Use of pattern recognition receptor (PRR) ligands to activate antigen presenting cells (APCs) is one of the pathway to augment the immunogenicity of peptide antigens. PRRs such as Toll-like receptors (TLRs), C-type lectin receptors (CLRs) etc. bind to lipopolysaccharide, lipopeptide, and other pathogen-associated molecules to facilitate APC maturation. We utilized Pam3CysSK4, a synthetic lipopeptide related to the N-terminus of Escherichia coli lipoprotein and a well-known TLR-2 ligand. The peptide portion of the molecule, SK4 was synthesized using solid phase peptide synthesis (SPPS) method using Fmoc strategy. 2-Chlorotrityl chloride resin was used for SPPS. It has been shown that peptides covalently linked to immuno-adjuvants are better at eliciting T-cell immune responses compared to their use as two different component in a mixture. Therefore, C-terminus of Pam3CysSK4 was coupled with azadibenzocyclooctyne-amine (DBCO-amine) in presence of propylphosphonic anhydride (T3P) to afford conjugatable strained-alkyne terminated immuno-adjuvant. We have synthesized a putative anticancer immunotherapeutic molecule consisting of a MUC-1 glyco-peptide bearing a Tumor Associated Carbohydrate Antigen (TACA) along with cytotoxic T-cell (CTL) epitope conjugated to Pam3CysSK4. A strain promoted alkyne-azide cycloaddition (SPAAC) was used to conjugate Pam3CysSK4 to the glycopeptide. The construct was incorporated into liposomes by the extrusion method. Liposomal delivery of synthesized construct in C57BL6 mice showed increased priming of both CD4+ and CD8+ T cells toward the MUC1-Tn cancer antigen. The primed CD8+ T-cells were capable of tumor cell killing. In addition, we synthesized a peptide-based immunotherapeutic construct with the goal of modulating immunity in type 1 diabetes (T1D). We prepared a single molecule constructs consisting of Pam3CysSK4 and GAD 546-554 peptide, a pathogenic CTL-inducing epitope of GAD 65 (Glutamic acid decarboxylate). When the pathogenic bioconjugate treated macrophage cell line was incubated with GAD 546-554 specific CD8 T cells, IFNg and TNFa release was increased significantly. Subtle modification of the pathogenic epitope (altered peptide ligand, APL) can induce tolerance in autoreactive T cells. Immunogenic epitope substitution with APL in bioconjugate platform can lead to a peptide based immunotherapy targeting the autoimmunity to prevent or reverse T1D.

Synthesis of lipopeptide-based immunotherapeutics using a conjugatable immuno-adjuvant and evaluation of immune responses

256th National Meeting (2018)

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