LCR9 Antibody

What is CCR9 and why is it important in immunological research?

CCR9 (C-C chemokine receptor 9, also known as CD199) is a chemokine receptor that plays a crucial role in the chemotaxis of immune cells and inflammatory responses. It serves as a receptor for C-C-chemokine ligand 25 (CCL25) and is expressed by various cell types including colonic epithelium, plasmacytoid dendritic cells, and transitional B cells. CCR9 is particularly important in mucosal immunity and lymphocyte trafficking to the small intestine. Furthermore, CCR9 has gained significant research interest due to its high expression in multiple tumor types, including several solid tumors and T-cell acute lymphoblastic leukemia, making it an attractive target for immunotherapeutic approaches .

What are the differences between anti-mouse CCR9 and anti-human CCR9 antibodies?

Anti-mouse CCR9 antibodies (such as Clone 9B1) and anti-human CCR9 antibodies target species-specific variants of the CCR9 receptor. The amino acid sequence of CCR9 differs between species, with human TLR9 sharing approximately 76% amino acid identity with mouse TLR9 over amino acids 64-189 . These differences necessitate species-specific antibodies for research. Mouse models typically require anti-mouse CCR9 antibodies like the Alexa Fluor 488 Rat Anti-Mouse CD199 (CCR9) for accurate detection . Anti-human CCR9 mAbs (such as clones 91R and 92R) have been developed specifically for human research and therapeutic applications, showing cytotoxicity against CCR9-positive human tumors through mechanisms like antibody-dependent cell-mediated cytotoxicity .

What fluorophores are commonly conjugated to CCR9 antibodies?

CCR9 antibodies are available with various fluorophore conjugations to accommodate different flow cytometry applications and multicolor panels. Common conjugates include:

• Alexa Fluor 488, which has spectral properties similar to FITC and is used in the BD PharmingenTM Rat Anti-Mouse CD199 (CCR9) antibody

• Alexa Fluor 750, utilized in antibodies such as the Human TLR9 Alexa Fluor 750-conjugated Antibody

The selection of fluorophore depends on the specific experimental design, instrument capabilities, and the need to minimize spectral overlap with other fluorochromes in multicolor experiments. Alexa Fluor 488 emission is collected at the same instrument settings as fluorescein isothiocyanate (FITC), making it compatible with standard flow cytometry configurations .

How can epitope mapping be performed for anti-CCR9 monoclonal antibodies?

Epitope mapping for anti-CCR9 monoclonal antibodies can be conducted using alanine scanning strategies via enzyme-linked immunosorbent assay (ELISA). In recent research, two different approaches were employed:

• Single alanine (1× Ala) substitution: 19 peptides where each amino acid of the target sequence is individually replaced with alanine

• Double alanine (2× Ala) substitution: 18 peptides with two consecutive amino acids replaced with alanine

The procedure involves:

• Synthesizing wild-type and alanine-substituted peptides of the target sequence

• Immobilizing these peptides on immunoassay plates (1 μg/mL)

• Blocking with bovine serum albumin in PBST

• Incubating with the monoclonal antibody of interest

• Detecting binding using appropriate secondary antibodies

• Analyzing which substitutions diminish antibody binding to identify critical epitope residues

This approach successfully determined the binding epitope of C9Mab-24, an anti-mouse CCR9 mAb developed via peptide immunization of the mCCR9 N-terminus (amino acids 1-19) .

What are the mechanisms by which anti-CCR9 antibodies exert antitumor activity?

Anti-CCR9 monoclonal antibodies demonstrate antitumor activity through several distinct mechanisms:

• Antibody-dependent cell-mediated cytotoxicity (ADCC): Anti-human CCR9 mAbs (clones 91R and 92R) recruit immune effector cells to target and eliminate CCR9-expressing tumor cells.

• Complement-dependent cytotoxicity (CDC): These antibodies can activate the complement system, leading to the formation of membrane attack complexes that lyse tumor cells.

• Tumor proliferation suppression: In mouse xenograft models, anti-CCR9 antibodies have been shown to suppress T-ALL (T-cell acute lymphoblastic leukemia) proliferation.

• CAR-T cell development: CCR9-specific monoclonal antibodies have been used to develop chimeric antigen receptor (CAR)-T cells, which demonstrated potent antitumor effects in cell lines and patient-derived xenograft mouse models of T-ALL .

These mechanisms highlight why CCR9 is considered an attractive therapeutic target for treating CCR9-positive malignancies.

How does CCR9 expression correlate with disease progression in different cancer types?

CCR9 is highly expressed in various tumor types, and its expression pattern has significant implications for disease progression. While specific correlation data is limited in the provided search results, research has demonstrated that CCR9 is notably expressed in:

• T-cell acute lymphoblastic leukemia (T-ALL)

• Several solid tumors

The therapeutic potential of targeting CCR9 in these malignancies is supported by preclinical studies showing that anti-CCR9 monoclonal antibodies exert significant antitumor activity. For instance, anti-human CCR9 mAbs exhibited cytotoxicity against CCR9-positive tumors through ADCC and CDC mechanisms, and suppressed T-ALL proliferation in mouse xenograft models .

The development of CCR9-specific CAR-T cells further demonstrates the importance of CCR9 as a target, as these engineered cells showed potent antitumor effects in both cell lines and patient-derived xenograft models of T-ALL .

What are the optimal conditions for using CCR9 antibodies in flow cytometry?

For optimal flow cytometry results with CCR9 antibodies, researchers should consider the following methodological guidelines:

• Titration: Since applications vary, each investigator should titrate the reagent to obtain optimal results for their specific cellular system .

• Isotype controls: An isotype control should be used at the same concentration as the antibody of interest to assess non-specific binding .

• Compensation assessment: When using fluorochrome-conjugated antibodies like Alexa Fluor 488 Rat Anti-Mouse CD199 (CCR9), researchers should assess fluorescence spillover (compensation). BD® CompBeads can be used as surrogates, but it's recommended to compare spillover on cells and CompBeads when using a reagent for the first time to ensure appropriateness for the specific cellular application .

• Instrument settings: For antibodies conjugated to Alexa Fluor 488, emission is collected at the same instrument settings as for fluorescein isothiocyanate (FITC) .

• Light protection: Fluorophore-conjugated antibodies should be protected from light to prevent photobleaching .

• Storage conditions: Antibodies should be stored at 2 to 8°C and should not be frozen to maintain optimal activity .

How should researchers approach validation of CCR9 antibody specificity?

Validating the specificity of CCR9 antibodies is crucial for reliable research results. A comprehensive validation approach includes:

• Cell line validation: Test the antibody on CCR9-overexpressing cell lines (like transfected CHO-K1 cells) versus control cells. The anti-mouse CCR9 mAb, C9Mab-24, was validated using mCCR9-overexpressed Chinese hamster ovary-K1 cells .

• Endogenous expression testing: Verify antibody performance on cell lines known to endogenously express CCR9, such as RL2 cells used for validating C9Mab-24 .

• Epitope mapping: Determine the precise binding epitope using techniques like alanine scanning via ELISA to confirm antibody specificity to the intended target region .

• Cross-reactivity assessment: Evaluate potential cross-reactivity with related receptors, particularly important when studying chemokine receptors that share structural similarities.

• Knockout/knockdown controls: Where possible, use CCR9 knockout or knockdown cells as negative controls to confirm specificity.

• Multiple detection methods: Confirm specificity across different applications (flow cytometry, Western blot, immunohistochemistry) if the antibody is to be used in multiple techniques.

What are the critical steps in developing new anti-CCR9 monoclonal antibodies?

The development of new anti-CCR9 monoclonal antibodies involves several critical steps, as exemplified by the creation of C9Mab-24 (rat IgG2a, kappa):

• Immunogen design: Synthesize a partial sequence of the target protein. For C9Mab-24, researchers used the N-terminal extracellular region of mCCR9 (amino acids 1-19) with cysteine at its C-terminus (mCCR9p1-19C) .

• Carrier protein conjugation: Conjugate the peptide to a carrier protein like keyhole limpet hemocyanin (KLH) to enhance immunogenicity (mCCR9p1-19C-KLH) .

• Animal immunization protocol: Implement a strategic immunization schedule. For C9Mab-24, a five-week-old Sprague–Dawley rat received an initial intraperitoneal injection of 100 μg mCCR9p1-19C-KLH peptide with Imject Alum, followed by three additional immunizations and a final booster .

• Hybridoma production: Harvest spleen cells after immunization and create hybridomas through cell fusion.

• Screening and selection: Screen hybridomas for specific antibody production using techniques like ELISA against the target peptide.

• Cloning and expansion: Clone positive hybridomas to ensure monoclonality and expand for antibody production.

• Characterization: Characterize the antibodies for specificity, affinity, and functionality in relevant applications (flow cytometry, Western blot, etc.).

• Epitope mapping: Determine the precise binding epitope using approaches like alanine scanning via ELISA .

How can CCR9 antibodies be integrated into multicolor flow cytometry panels?

Integrating CCR9 antibodies into multicolor flow cytometry panels requires careful consideration of spectral compatibility. Researchers should:

• Select appropriate fluorophore conjugates: Choose CCR9 antibody conjugates that minimize spectral overlap with other markers in your panel. For example, Alexa Fluor 488-conjugated CCR9 antibodies can be used in panels containing PE, APC, and other non-green fluorophores .

• Perform proper compensation: Since fluorochromes can have small differences in spectral emissions compared to cells, resulting in spillover values that differ when compared to biological controls, it's crucial to compare spillover on cells and compensation beads (like BD® CompBeads) when using a reagent for the first time .

• Reference spectral compatibility resources: For fluorochrome spectra and suitable instrument settings, refer to resources like BD Biosciences' Multicolor Flow Cytometry web page .

• Use isotype controls: Include appropriate isotype controls at the same concentration as the CCR9 antibody to accurately assess non-specific binding .

• Optimize antibody concentration: Titrate the CCR9 antibody to determine the optimal concentration for your specific cellular application, as applications may vary .

• Consider staining buffer components: Be aware that sodium azide, commonly used in antibody preparations, yields highly toxic hydrazoic acid under acidic conditions, which may affect certain cell types or interact with other staining components .

What are the key considerations when using CCR9 antibodies for studying immune cell trafficking?

When using CCR9 antibodies to study immune cell trafficking, researchers should consider the following:

• Functional role context: CCR9 is crucial in the chemotaxis of immune cells, particularly in gut-associated lymphoid tissue. It responds to CCL25, facilitating the migration of specific lymphocyte subsets to the small intestine .

• Cell type specificity: CCR9 is expressed by specific immune cell populations, including transitional B cells and plasmacytoid dendritic cells. Proper identification of these populations requires careful gating strategies and inclusion of additional lineage markers .

• In vivo versus in vitro applications: Consider whether you're tracking cells in vivo or studying migration in vitro, as different antibody formats and experimental approaches may be required.

• Blocking versus detection applications: Determine if you need the antibody for detection only (flow cytometry, immunohistochemistry) or for functional blocking studies to inhibit CCR9-CCL25 interactions.

• Species considerations: Human and mouse CCR9 share similarities but have distinct sequences and potentially different trafficking patterns. Ensure you're using the appropriate species-specific antibody for your research model .

• Developmental and inflammatory context: CCR9 expression and function may vary during development and under inflammatory conditions, necessitating careful experimental design and appropriate controls.

How do researchers utilize CCR9 antibodies in cancer immunotherapy research?

CCR9 antibodies have become valuable tools in cancer immunotherapy research due to CCR9's high expression in various tumors. Researchers utilize these antibodies in several ways:

• Direct therapeutic application: Anti-human CCR9 monoclonal antibodies (clones 91R and 92R) have shown promising results in preclinical studies, exhibiting cytotoxicity against CCR9-positive tumors through antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) mechanisms .

• Xenograft model studies: These antibodies have been used to suppress T-cell acute lymphoblastic leukemia (T-ALL) proliferation in mouse xenograft models, providing valuable insights into potential therapeutic applications .

• CAR-T cell development: CCR9-specific monoclonal antibodies have been utilized to develop chimeric antigen receptor (CAR)-T cells. In one notable study, researchers developed a CCR9-specific mAb by gene-gun vaccination of rats with a plasmid encoding human CCR9, then produced CAR-T cells that demonstrated potent antitumor effects in both cell lines and patient-derived xenograft mouse models of T-ALL .

• Tumor targeting and diagnostics: CCR9 antibodies can be used to identify and potentially target CCR9-expressing tumors, offering both diagnostic and therapeutic possibilities.

• Combination therapy research: Researchers are exploring how anti-CCR9 approaches might complement other immunotherapies or conventional cancer treatments.

This emerging field highlights CCR9's position as an attractive target for tumor therapy, with antibody-based approaches showing considerable promise in preclinical settings .