KLK peptide (KLKL5KLK) and KPK peptide (KPKP5KPK) were synthesized by CPC Scientific, San Jose, CA, USA.
Abstract
Endosomal Toll-like receptors (TLR) such as TLR3, 7, 8 and 9 recognize pathogen associated nucleic acids. While DNA sequence does influence degree of binding to and activation of TLR9, it also appears to influence the ability of the ligand to reach the intracellular endosomal compartment. The KLK (KLKL5KLK) antimicrobial peptide, which is immunostimulatory itself, can translocate into cells without cell membrane permeabilization and thus can be used for endosomal delivery of TLR agonists, as has been shown with the IC31 formulation that contains an oligodeoxynucleotide (ODN) TLR9 agonist. We evaluated the adjuvant activity of KLK combined with CpG or non-CpG (GpC) ODN synthesized with nuclease resistant phosphorothioate (S) or native phosphodiester (O) backbones with ovalbumin (OVA) antigen in mice. As single adjuvants, CpG(S) gave the strongest enhancement of OVA-specific immunity and the addition of KLK provided no benefit and was actually detrimental for some readouts. In contrast, KLK enhanced the adjuvant effects of CpG(O) and to a lesser extent of GpC (S), which on their own had little or no activity. Indeed while CD8 T cells, IFN-γ secretion and humoral response to vaccine antigen were enhanced when CpG(O) was combined with KLK, only IFN-γ secretion was enhanced when GpC (S) was combined to KLK. The synergistic adjuvant effects with KLK/ODN combinations were TLR9-mediated since they did not occur in TLR9 knock-out mice. We hypothesize that a nuclease resistant ODN with CpG motifs has its own mechanism for entering cells to reach the endosome. For ODN without CpG motifs, KLK appears to provide an alternate mechanism for accessing the endosome, where it can activate TLR9, albeit with lower potency than a CpG ODN. For nuclease sensitive (O) backbone ODN, KLK may also provide protection from nucleases in the tissues.
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CPC Scientific at DCAT 2025!
GMP Peptide Manufacturing
What an amazing time we had at DCAT 2025! It was fantastic to meet with colleagues, clients, and friends, and showcase CPC Scientific’s cutting-edge capabilities in synthetic API development and commercial production.
A heartfelt […]
We are excited to share that CPC Scientific Inc. has officially received the Good Manufacturing Practice (GMP) Certificate in accordance with the ISO 22716:2007(E) Cosmetic Guidelines.
This certification highlights our unwavering commitment to delivering the highest quality peptide ingredients for cosmetic use, guaranteeing:
- Superior Quality Control – Striving for excellence in safety, consistency, and purity.
[…]
In Part 1 of our Minimal Protection Group Strategies for SPPS, we discussed methods for eliminating sidechain protection on hydroxy-bearing amino acids such as serine, threonine, tyrosine, and hydroxyproline. By omitting t-butyl protection, we enhanced atom economy and avoided the use of hazardous solvents typically required to remove these protection groups. In Part 2, we present a new case study, expanding our approach to include the unprotected side chains of histidine, tryptophan, and arginine. We demonstrate the synthesis of a Goserelin peptide API impurity, showcasing how a convergent peptide fragment strategy can be used to eliminate the need for TFA and diethyl ether, eliminate side chain protection of Arginine, Histidine, and Tryptophan.
The synthesis of the linear RP-182 analog, bicyclo[6.1.0]non-4-yn-9-ylmethyloxycarbonyl-PEG2-Lys-Phe-Arg-Lys-Ala-Phe-Lys-Arg-Phe-Phe-Lys(azido-PEG)-NH2, was achieved using standard solid-phase peptide synthesis (SPPS) protocols. After cleaving the linear peptide from the resin, macrocyclization was performed in the liquid phase through a strain-promoted click reaction. BCN introduces extra ring strain due to its fused cyclopropane structure. The combined effect of ring strain, the selection of BCN, and copper catalysis significantly increases the macrocyclization efficiency of longer peptides like RP-182.
Nakagawa, Mayumi, Teresa Evans, Milan Bimali, Hannah Coleman, Jasmine Crane, Nadia Darwish, Jennifer L. Faulkner et al. MedRxiv (2025): 2025-01.
The vaccine consisted of four current good manufacturing production-grade synthetic peptides covering the HPV 16 E6 protein [amino acid (aa)1-45, 46-80, 81-115, and 116-158] (CPC Scientific, San Jose, CA [..]
Stapled peptides have emerged as a powerful tool in drug discovery and therapeutic development due to their ability to overcome the limitations associated with traditional peptide drugs, such as poor stability and low cell permeability. By introducing staples into the peptide backbone, researchers can stabilize peptide conformations and enhance their interactions with target proteins, resulting in improved efficacy and specificity. This approach not only addresses the challenges of peptide drug design but also opens new avenues for targeting challenging biomolecular interactions that are difficult to modulate with small molecules or antibodies. The development of stapled peptides has led to significant advancements in targeting protein-protein interactions, addressing previously intractable diseases, and enhancing the precision of therapeutic interventions.
Peptide receptor radionuclide therapy (PRRT) is a targeted therapeutic approach that utilizes peptides to deliver cytotoxic radiation to specific receptors overexpressed on cancer cells. Peptides offer several advantages as therapeutic vectors in PRRT due to their small size, favorable pharmacokinetics, high binding affinity, low immunogenicity and toxicity, and minimal off-target effects. Tumor-targeting peptides conjugated to radionuclide chelates represent a promising class of cancer therapeutics.
Singh, S.S., Calvo, R., Kumari, A., Sable, R.V., Fang, Y., Tao, D., Hu, X., Castle, S.G., Nahar, S., Li, D. and Major, E. Molecular Cancer Therapeutics (2024).
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[..] assembling the peptide on the Rink Amide resin and attaching the PEG azide moiety to the N-terminal Lys, the Dde group was removed as previously shown and coupled to the Fmoc-PEG2-acid. Removal of the Fmoc followed by simultaneously click/coupling to bicyclo[6.1.0]non-4-yn-9-ylmethyl (2,5-dioxopyrrolidin-1-yl) carbonate gave 1c which was deprotected and cleaved from the resin to give 1c.
Most proteinogenic peptides, except for certain hydrophobic sequences, can be synthesized in a linear fashion using solid-phase peptide synthesis (SPPS) methods. However, longer sequences, particularly those exceeding 70 amino acids, often require alternative techniques due to challenges like steric hindrance. Other issues, such as poor solvation of the protected peptide during synthesis and the formation of intermolecular hydrogen bonds (e.g., β-sheets) between fragments, can also result in inefficient coupling and deprotection.