Obviousness — US Patent 11566276

Technical Analysis of Obviousness for U.S. Patent 11,566,276 under 35 U.S.C. § 103

Date of Analysis: April 26, 2026
Patent at Issue: US 11,566,276 B2 ("the '276 patent")
Relevant Legal Standard: A patent claim is invalid as obvious under 35 U.S.C. § 103 if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art (a "PHOSITA"). Obviousness can be shown by combining elements from multiple prior art references, provided there was a motivation to combine them to achieve a predictable result.

I. Core Invention of the '276 Patent

The core invention claimed in the '276 patent is a method and composition for detecting a large number of different analytes (e.g., proteins, RNA) simultaneously within a biological sample while preserving spatial information. This is achieved by using a set of "detection reagents," where each reagent consists of a probe (e.g., an antibody) that binds to a specific target analyte, and a unique, pre-assigned nucleic acid sequence that acts as a barcode. After the probes bind to their targets in situ, the locations are fixed, and the nucleic acid barcodes are amplified and sequenced directly within the sample. The identity and location of the original analytes are then determined by reading these sequenced barcodes.

The independent claims (1, 19, and 28) protect this overall method, the composition of detection reagents, and a kit containing these reagents. A key feature specified in the claims for the composition and kit is that the nucleic acid labels are not naturally present in the biological sample, so as to avoid confusion with the sample's endogenous nucleic acids.

II. Analysis of Obviousness based on Prior Art Combinations

Based on the prior art cited during the prosecution of the '276 patent and other relevant references pre-dating the priority date (December 22, 2011), the claims of the '276 patent would have been obvious to a PHOSITA. The primary combination rendering the claims obvious is Larman et al., US 2010/0047814 ("Larman") in view of either Mir, US 2005/0260633 ("Mir") or Mitra et al., "Fluorescent in situ sequencing of RNA in cells," (2010) ("Mitra").

A. Obviousness of Method Claim 1

Independent claim 1 recites a method for detecting a plurality of analytes by (a) contacting a sample with detection reagents having analyte-specific probes and unique nucleic acid labels, (b) fixing the location of the reagents, (c) amplifying the labels, and (d) sequencing the labels to identify the analytes and their locations.

Larman (US 2010/0047814): This reference teaches a method for highly multiplexed molecular profiling, particularly of proteins. Larman explicitly discloses the use of detection reagents comprised of antibodies (probes) conjugated to unique DNA oligonucleotide barcodes (nucleic acid labels). It further teaches amplifying these barcodes via PCR and identifying them using high-throughput sequencing to determine the presence and quantity of the target proteins. Thus, Larman discloses the core elements of multiplexed detection using nucleic acid-barcoded probes with a sequencing readout.
What Larman Lacks: Larman's primary embodiment describes a process where the sample is typically lysed, and the barcodes are analyzed in solution, thereby losing the crucial spatial information about where the analytes were located within the original sample. Claim 1 of the '276 patent specifically requires "fixing a location" and determining the identity of the analyte "at the location."
Mir (US 2005/0260633) and Mitra (2010): Both Mir and Mitra teach methods for performing sequencing reactions in situ.
- Mir discloses methods for analyzing single nucleic acid molecules on a solid surface or within a fixed cell. It explicitly describes immobilizing or "fixing" molecules and then performing sequencing-by-synthesis directly on the immobilized molecules, thereby preserving their spatial coordinates.
- Mitra describes a specific implementation of this concept, demonstrating "fluorescent in situ sequencing" of RNA molecules directly inside fixed cells. This work shows that sequencing chemistry is compatible with the environment of a fixed biological sample.
Motivation to Combine: A person of ordinary skill in the art in 2011 would have been well aware of the limitations of existing multiplexing technologies. Methods like those in Larman offered high multiplexing but no spatial context. Conversely, established spatial techniques like immunohistochemistry (IHC) and in situ hybridization (ISH) provided location data but were severely limited in the number of analytes that could be detected simultaneously due to spectral overlap of fluorophores.

The motivation to combine Larman with Mir or Mitra would have been strong and straightforward: to create a single method that achieves both high-plex molecular profiling and spatial resolution. A PHOSITA, seeing the powerful barcoding and sequencing readout from Larman, would look to known techniques for spatial analysis to overcome Larman's limitations. Mir and Mitra provide an explicit roadmap for performing sequencing reactions in situ on fixed samples. Combining Larman's barcoding approach with Mir's or Mitra's in situ sequencing platform would have been a predictable fusion of known technologies to solve a well-known problem. The result—spatially resolved, highly multiplexed analyte detection—would have been the expected outcome of this combination.

B. Obviousness of Composition Claim 19 and Kit Claim 28

Independent claim 19 covers the composition of detection reagents, and claim 28 covers a kit including them. The central elements are the analyte-specific probes and the unique, pre-assigned nucleic acid labels. A further limitation is that these labels are "not naturally present in the biological sample."

Larman teaches the fundamental composition: a plurality of reagents, each with a probe and a unique nucleic acid barcode.
The limitation that the nucleic acid labels are "not naturally present" would have been an obvious and necessary design principle for a PHOSITA. In any assay involving custom nucleic acid tags, probes, or primers, it is standard practice to design sequences that have minimal homology to the genome or transcriptome of the sample organism. This is done to prevent non-specific binding, cross-hybridization, and amplification of endogenous sequences, which would create background noise and invalidate the assay's results. This principle is so fundamental to molecular biology that it constitutes a routine design consideration. For example, Sampson et al., US 2009/0233822, which also describes using oligonucleotide tags for molecular identification, explicitly teaches selecting tag sequences to have minimal sequence identity to the target genome to ensure specificity.

Therefore, a PHOSITA implementing the method of Larman would have, as a matter of standard practice and obvious design, created barcode sequences that were not present in the sample to ensure the assay worked as intended. This renders the compositions of claims 19 and 28 obvious over Larman, potentially in view of the common knowledge in the art as exemplified by Sampson.

III. Conclusion

The claims of the '276 patent would have been obvious to a person of ordinary skill in the art at the time of the invention. The foundational concept of using nucleic acid barcodes to identify analytes via sequencing was taught by Larman. The novel contribution of adding in situ spatial resolution was a predictable extension of this work, motivated by the desire to combine the multiplexing power of sequencing with the spatial context of established in situ techniques, for which a clear path was provided by references like Mir and Mitra. The remaining claim limitations, such as using non-natural sequences for the barcodes, represent routine and obvious design choices for ensuring assay specificity.