Granted Patent B1

US 12,449,419 · App. 17/172,868 · Granted Oct 21, 2025

Inventors: Michael John Terry Stubbington (Pleasanton, CA); Luz Montesclaros (Pleasanton, CA); Katherine Pfeiffer (Oakland, CA); Sarah E. B. Taylor (Pleasanton, CA); Alvaro Martinez Barrio (Pleasanton, CA)

Assignee: 10x Genomics, Inc.

G01N33/56977C12N9/22G01N1/30B01L3/502784B01L2200/0673B01L2300/0829C12Y301/22001G01N2001/305G01N2333/70539

Abstract

Claims (34)

1. A method for detection of binding of a peptide to a T cell comprising:

(a) providing a peptide Major Histocompatibility Complex conjugate (pMHC) complex comprising a MHC monomer and a peptide, and wherein the MHC monomer comprises a reporter polynucleotide covalently attached thereto, wherein the reporter polynucleotide comprises a reporter sequence that identifies the peptide of the pMHC;

(b) presenting the pMHC monomers to T cells to generate a pMHC bound to a T cell, wherein a T cell receptor (TCR) of the T cell binds a pMHC monomer to form a pMHC-TCR complex, wherein the pMHC-TCR complex is stabilized by:

(i) using a chemical fixative;

(ii) reducing diffusion of the pMHC monomer from the TCR; and/or

(iii) retaining and/or increasing the rate of internalization of the pMHC-TCR complex into T cells;

(c) partitioning the T cell and a plurality of nucleic acid barcode molecules comprising a cell barcode sequence into a partition; and

(d) generating a barcoded nucleic acid molecule comprising (i) the reporter sequence or a reverse complement thereof and (ii) the cell barcode sequence or a reverse complement thereof.

2. The method of claim 1 , wherein the T cell comprises a template nucleic acid molecule comprising a sequence corresponding to an immune receptor; and further comprising, subsequent to (c), generating an additional barcoded nucleic acid molecule comprising (i) a sequence of a V(D)J region of the immune receptor or a reverse complement thereof and (ii) the cell barcode sequence or a reverse complement thereof.

3. The method of claim 2 , further comprising, lysing the T cell in the partition to release the template nucleic acid molecule.

4. The method of claim 3 , wherein the template nucleic acid molecule is a messenger RNA (mRNA) molecule.

5. The method of claim 1 , wherein the pMHC complex comprises a carrier, and wherein said pMHC monomer and said polynucleotide are attached to said carrier.

6. The method of claim 1 , wherein the reporter polynucleotide comprises an adapter sequence, wherein at least some of said plurality of nucleic acid barcode molecules comprise a capture sequence complementary to said adapter sequence, and wherein (d) comprises hybridizing said adapter sequence to a first nucleic acid barcode molecule and performing one or more nucleic acid reactions to generate the barcoded nucleic acid molecule.

7. The method of claim 1 , further comprising sequencing the barcoded nucleic acid molecule or a derivative thereof and/or the additional barcoded nucleic acid molecule or a derivative thereof to generate sequencing data corresponding to the barcoded nucleic acid molecule and the additional barcoded nucleic acid molecule.

8. The method of claim 1 , wherein the chemical fixative in step (b) is not a thiol-based fixative.

9. The method of claim 1 , wherein the chemical fixative is selected from the group consisting of ethylene glycol bis(succinimidyl succinate) (EGS), PEGylated bis(sulfosuccinimidyl)suberate (BS(PEG)9), BS(PEG)5, sulfo-EGS, bis-dPEG5-PFP, and bis(sulfosuccinimidyl)suberate (BS3).

10. The method of claim 1 , wherein reducing diffusion of the pMHC monomer in step (c) comprises physiochemical alterations to the pMHC monomer and/or physiochemical alterations to the composition of the binding buffer.

11. The method of claim 1 , wherein increasing the rate of internalization of the pMHC-TCR complex into T cells comprises stimulating protein kinase activity; and/or wherein retaining internalization of the pMHC-TCR complex comprises not using a protein kinase inhibitor.

12. The method of claim 1 , wherein retaining and/or increasing the rate of internalization of the pMHC-TCR complex into T cells in step (b) further comprises inhibiting lysosomal degradation of the pMHC monomers in the T cells.

13. The method of claim 12 , wherein inhibiting lysosomal degradation of the pMHC monomers comprises inhibiting DNAse II activity.

14. The method of claim 1 , wherein the reporter polynucleotide comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nuclease-resistant internucleoside linkages.

15. The method of claim 1 , wherein the plurality of nucleic acid barcode molecules are attached to a bead.

16. The method of claim 1 , wherein the partition is a droplet in an emulsion or a well in a microwell array.

17. A system for detection of binding of a peptide to a T cell comprising:

(a) a peptide-MHC conjugate (pMHC) complex comprising an MHC monomer and a peptide, and wherein the MHC monomer comprises a reporter polynucleotide covalently attached thereto, wherein the reporter polynucleotide comprises a reporter sequence that identifies the peptide of the pMHC; and

(b) a pMHC-TCR stabilization agent comprising:

(i) a chemical fixative;

(ii) an agent configured to reduce diffusion of the pMHC monomer from a TCR; and/or

(iii) an agent capable of retaining and/or increasing the rate of internalization of the pMHC-TCR complex into a T cell.

18. The system of claim 17 , further comprising:

(c) a plurality of nucleic acid barcode molecules comprising a cell barcode sequence; and

(d) a device configured to partition a plurality of cells into a plurality of partitions.

19. The system of claim 18 , wherein the device is a microwell array comprising a plurality of microwells.

20. The system of claim 18 , wherein the device is a microfluidic device configured to generate a plurality of droplets in an emulsion.

Assignments (4)

Continuity (1)

References Cited (400)