The Sticky Side of Immunotherapy

When DNA Mimics Disrupt Cellular Anchors

Introduction: The Promise and Peril of Synthetic DNA

Imagine a molecular soldier designed to rally immune defenses against cancer or infections—only to discover it's also sabotaging your cells' ability to stay anchored. This paradox lies at the heart of phosphorothioate-modified CpG oligodeoxynucleotides (PTO-CpG ODNs), synthetic DNA fragments engineered to mimic bacterial invaders and activate immune responses.

While celebrated as potent vaccine adjuvants and cancer immunotherapies 7 , these compounds harbor a dark side: they can unexpectedly disrupt cellular adhesion and migration. Recent research reveals how a simple chemical tweak—swapping oxygen for sulfur in their molecular backbone—triggers effects far beyond immune activation, forcing scientists to rethink therapeutic design 1 4 .

DNA structure
Key Points
  • PTO-CpG ODNs are synthetic DNA fragments
  • Used in cancer immunotherapy and vaccines
  • Can disrupt cellular adhesion

Decoding the CpG Toolkit

Key Concepts and Terminology

  • CpG Motifs: Short unmethylated cytosine-guanine sequences in bacterial/viral DNA that alert the immune system. Synthetic versions ("CpG ODNs") are therapeutic tools 7 .
  • Phosphorothioate (PTO) Modification: Replacement of a non-bridging oxygen atom with sulfur in the DNA backbone. Enhances stability against enzymes but introduces "stickiness" to proteins and cells 1 4 .
  • Toll-like Receptor 9 (TLR9): The immune sensor that detects CpG motifs, triggering cytokine production. Found in B cells and plasmacytoid dendritic cells (pDCs) 4 .
Why PTO Backbones?

Natural DNA is rapidly chewed up by nucleases. PTO modification creates a nuclease-resistant scaffold, allowing synthetic CpG ODNs to persist long enough to activate immune cells. By 2021, PTO-based CpG adjuvants were FDA-approved for hepatitis B vaccines and in >100 cancer clinical trials 7 .

When Anchors Fail: The Adhesion Crisis

Neuronal Detachment: An Accidental Discovery

In 2009, neuroscientists made a startling observation: embryonic neurons grown on polyethylenimine (PEI)—a common lab coating—detached and clumped into aggregates after PTO-CpG treatment. Crucially, this occurred even with non-stimulatory control sequences, proving the effect was independent of TLR9 signaling 1 .

The Substrate Paradox
  • Cells on PEI or poly-D-lysine showed severe detachment.
  • Cells on poly-L-ornithine remained intact 1 .

This highlighted the role of extracellular matrix (ECM) chemistry in mediating PTO toxicity.

Gene Expression Whiplash

PTO-modified and unmodified CpG ODNs triggered divergent gene expression profiles in neurons. For example:

  • Unmodified CpG or poly(I:C) (a viral RNA mimic) induced overlapping transcriptional changes.
  • PTO-CpG activated a unique set of genes tied to stress responses and adhesion loss 1 .
Growth Substrate Unmodified CpG PTO-Modified CpG Key Observations
Polyethylenimine (PEI) No detachment Severe detachment Axon bundling, cell clustering
Poly-L-ornithine No detachment No detachment Normal morphology
Laminin Mild detachment Moderate detachment Reduced neurite outgrowth
Table 1: Adhesion Effects of CpG Backbones in Neural Cells

Immune Cell Misfires: Beyond the Neuron

Amplified Inflammation

In immune cells, PTO backbones supercharge responses to bacterial toxins. When paired with lipopolysaccharide (LPS):

  • PTO ODNs boosted TNF-α production by 410% in human monocytes.
  • Unmodified or partially modified ODNs had no effect 3 .

This synergy risked "cytokine storms" in infections.

Migration Sabotage in Cancer

Tumors exploit PTO side effects. In head and neck cancer:

  • Tumor supernatants impaired pDC interferon-α production in response to CpG.
  • Paradoxically, they enhanced pDC migration toward chemokines like SDF-1, diverting immune cells into dysfunctional niches 5 .
Parameter Phosphodiester (PD) Backbone Phosphorothioate (PTO) Backbone
TLR9 Activation Efficiency Moderate High (but sequence-biased)
TNF-α Amplification with LPS None Up to 410% increase
Interferon-α Induction Strong in multimeric forms Weak unless multimerized
Nuclease Resistance Low High
Table 2: Immune Impacts of PTO vs. Phosphodiester (PD) Backbones

The Binding Dilemma: TLR9's Backbone Bias

Structural Snobbery

In 2017, crystallography studies revealed TLR9 discriminates against PTO linkages within CpG motifs. Key residues (W47, W96, K690) "sense" backbone chemistry:

  • Phosphodiester bonds fit perfectly into TLR9's binding groove.
  • Phosphorothioate bonds distort interactions, reducing activation 4 .
Hybrid Solutions

Researchers designed "backbone-swapped" ODNs:

  • H75PTO-CG1PD: PTO backbone except PD in the first CpG motif.
  • Result: 10-fold greater activation of human B cells than fully PTO versions 4 .
Molecular structure
TLR9 binding with different backbone modifications 4

Spotlight Experiment: Rescuing TLR9 Activation with Backbone Engineering

Aim

Test if restoring phosphodiester bonds in CpG motifs improves immune function.

Methods 4 :
  1. Oligonucleotide Design: Created variants of the immunostimulant "H75":
    • Fully PTO (H75PTO)
    • Hybrids: PTO backbone with PD in CpG1 (H75PTO-CG1PD), CpG2 (H75PTO-CG2PD), or both (H75PTO-CGPD).
  2. Cell Stimulation:
    • Human B cells (Ramos Blue) and primary pDCs from blood.
    • Mouse bone marrow-derived dendritic cells (BMDCs).
  3. Readouts:
    • TLR9 activation (NF-κB reporter).
    • Cytokine secretion (ELISA for IL-6, TNF-α, IFN-α).
Results:
  • Human cells: H75PTO-CG1PD outperformed H75PTO by 10-fold in TLR9 activation.
  • Mouse cells: H75PTO-CG2PD was optimal, highlighting species-specific preferences.
ODN Type IL-6 (pg/mL) TNF-α (pg/mL) IFN-α (pg/mL)
H75PTO 850 ± 120 210 ± 40 15 ± 3
H75PTO-CG1PD 2200 ± 180 490 ± 60 40 ± 5
Fold Change 2.6x ↑ 2.3x ↑ 2.7x ↑
Table 3: Cytokine Production in Primary Human pDCs
Analysis:

Hybrid ODNs balanced nuclease resistance with natural backbone recognition, offering a blueprint for safer designs.

The Scientist's Toolkit: Key Reagents for CpG Research

Reagent Function Example Use Case
Polyethylenimine (PEI) Cationic cell culture coating Detects PTO-induced neuronal detachment 1
Allylamine-Modified Nanoparticles Non-toxic siRNA/CpG carriers Delivers CpG to endosomes without polycation toxicity 6
Heparin Polyanion inhibitor Reverses PTO-enhanced TNF-α production 3
TLR9-Knockout Cells Controls for TLR9-specific effects Confirms non-TLR9 effects of PTO backbones 4
Backbone-Swapped ODNs Hybrid PD/PTO oligonucleotides Improves TLR9 activation while maintaining stability 4
Table 4: Essential Tools for Studying CpG Side Effects

Therapeutic Crossroads: Navigating Side Effects

Cancer Therapy Double Bind

In melanoma models, intratumoral PTO-CpG (e.g., CpG2018B):

  • Shrank tumors and attracted CD8+ T cells ("cold" to "hot" transition).
  • Risked off-target adhesion effects in bystander tissues 7 .
Delivery Innovations
  • Lipid Nanoparticles (LNPs): Encapsulate CpG + mRNA vaccines, reducing systemic exposure 7 .
  • Quantum Dot Silicon NPs: Control CpG multimerization to steer cytokine responses (e.g., force IFN-α from class B CpG) 6 .

Conclusion: Toward Smarter Immunostimulants

The phosphorothioate saga underscores a core truth in drug design: every chemical modification ripples beyond its intended function. As researchers refine CpG ODNs—through backbone hybrids, nanoparticle delivery, or substrate-aware assays—the goal remains clear: harness immunity without collateral damage. For now, these "sticky" side effects are not dead ends but detours, guiding us toward safer therapeutic paths.

Adapted from Okun et al., Cell Adhesion & Migration (2009) 1

References