CPL4 Antibody

What are endolysins and how do they function as antibacterial agents?

Endolysins like Pal and Cpl-1 are bacteriolytic enzymes derived from bacteriophages (Pal from Streptococcus phage Dp-1 and Cpl-1 from Streptococcus phage Cp-1) that function as promising antibacterial agents. These enzymes degrade bacterial cell walls, leading to bacterial lysis and death. Their activity is targeted primarily against specific bacterial species, making them potential alternatives to conventional antibiotics .

The effectiveness of these enzymes depends on their ability to maintain proper tertiary structure and enzymatic activity while targeting specific bacterial cell wall components. When designing experiments with these enzymes, researchers should evaluate their activity across a range of concentrations and against appropriate bacterial strains to establish dose-response relationships .

Why do bacteriolytic enzymes induce immune responses and how does this impact their therapeutic potential?

Bacteriolytic enzymes like Cpl-1 and Pal can induce typical immune responses when administered in vivo, leading to the production of antibodies that may neutralize their activity. This immunogenicity presents a significant challenge for therapeutic applications, particularly for repeated administrations where neutralizing antibodies can render the enzymes ineffective .

The immune system recognizes specific epitopes (immunogenic regions) on these proteins, generating antibodies that bind to these regions and potentially inhibit enzymatic activity either directly (by blocking the catalytic site) or indirectly (by causing conformational changes or steric hindrance). Understanding and addressing this immunogenicity is crucial for developing these enzymes as effective therapeutic agents .

How do anti-CTLA-4 antibodies modulate immune responses in cancer research?

Anti-CTLA-4 antibodies function by blocking CTLA-4 (Cytotoxic T-Lymphocyte Associated Protein 4), an immune checkpoint receptor that normally downregulates immune responses. In cancer research, these antibodies enhance anti-tumor immunity by preventing inhibitory signals that suppress T-cell activity, effectively "removing the brakes" from the immune system .

The methodology for studying these effects typically involves administering the antibodies to experimental models with established tumors and evaluating both tumor response and autoimmune manifestations through histopathological analysis, flow cytometry of immune cell populations, and functional assays .

How can epitope scanning be used to engineer endolysins with altered immunogenicity?

Epitope scanning represents a sophisticated approach to modifying the immunogenicity of proteins while maintaining their functional activity. For endolysins like Pal and Cpl-1, this process involves:

• Identifying key immunogenic amino acids through in silico analysis and experimental validation

• Designing variants by substituting these amino acids with residues that differ in electrostatic charge and chemical structure

• Selecting substitutions that minimize increases in protein folding energy (ΔΔG)

• Preferring smaller amino acids to minimize steric tensions

• Avoiding modifications to the catalytic domain to preserve enzymatic activity

This approach allows for the creation of variants with either decreased immunogenicity (e.g., Pal variant 257-259 MKS → TFG) or increased immunogenicity (e.g., Pal variant 257-259 MKS → TFK), depending on the research objectives. Most significantly, variants like Pal 280-282 DKP → GGA can demonstrate significantly increased antibacterial activity while escaping cross-neutralization by antibodies induced by the wild-type enzyme .

What methodological approaches can be used to evaluate cross-neutralization between antibodies and engineered enzyme variants?

Evaluating cross-neutralization between antibodies and engineered enzyme variants requires a multi-faceted approach:

• Antibody generation: Challenge experimental animals (e.g., C57BL6/J mice) with the wild-type enzyme or its variants (typically without adjuvants to mimic clinical situations), collect serum after an appropriate interval (e.g., 28 days), and separate it through double centrifugation .

• Antibacterial activity assessment: Test the enzyme variants against sensitive bacteria (e.g., pneumococci for Pal and Cpl-1) across a range of concentrations, comparing their activity to that of the wild-type enzyme .

• Cross-neutralization assays: Pre-incubate the engineered variants with antibodies generated against the wild-type enzyme (and vice versa) and measure residual enzymatic or antibacterial activity .

This methodological approach allows researchers to identify variants that not only maintain (or enhance) enzymatic activity but also escape neutralization by antibodies generated against the original protein .

How does structural modeling contribute to the design of engineered enzyme variants?

Structural modeling plays a critical role in the design of engineered enzyme variants by providing a framework for rational amino acid substitutions. For proteins with known crystal structures (like Cpl-1, PDB Code: 2IXU), the three-dimensional structure can be downloaded directly from the Protein Data Bank. For proteins without determined structures (like Pal), homology modeling using software such as I-Tasser can generate structural predictions .

Once a structural model is obtained, software like FoldX can calculate the differences in free folding energy (ΔΔG) for each potential amino acid substitution, helping researchers select modifications that maintain protein stability. This approach guides the engineering process by:

What expression and purification strategies are optimal for engineered endolysin variants?

Based on the search results, the following expression and purification protocol has proven effective for engineered endolysin variants:

• Vector construction: Clone the coding sequences into appropriate expression vectors (e.g., pBAD24 or pBAD_HisA) with a C-terminal 6xHis tag. For novel variants, synthesize the genes de novo or introduce mutations via PCR with primers coding for the mutagenized sites .

• Expression system: Transform the vectors into E. coli B834(DE3) cells and grow them at 37°C with shaking in LB broth supplemented with appropriate antibiotics (e.g., ampicillin at 50 mg/L) until reaching the desired optical density (OD600 ≈ 1.0) .

• Induction: Induce protein expression by adding arabinose to a final concentration of 2.5 g/L (0.25%) and incubate overnight at a reduced temperature (22°C) with intensive shaking to maximize protein solubility .

• Harvest and purification: Harvest bacteria by centrifugation (7,000 × g, 5 min), resuspend in an appropriate buffer, and purify the His-tagged proteins using standard affinity chromatography followed by quality assessment via SDS-PAGE and activity assays .

This methodological approach ensures consistent production of soluble, active enzyme variants for subsequent analysis.

What methods can be used to evaluate the binding properties of antibodies to their targets?

Several complementary methods can be used to evaluate antibody binding:

• ELISA (Enzyme-Linked Immunosorbent Assay): Pre-coat plates with the target protein (e.g., His-tagged CTLA-4 at 1 μg/mL), incubate with the antibody of interest, and detect binding using an HRP-conjugated secondary antibody. This provides quantitative information about relative binding affinities .

• Flow cytometry: Incubate cells expressing the target protein (e.g., CHO cells expressing CTLA-4) with the antibody, detect binding with a fluorophore-conjugated secondary antibody, and analyze using flow cytometry. This approach evaluates binding to the protein in its native conformation on the cell surface .

• SDS-PAGE and Western blotting: For antibody-drug conjugates or modified antibodies, SDS-PAGE can confirm size shifts after modification, while Western blotting can verify target specificity .

How can researchers assess the in vivo effects of antibody treatments on immune cell populations?

Assessing the in vivo effects of antibody treatments on immune cell populations requires a comprehensive approach:

• Experimental design: Treat experimental animals with the antibody of interest (e.g., 100 μg/mouse administered intraperitoneally) according to a defined schedule (e.g., every three days) .

• Sample collection: Collect blood samples at appropriate timepoints, and harvest tissues of interest (e.g., bone marrow, lymph nodes, tumor tissue) for analysis .

• Flow cytometry analysis: Process the samples and perform multi-parameter flow cytometry to identify and quantify specific cell populations (e.g., regulatory T cells defined as CD4+ Foxp3+, B cells defined as B220+, or more specific subpopulations) .

• Functional assays: Assess cell proliferation (e.g., using Ki67 staining), activation status (via surface markers), and cytokine production to evaluate functional changes in addition to numerical alterations .

This methodological approach provides insights into both the quantitative and qualitative changes in immune cell populations following antibody treatment, critical for understanding mechanisms of action and potential side effects.

How can researchers interpret unexpected variations in antibacterial activity among engineered endolysin variants?

When interpreting unexpected variations in antibacterial activity among engineered endolysin variants, researchers should consider several factors:

• Structural effects beyond the substitution site: Even mutations designed to alter only immunogenicity may have subtle effects on protein folding, leading to changes in catalytic site geometry or substrate accessibility. Comprehensive structural analysis using techniques like circular dichroism spectroscopy can help evaluate these effects .

• Substrate binding affinity: Modifications might alter the binding affinity for cell wall components, even if they are distant from the binding domain. Surface plasmon resonance or other binding assays can quantify these effects .

• Protein stability and solubility: Some variants may have altered stability or solubility in physiological conditions, affecting their effective concentration and activity. Thermal shift assays and solubility testing under various conditions can address these possibilities .

• Unexpected synergistic effects: In some cases, mutations intended to reduce immunogenicity may serendipitously enhance activity by improving protein dynamics or reducing inhibitory interactions, as observed with the Pal variant 280-282 DKP → GGA, which showed significantly increased antibacterial activity .

These considerations inform both the interpretation of current results and the design of next-generation variants with optimized properties.

What approaches can be used to develop combination antibody therapies that enhance efficacy while reducing adverse effects?

Developing effective combination antibody therapies requires:

• Mechanistic understanding: Thoroughly investigate the mechanisms of action of individual antibodies to identify potential synergistic or antagonistic pathways. For example, combining anti-CTLA-4 and anti-4-1BB antibodies enhanced cancer immunity while unexpectedly reducing autoimmunity, contrary to what might be predicted from the effects of anti-CTLA-4 alone .

• Cell-specific effects: Analyze how different antibody combinations affect specific cell populations. The combination of anti-CTLA-4 and anti-4-1BB led to CD8 T-cell-mediated rejection of established tumors while enhancing regulatory T cell function, providing a mechanistic explanation for the reduced autoimmunity .

• Tumor-specific responses: Evaluate combination therapies across different tumor models, as efficacy may vary. The anti-CTLA-4/anti-4-1BB combination was effective against MC38 tumors but not against B16 melanoma, highlighting the importance of tumor-specific factors .

• Dosing and scheduling optimization: Systematically evaluate different doses and treatment schedules to identify optimal therapeutic windows that maximize efficacy while minimizing adverse effects .

This integrated approach can lead to counterintuitive but highly effective combination strategies that simultaneously enhance cancer immunity while reducing autoimmune manifestations.

How does antibody-mediated modulation of immune cells impact the tumor microenvironment?

Antibody-mediated modulation of immune cells can profoundly impact the tumor microenvironment through several mechanisms:

• Direct effects on tumor-infiltrating lymphocytes: Antibodies like anti-CTLA-4 can enhance the activation and proliferation of tumor-specific T cells, leading to increased infiltration and cytotoxic activity within the tumor .

• Modulation of regulatory T cells: Certain antibodies or antibody-drug conjugates can deplete or functionally impair regulatory T cells within the tumor microenvironment, reducing their immunosuppressive effects. For example, CTLA-4 antibody-drug conjugates can impair regulatory T-cells in the tumor microenvironment .

• Effects on B cell populations: Some antibody treatments can lead to B cell depletion, which might alter antigen presentation and antibody production within the tumor microenvironment. CTLA-4 antibody-drug conjugates have been shown to cause B-cell depletion, potentially affecting tumor immunity .

• Increased cytotoxic T cell infiltration: Humanized antibodies against targets like CKAP4 not only directly suppress cancer cell growth by inhibiting specific signaling pathways but also induce increased infiltration of cytotoxic T cells into the tumor microenvironment, enhancing anti-tumor immune responses .

This complex interplay between different immune cell populations ultimately determines the efficacy of antibody-based immunotherapies and provides opportunities for rational combination approaches.