross-reactivity with homologous chemokine receptors should be evaluated, particularly those sharing structural similarity with CXCR4. Consider the specific epitope recognized by the antibody, as those targeting different domains (N-terminus versus ECL2) may exhibit different specificity profiles . Validation across multiple detection platforms (flow cytometry, Western blot, immunohistochemistry) provides additional confidence in antibody specificity. For advanced applications, recombinant expression systems using cell lines engineered to express only CXCR4 can serve as definitive positive controls. These approaches collectively ensure that experimental results genuinely reflect CXCR4 biology rather than cross-reactivity artifacts.

What factors influence CXCR4 antibody performance in different experimental applications?

Multiple factors can significantly impact CXCR4 antibody performance across different experimental applications. The conformational state of CXCR4 represents a critical factor, as the receptor exists in multiple conformational subpopulations in equilibrium on the cell surface . These conformational variations affect epitope accessibility and can result in application-specific performance differences. The specific epitope recognized by the antibody is equally important; antibodies targeting the N-terminus versus ECL2 show different binding characteristics and functional properties . Sample preparation methods, particularly fixation and permeabilization protocols for flow cytometry and immunohistochemistry, can alter epitope structure and accessibility . Antibody format (monoclonal vs. polyclonal) and conjugation type affect performance in specific applications, with some conjugates being more suitable for particular techniques. The presence of small molecule inhibitors like AMD3100 can disrupt the conformation of specific CXCR4 domains, potentially affecting antibody binding . Cell type and activation state can influence CXCR4 expression levels and conformational distribution. Buffer conditions including pH, ionic strength, and detergent composition can impact antibody-epitope interactions, particularly in biochemical assays like Western blotting.

What are the best validation practices for new CXCR4 antibody clones?

Comprehensive validation of new CXCR4 antibody clones requires a systematic, multi-parameter approach. Begin with epitope mapping to determine which region of CXCR4 the antibody recognizes (N-terminus, ECL2, or other domains) . Quantitatively assess binding characteristics by measuring affinity (Kd) and determining maximal binding levels to CXCR4-expressing cells, as these parameters correlate with functional efficacy . Evaluate recognition of different CXCR4 conformational states, as conformational subpopulations exist in equilibrium on the cell surface . Functional validation should include SDF-1 competition assays, signaling pathway inhibition assessment (monitoring downstream effectors like Akt, Erk1/2), and cell migration/proliferation assays . For antibodies targeting CXCR4 as an HIV coreceptor, HIV-1 entry inhibition should be assessed using multiple viral isolates . Cross-platform validation across flow cytometry, Western blot, immunohistochemistry, and other relevant applications ensures versatility . Specificity testing should evaluate cross-reactivity with related receptors and performance across different species. Reproducibility assessment across different lots and experimental conditions confirms reliability. Finally, test compatibility with known CXCR4 inhibitors like AMD3100, which can affect binding to different epitopes .

How do CXCR4 antibodies inhibit cancer cell signaling pathways?

CXCR4 antibodies disrupt cancer cell signaling through multiple coordinated mechanisms. Primary blockade occurs through competitive inhibition, where antibodies like hz515H7 effectively compete with SDF-1 for binding to CXCR4, preventing ligand-induced receptor activation . Upon binding, these antibodies can induce conformational changes in CXCR4 homodimers, altering receptor function and preventing normal signaling processes . At the signal transduction level, CXCR4 antibodies inhibit receptor-mediated G-protein activation, a critical early step in the signaling cascade. Concurrently, they prevent β-arrestin-2 recruitment following CXCR4 activation, interfering with receptor internalization and sustained signaling . These upstream disruptions culminate in reduced phosphorylation of key downstream effectors involved in cancer cell survival and proliferation, including Akt (survival pathways), Erk1/2 (proliferation signaling), p38 (stress responses), and GSK3β (multiple cellular processes) . The functional consequences include inhibition of cancer cell migration and metastatic potential, reduction in cellular proliferation rates, and potential induction of apoptotic pathways in neoplastic cells. These comprehensive signaling disruptions make CXCR4 antibodies promising therapeutic agents for targeting cancers dependent on the CXCR4/SDF-1 axis.

Why is CXCR4 a significant target in hematologic malignancies?

CXCR4 represents a particularly valuable therapeutic target in hematologic malignancies due to several key biological properties. CXCR4 is expressed in a large variety of human cancers, including many hematologic malignancies, where this receptor and its ligand SDF-1 play crucial roles in disease progression . The CXCR4/SDF-1 axis contributes to the homing and retention of malignant cells within protective bone marrow niches, shielding them from conventional therapies and promoting treatment resistance. In acute myeloid leukemia and multiple myeloma, primary tumor cells express CXCR4 and respond to therapeutic antibodies like hz515H7, highlighting the clinical relevance of this target . The dual mechanisms of action available through antibody targeting—signaling blockade and immune effector functions—are particularly advantageous for hematologic malignancies. Antibodies like hz515H7 induce both antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against neoplastic cells while preserving normal blood cells, providing selective targeting of malignant populations . In mouse xenograft models, anti-CXCR4 antibodies demonstrate significant antitumor activities with multiple hematologic tumor cell lines, with Fc-mediated effector functions proving essential for efficacy . These combined features make CXCR4 antibodies promising therapeutic agents for hematologic malignancies resistant to conventional treatments.

What techniques can assess CXCR4 expression in patient samples?

Accurate assessment of CXCR4 expression in patient samples requires sophisticated methodological approaches tailored to clinical specimens. Flow cytometry represents the gold standard for quantifying CXCR4 on hematologic malignancies, allowing analysis of expression levels on specific cell populations identified through additional markers . For intracellular CXCR4 detection, proper fixation and permeabilization protocols are essential, as demonstrated with dendritic cells, platelets, and PBMC monocytes . Immunohistochemistry on formalin-fixed paraffin-embedded (FFPE) or frozen tissue sections enables visualization of CXCR4 expression patterns within the tissue microenvironment, providing spatial context that is particularly valuable for solid tumors and lymph node specimens . Multiplexed immunofluorescence can simultaneously assess CXCR4 alongside other biomarkers to characterize specific cell populations within heterogeneous samples. For molecular quantification, quantitative RT-PCR measuring CXCR4 mRNA provides complementary data to protein-level assessments. When analyzing patient samples, researchers should be aware of the conformational heterogeneity of CXCR4, which may affect antibody binding . Using multiple antibody clones targeting different epitopes can provide a more complete picture of CXCR4 expression. Finally, functional assays measuring SDF-1 responses in primary cells can complement expression data to assess receptor functionality.