# Proposed next study: linked canine biological evidence

## What the computational work supports

The public-data evaluation checks sequence construction, matched tumour RNA support,
canine presentation ranking against artificial backgrounds, and human assay-panel ranking.
It does not establish patient-specific canine DLA nominations, mutant peptide display,
immune recognition, tumour killing or treatment benefit. Mixed and negative results remain
part of the evidence.

## Proposed collaboration questions

A veterinary biobank, sequencing facility and qualified immunology laboratory could assess
whether a new cohort can link matched tumour/normal DNA, tumour RNA, DLA typing and
expression to binding/stability and feasible functional assays. OVC Veterinary Biobank
and the University of Guelph AAC are prospective routes; participation, access rights,
exclusivity, funding and assay agreements are not established by this brief.

| Step | Question | Feasibility and evidence needed |
|---|---|---|
| Specimen inventory | Which samples support linked analysis? | Identity, tumour/normal matching, RNA quality, available material and access/consent terms |
| DLA characterization | Which DLA alleles are present and expressed? | Qualified typing resolution, expression strategy and uncertainty reporting |
| Mutant RNA | Is the mutated sequence supported in the tumour? | Prespecified coverage/quality rules, reference context and low-coverage reporting |
| Binding/stability | Does the peptide bind the intended DLA and remain bound? | Qualified assays, appropriate reagents, matched normal-peptide controls and distinct endpoints |
| Functional recognition | Is there a peptide-specific immune response? | Suitable viable immune material, assay-specific controls and laboratory feasibility assessment |
| Tumour recognition | Does a response extend to tumour cells and killing? | Suitable target cells and a separately qualified functional system; not inferred from peptide reactivity |

A molecular cohort and a smaller functional-assay subset may have different eligibility
criteria. Request a case-by-modality inventory before choosing cohort size or targets.
Plasma or stored tissue alone does not establish viable T-cell availability. The assay
laboratory should specify collection, processing and preservation requirements before
new sampling. This is a discussion brief, not a laboratory protocol.

## Study-design decisions to settle with collaborators

- Cohort size, specimen and assay eligibility, sample identity and batch handling.
- Prespecified endpoints, controls, comparison candidates and interpretation criteria.
- Separation of method development from held-out biological evaluation.
- Recording of every selected/assayed candidate, failed assay and negative outcome.
- Budget, responsibilities, timelines and publication arrangements.
- Data use, commercial use, ownership and access rights; no automatic exclusivity assumed.

An immune assay would not itself qualify an mRNA–LNP product or authorize clinical
administration. Formulation and in-vivo work require separate readiness evidence and
applicable permissions. No laboratory result, partner endorsement or clinical approval
is claimed here.