Clonal vs. Subclonal Neoantigens: Why Tumor Evolution Determines Immunotherapy Response

By Lociven · NeoantigenLab · July 2026

Figure 1. CCF distribution (left) and objective response rate by clonal neoantigen load (right).

Two patients have the same TMB. One responds to checkpoint immunotherapy. One doesn't. Why?

Part of the answer lies in whether their mutations are clonal or subclonal. This distinction — where in tumor evolution a mutation occurred — turns out to matter as much as how many mutations exist. This post explains the biology, how to determine clonality from sequencing data, and why it should inform how you prioritize neoantigen candidates.

Clonal vs. subclonal: the basic distinction

Tumors are not genetically uniform. They evolve through successive rounds of mutation, selection, and clonal expansion. Mutations that occurred early — before the major clonal expansion — are present in virtually every tumor cell. These are clonal mutations.

Mutations that accumulated later, after the tumor had already diversified into subpopulations, are present in only a fraction of cells. These are subclonal mutations.

The practical consequence: neoantigens derived from clonal mutations are displayed on every tumor cell. Neoantigens from subclonal mutations may only be on 10–30% of cells — or less.

Why clonality predicts immunotherapy response

The landmark McGranahan et al. paper in Science (2016) showed that high clonal neoantigen burden — not total TMB — was the strongest predictor of response to checkpoint inhibitors in lung cancer and melanoma. Patients with many subclonal neoantigens but few clonal neoantigens did not respond well, even when total TMB was high.

The immune logic is straightforward:

  • A T cell that recognizes a clonal neoantigen can kill every tumor cell — it's a universal target
  • A T cell that recognizes a subclonal neoantigen can only kill the subclone expressing that antigen — the rest of the tumor survives and can repopulate
  • Under immune pressure, subclonal neoantigen-expressing cells can be eliminated while antigen-loss variants (without the mutation) expand — a form of immune editing

How to determine clonality from sequencing data

Clonality is estimated from variant allele frequency (VAF) in tumor WES, corrected for tumor purity and local copy number.

The key formula:

Cancer cell fraction (CCF) = VAF × (copy number / tumor purity)

A mutation with CCF ≈ 1.0 is clonal — present in essentially all tumor cells. A mutation with CCF < 0.5 is subclonal.

Tools that automate this:

  • ABSOLUTE: Estimates tumor purity, ploidy, and CCF per mutation from WES data
  • PyClone / PyClone-VI: Clusters mutations by CCF to identify clonal architecture
  • TITAN / FACETS: Copy number analysis needed as input for CCF calculation

Clonality in vaccine design

For neoantigen vaccines, targeting clonal neoantigens is even more critical than for checkpoint immunotherapy. A vaccine primes T cells against specific epitopes — if those epitopes are only on a subclone, the vaccine creates T cells that eliminate a small fraction of the tumor and exert selection pressure for antigen loss in the rest.

The mRNA-4157 pipeline uses VAF as one of the ranking criteria for this reason. High-VAF, high-CCF mutations get prioritized. In practice, tumors with predominantly clonal architecture (early-stage or surgically resected tumors that haven't undergone extensive subclonal diversification) are better vaccine candidates than heavily treated, highly heterogeneous tumors.

Tumor heterogeneity and the biopsy problem

Single-site biopsies only sample one region of the tumor. Subclonal mutations in the sampled region may be clonal in another region — and vice versa. This is the intratumor heterogeneity (ITH) problem.

Multi-region sequencing studies (TRACERx in lung cancer) have shown that clonal neoantigen burden estimated from a single biopsy correlates reasonably well with multi-region estimates — because truly clonal mutations are present everywhere by definition. But subclonal architecture is highly variable across biopsy sites.

For practical purposes: single-site WES is sufficient to identify clonal candidates. If you are designing a clinical trial and need comprehensive neoantigen coverage, liquid biopsy (ctDNA) can complement tissue sequencing to capture spatial heterogeneity.

The next post reviews the current landscape of neoantigen vaccine clinical trials beyond mRNA-4157 — what other platforms are in development and what the trial results tell us so far.

Tags: clonal neoantigen, subclonal mutation, tumor heterogeneity, VAF, cancer cell fraction, checkpoint inhibitor, neoantigen vaccine, TRACERx

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