阅读说明：本技术 用于鉴定抗原特异性t细胞的组合物和方法 (Compositions and methods for identifying antigen-specific T cells ) 是由 彭松明 博伊·布莱恩特·夸克 安朵 罗伯特·包晓研 亚历克西斯·弗兰佐索夫 芭芭拉·森尼诺 于 2020-02-12 设计创作，主要内容包括：本文公开了抗原肽-MHC复合物,称为comPACT多肽和comPACT多核苷酸,以及产生这种复合物的方法。本文还公开了产生comPACT多核苷酸和多肽的文库的方法,以及它们在以高精度捕获癌症新表位反应性T细胞中的示例性用途。提供了用于检测具有改进的灵敏度和特异性的新抗原特异性T细胞的双颗粒检测方法。还提供了分离的T细胞的信噪比分析,其用于检测具有改进的T细胞的新抗原特异性T细胞。(Disclosed herein are antigenic peptide-MHC complexes, referred to as comPACT polypeptides and comPACT polynucleotides, and methods of producing such complexes. Also disclosed herein are methods of generating libraries of comPACT polynucleotides and polypeptides, and their exemplary uses in capturing cancer neo-epitope reactive T cells with high accuracy. A dual particle detection method for detecting neoantigen-specific T cells with improved sensitivity and specificity is provided. Also provided is a signal to noise ratio assay of isolated T cells for detecting neoantigen-specific T cells with improved T cells.)

1. A method, comprising:

a. contacting the sample with a plurality of different sets of particles,

i. wherein each particle comprises a unique antigenic peptide, an operably associated barcode, and at least one identifying label,

wherein the sample comprises T cells, and

Wherein said contacting comprises providing conditions suitable for binding of a single T cell to a unique antigenic peptide of at least one collection of particles;

b. isolating the one or more T cells bound to the particle;

c. identifying barcodes of particles bound to the isolated T cells;

d. the ratio of each barcode is determined.

2. The method of claim 1, wherein the ratio is calculated by identifying the copy number of the first barcode and the copy number of the second barcode and dividing the copy number of the first barcode by the copy number of the second barcode.

3. The method of any one of claims 1-2, wherein the unique antigenic peptide is the same for each different set of particles.

4. The method of any one of claims 1-3, wherein each distinct set of particles comprises at least one or more barcodes, wherein each barcode is associated with an identity of the antigenic peptide.

5. The method of any one of claims 1-4, wherein the ratio of each barcode corresponds to the antigen specificity of the isolated T cells.

6. The method of any one of claims 1-5, wherein the isolated T cells are identified as antigen-specific T cells if the ratio of the first barcodes is above a threshold.

7. The method of claim 6, wherein the threshold is at least or greater than 2,3,4,5,6,7,8,9,10,2-5,3-6,4-7,5-8,5-10,7-10, or greater than 10.

8. The method of any one of claims 1-7, wherein identifying the barcode comprises a nucleotide-based assay.

9. The method of claim 8, wherein the nucleotide-based assay is a PCR, RT-PCR, sequencing, or hybridization assay.

10. The method of any one of claims 8 or 9, wherein the nucleotide-based assay determines (a) the sequence per barcode and/or (b) the copy number per barcode.

11. The method of any one of claims 1-10, further comprising obtaining a T Cell Receptor (TCR) CDR sequence.

12. The method of any of the above claims, further comprising obtaining a TCR gene sequence.

13. The method of claim 12, wherein the TCR sequence is a TCR α or TCR β chain sequence.

14. The method of any one of the preceding claims, for identifying the antigen specificity of T cells.

15. The method of claim 14, wherein the antigen specificity of the T cell comprises the sequence of the antigenic peptide and the TCR sequence of the bound T cell.

16. The method of any one of claims 1-15, wherein the at least one identifying mark is the same in each distinct set of particles.

17. The method of any one of claims 1-16, comprising at least two different identifying marks.

18. The method of any one of claims 1-17, wherein the at least one identifying label is a fluorophore.

19. The method of claim 18, wherein the fluorophore is selected from the group consisting of Allophycocyanin (APC) and Phycoerythrin (PE).

20. The method of claim 17, wherein the at least two different signature markers are fluorophores, wherein the fluorophores are selected from the group consisting of Allophycocyanin (APC) and Phycoerythrin (PE).

21. The method of any one of claims 1-20, wherein the antigenic peptide is selected from the group consisting of: tumor antigens, neoantigens, tumor neoantigens, viral antigens, bacterial antigens, phospho-antigens, and microbial antigens.

22. The method of claim 21, wherein the neoantigen is identified from tumor sequencing data of the subject.

23. The method of claim 22, wherein a computer predictive algorithm is used to determine neoantigens.

24. The method of claim 23, wherein the prediction algorithm further comprises an MHC binding algorithm to predict binding between a neoantigen and an MHC peptide.

25. The method of any one of claims 1-24, wherein the sample is selected from a blood sample, a bone marrow sample, a tissue sample, a tumor sample, or a Peripheral Blood Mononuclear Cell (PBMC) sample.

26. The method of any one of claims 1-25, wherein the T cell is a human T cell.

27. The method of claim 26, wherein the T cell is a CD8+ T cell.

28. The method of any one of claims 1-27, wherein the method comprises a library of different collections of particles.

29. The method of claim 28, wherein the library comprises a collection of 2 to 500 distinct particles.

30. The method of any one of claims 1-29, wherein each particle comprises an MHC peptide.

31. The method of claim 30, wherein the MHC peptide is a human MHC peptide.

32. The method of claim 30, wherein the MHC peptide is a class I HLA peptide.

33. The method of claim 30, wherein the HLA peptides comprise HLA-A, HLA-B or HLA-C peptides.

34. The method of claim 33, wherein the HLA peptide comprises HLA-A01: 01, HLA-A02: 01, HLA-A03: 01, HLA-A24: 02, HLA-A30: 02, HLA-A31: 01, HLA-A32: 01, HLA-A33: 01, HLA-A68: 01, HLA-A11: 01, HLA-A23: 01, HLA-A30: 01, HLA-A33: 03, HLA-A25: 01, HLA-A26: 01, HLA-A29: 02, HLA-A68: 02, HLA-B07: 02, HLA-B14: 02, HLA-B18: 01, HLA-B02: 27, HLA-B01, HLA-A18: 44, HLA-B33: 44, HLA-B46: 01, HLA-B50: 01, HLA-B57: 01, HLA-B58: 01, HLA-B08: 01, HLA-B15: 03, HLA-B35: 01, HLA-B40: 02, HLA-B42: 01, HLA-B44: 03, HLA-B51: 01, HLA-B53: 01, HLA-B13: 02, HLA-B15: 07, HLA-B27: 05, HLA-B35: 03, HLA-B37: 01, HLA-B38: 01, HLA-B41: 02, HLA-B44: 05, HLA-B49: 49, HLA-C55, HLA-B55: 55, HLA-C05: 01, HLA-C07: 01, HLA-C01: 02, HLA-C04: 01, HLA-C06: 02, HLA-C07: 02, HLA-C16: 01, HLA-C03: 03, HLA-C07: 04, HLA-C08: 01, HLA-C08: 02, HLA-C12: 03, HLA-C14: 02, HLA-C15: 02 or HLA-C17: 01.

35. The method of any one of claims 1-34, wherein each particle comprises an HLA peptide and a β 2M peptide.

36. The method of claim 35, wherein the β 2M peptide is a human β 2M peptide.

37. The method of claim 36, wherein the β 2M peptide comprises a mutation.

38. The method of claim 37, wherein the mutation is S88C.

39. The method of any one of claims 1-38, wherein each particle comprises a polypeptide comprising in the amino to carboxy terminal direction (i) an antigenic peptide, (ii) a β 2M peptide, and (iii) an MHC peptide.

40. The method of any one of claims 1-39, wherein the antigenic peptide is 7-15 amino acids, 7-10,8-9,7,8,9,10,11,12,13,14, or 15 amino acids in length.

41. The method of claim 39 or 40, wherein the polypeptide is biotinylated.

42. The method of any one of claims 1-41, wherein the particles are selected from the group consisting of: magnetic beads, agarose beads, styrene polymer particles, and dextran polymer particles.

43. The method of any one of claims 1-42, wherein the particles are coated with streptavidin.

44. The method of any one of claims 1-43, which is used to monitor an immune repertoire in a subject.

45. The method of claim 44, further comprising monitoring the subject for changes in antigen-specific T cells.

46. The method of claim 44 or 45, comprising administering immunotherapy to the subject.

47. The method of claim 46, wherein the immunotherapy is an adoptive cell transfer or checkpoint inhibitor.

48. The method of any one of claims 1-47, which is used to identify at least one TCR sequence.

49. The method of claim 48, wherein the at least one TCR sequence is a TCR α sequence, a TCR β sequence, or a combination thereof.

50. The method of claim 48 or 49, further comprising making a soluble TCR polypeptide.

51. A library comprising at least two sets of particles, each set of particles comprising an antigenic peptide, a barcode operably associated with the identity of the antigenic peptide, and at least one identifying tag.

52. The library of claim 51, wherein the at least one identifying tag is the same in each collection of particles.

53. The library of claim 51 or 52, comprising at least two different identifying labels in each different set of particles.

54. The library of any one of claims 51-53, wherein the at least one identifying label is a fluorophore.

55. The library of claim 54, wherein the fluorophore is selected from the group consisting of Allophycocyanin (APC) and Phycoerythrin (PE).

56. The library of claim 53, wherein the at least two different signature tags are fluorophores, wherein the fluorophores are selected from the group consisting of Allophycocyanin (APC) and Phycoerythrin (PE).

57. A particle comprising at least one polypeptide, a barcode, and an identifying label, wherein the polypeptide comprises an antigenic peptide, a β 2M peptide, and an MHC peptide, and wherein the barcode is operably associated with the identity of the antigenic peptide.

58. The particle of claim 59, selected from the group consisting of: magnetic beads, agarose beads, styrene polymer particles, and dextran polymer particles.

59. The particle of claim 57 or 58, wherein the identifying label is a fluorophore.

60. The particle of any one of claims 57-59, which is coated with streptavidin.

61. The particle of any one of claims 57-60, wherein the polypeptide is labeled.

62. A method of treating cancer in a subject, comprising:

a. preparing a plurality of particles, each particle comprising a plurality of labeled polypeptides, wherein the polypeptides comprise an antigenic peptide, a β 2M sequence, an HLA sequence, and a detectable label;

b. Contacting the plurality of particles with a plurality of T cells from a subject under conditions suitable for antigen-specific binding of the T cells to the particles;

c. isolating the T cells bound to the particles and identifying the TCR gene sequence of the isolated T cells;

d. preparing a polynucleotide comprising homology arms and at least one TCR gene sequence, wherein the TCR gene sequence is located between the homology arms;

e. recombining said polynucleotide into an endogenous locus of said subject's T cells;

f. culturing the modified T cells to produce a population of T cells; and

g. administering to the subject a therapeutically effective amount of the modified T cell thereby treating the cancer.

63. A method of modifying a cell, comprising:

a. introducing into the cell a Homologous Recombination (HR) template nucleic acid sequence comprising:

i. first and second homology arms homologous to first and second endogenous sequences of the cell;

a T Cell Receptor (TCR) gene sequence obtained by the method of any one of claims 1-50, wherein the TCR gene sequence is located between the first and second HR arms; and

a first 2A coding sequence located upstream of the TCR gene sequence and a second 2A coding sequence located downstream of the TCR gene sequence, wherein the first and second 2A coding sequences encode identical amino acid sequences whose codons diverge from each other;

b. Recombining said HR template nucleic acid into an endogenous locus of a cell, wherein said cell comprises first and second endogenous sequences homologous to first and second homology arms of HR template nucleic acid.

64. A composition comprising a modified cell, wherein the modified cell comprises an exogenous nucleic acid sequence integrated into an endogenous locus, the exogenous nucleic acid sequence comprising:

a. a TCR gene sequence identified by the method of any one of claims 1-50, and

b. a first 2A coding sequence located upstream of the TCR gene sequence and a second 2A coding sequence located downstream of the TCR gene sequence, wherein the first and second 2A coding sequences encode the same amino acid sequence that is codon divergent from each other.

Background

T cells are the primary mediator of adaptive immunity. Under the direction of the specificity of the T Cell Receptor (TCR) unique to each T cell, T cells modulate autoimmunity, help activate B cells and innate effectors, and directly kill infected and cancer cells in a precisely targeted manner. Each TCR recognizes a ligand presented by a Major Histocompatibility Complex (MHC) molecule on a target cell. The identification of related peptide-MHC complex ligands plays a role in understanding the immune response to tumors and pathogens. MHC complex ligands are also valuable for understanding responses to self and dietary antigens. This understanding enables clinically beneficial immunotherapy (e.g., TCR gene transfer and vaccines) that initiate, amplify or attenuate an immune response to a target antigen.

Mutated "neo-epitopes" are important targets for endogenous and engineered immune responses against cancer. New epitope-reactive Tumor Infiltrating Leukocytes (TILs) are present in the endogenous pool and regress the tumor after adoptive transfer. Likewise, tumor mutation burden predicts the clinical effectiveness of CTLA-4 or PD-1 blockade, suggesting that these checkpoint inhibition strategies affect tumor regression by releasing neoepitope-reactive T cells. Since neo-epitopes are caused by somatic mutations in tumor cells, they are not normally presented by thymic epithelial cells to induce central tolerance. Thus, T cell responses to these neo-epitopes are tumor specific, possibly highly avidity, and patient specific (i.e., private). From a clinical perspective, this is both an opportunity and a challenge: the neoepitope is an excellent target for immunotherapy, but the TCR isolation method should have a sufficiently high throughput to enable therapeutic applications on a clinically useful scale.

There is an unmet need for rapid and robust TCR ligand discovery techniques for both basal and transformation studies. peptide-MHC multimers are capable of sorting T cells based on the antigen specificity of the TCR, an important step in the isolation of tumor-specific TCRs for gene therapy. The current typical peptide-MHC production protocol begins with solid phase synthesis of the peptide ligand of interest. At the same time, the universal β 2-microglobulin and related MHC class I molecules are expressed heterologously in e. Each peptide was added to a refolding reaction containing β 2-microglobulin and associated MHC class I molecules. Finally, the properly refolded portion of the ternary complex can be purified and formulated for peptide-MHC multimer production. To facilitate parallel production of specific MHC molecules with many different peptide ligands, Schumacher and co-workers designed a photocleavable peptide that binds to specific MHC molecules as conditional ligands. A single refolding reaction is performed to generate MHC molecules bound to their conditional ligands. Upon exposure to ultraviolet light, the conditional ligand is cleaved and exchanged for the desired peptide present in excess. Many such crossover reactions can be performed in parallel, enabling the construction of a pMHC library for that particular MHC allele. Even so, this state of the art technique has challenging limitations. First, the production, purification and refolding of MHC molecules expressed in e.coli inclusion bodies is laborious and the yield of correctly folded peptide-MHC complexes is low. Second, the turn-around time (weeks) for commercial peptide synthesis is not consistent with the optimal time scale for personalized on-demand TCR gene therapy for patient-specific neo-epitopes. Third, many predicted ligands cannot be used to screen T cells by this method because the biophysical properties of the peptide (e.g., hydrophobicity) prevent its synthesis or exchange. Fourth, the exchange efficiency is usually poor (most predicted exchange efficiency for HLA-binding peptides < 50%). The resulting mixture of correctly folded, exchanged MHC and misfolded, unliganded MHC results in staining of multimers with low signal-to-noise ratios, a problem that is exacerbated when T cells are screened with multiplexed (multiplexed) pools of peptide-MHC reagents. Fifth, designing and validating conditional ligands for each new MHC allele is a laborious and unreliable task. Since the MHC locus is the most multiallelic locus in the human genome, this is a major obstacle to the implementation of new epitope-targeted gene therapy in different MHC haplotype patients. In summary, these limitations highlight the need for new technologies in this area. Disclosed herein are various compositions and methods for generating peptide-MHC multimers that address these limitations.

Summary of The Invention

The present disclosure provides compositions and methods for identifying neoepitopes, identifying and isolating T cell receptors, engineering primary cells to express specific T cell receptors, expanding engineered T cells, and treating disorders using cell therapy. In various embodiments, the present invention provides improved cell therapies and compositions for identifying neoepitopes, identifying and isolating T cell receptors, engineering primary cells to express specific T cell receptors, expanding engineered T cells, and for treating proliferative diseases, disorders, and conditions.

In one aspect, provided herein is a method for identifying antigen specificity of T cells, comprising providing two or more distinct sets of particles, each distinct set of particles comprising a unique antigenic peptide and at least one defined barcode operably associated with an antigenic peptide identity, and each set comprising a first particle comprising a first identifying label and a second particle comprising a second identifying label different from the first identifying label; providing a sample known or suspected to comprise one or more T cells; contacting the sample with two or more collections of particles, wherein the contacting comprises providing conditions sufficient for binding of a single T cell to a unique antigen of at least one collection of particles; separating the one or more T cells bound to the collection of particles by their associated first and second signature labels; performing an assay to identify one or more barcodes that bind to a collection of particles bound to isolated T cells; determining a ratio of barcodes bound to isolated T cells, wherein the ratio is calculated by identifying a first copy number of a primary barcode and a second copy number of a different barcode and dividing the first copy number by the second copy number; and identifying the antigen specificity of the T cells based on the ratio.

In one aspect, provided herein is a method for identifying antigen specificity of a T cell, comprising: obtaining or having obtained at least one antigen-specific T cell bound to two or more distinct sets of particles, each distinct set of particles comprising a unique antigenic peptide and at least one defined barcode operably associated with the identity of the antigenic peptide, wherein each set comprises a first particle comprising a first identifying label and a second particle comprising a second identifying label different from the first identifying label; performing or having performed at least one assay to identify one or more barcodes that detectably bind to a collection of particles that bind to T cells; and determining or having determined a ratio of barcodes that bind to T cells that are antigen specific for the identified T cells, wherein the ratio is calculated by identifying a first copy number of the primary barcode and a second copy number of the different barcodes and dividing the first copy number by the second copy number.

In one aspect, provided herein is a method for identifying antigen specificity of a T cell, comprising: obtaining or having obtained a data set comprising data associated with one or more barcodes detectably bound to a collection of particles that bind directly or indirectly to T cells, wherein each of the one or more barcodes is operably associated with a unique antigenic peptide; and determining or having determined a ratio of barcodes bound to T cells that identify T cell antigen specificity, wherein the ratio is calculated by identifying a first copy number of a primary barcode and a second copy number of a different barcode and dividing the first copy number by the second copy number.

In some embodiments, the data set includes one or more barcodes and one or more barcode copy numbers.

In some embodiments, the unique antigenic peptides are the same for each different set of particles.

In some embodiments, the first particle comprises a first barcode and the second particle comprises a second barcode different from the first barcode, wherein the first and second barcodes are associated with the identity of the antigen.

In some embodiments, the ratio of barcodes corresponds to the antigen specificity of the isolated T cells.

In some embodiments, the isolated T cells are identified as antigen-specific T cells if the ratio of barcodes is above a threshold.

In some embodiments, the threshold is at least 2 or greater than 2.

In some embodiments, the threshold is at least 5 or greater than 5.

In some embodiments, the threshold is at least 10 or greater than 10.

In some embodiments, the threshold is between 2 and 5.

In some embodiments, the threshold is between 5 and 10.

In some embodiments, the threshold is at least or greater than 2,3,4,5,6,7,8,9,10,2-5,3-6,4-7,5-8,5-10,7-10, or greater than 10.

In some embodiments, the assay is a nucleotide-based assay.

In some embodiments, the nucleotide-based assay is a PCR assay, RT-PCR assay, sequencing assay, or hybridization assay.

In some embodiments, the assay determines the sequence of one or more barcodes.

In some embodiments, the assay determines the sequence and copy number of one or more barcodes.

In some embodiments, the method further comprises obtaining a T Cell Receptor (TCR) CDR sequence.

In some embodiments, the method further comprises obtaining TCR α and β chain sequences.

In some embodiments, the antigen specificity of the T cell includes each of (a) the sequence of the antigenic peptide and (b) the TCR sequence of the bound T cell.

In some embodiments, the first identifying indicia of each first particle is the same in each set.

In some embodiments, the second identifying indicia of each second particle is the same in each set.

In some embodiments, the first identifying mark of each first particle is the same in each set, and wherein the second identifying mark of each second particle is the same in each set.

In some embodiments, the first and second identifying labels are fluorophores.

In some embodiments, the first fluorophore is Allophycocyanin (APC).

In some embodiments, the second fluorophore is Phycoerythrin (PE).

In some embodiments, the set of particles comprises a third particle comprising a third barcode different from the first and second barcodes, wherein the first, second, and third barcodes are associated with the identity of the antigen.

In some embodiments, the unique antigenic peptide is selected from the group consisting of: tumor antigen peptides, neoantigen peptides, tumor neoantigen peptides, viral antigen peptides, bacterial antigen peptides, phosphate antigen peptides, and microbial antigen peptides.

In some embodiments, the unique antigenic peptide is a neoantigenic peptide.

In some embodiments, wherein the neoantigen is derived from tumor sequencing data of the subject for identifying one or more somatic mutations present in the data relative to wild-type.

In some embodiments, the neoantigen is determined using a computer predictive algorithm.

In some embodiments, the prediction algorithm further comprises an MHC binding algorithm to predict binding between the neoantigen and the MHC peptide.

In some embodiments, the sample is selected from a blood sample, a bone marrow sample, a tissue sample, a tumor sample, or a Peripheral Blood Mononuclear Cell (PBMC) sample.

In some embodiments, the sample is a PBMC sample.

In some embodiments, the sample is a tumor sample.

In some embodiments, the sample is a bone marrow sample.

In some embodiments, the T cell is a human T cell.

In some embodiments, the T cell is a CD8+ T cell.

In some embodiments, the CD8+ T cells are human CD8+ T cells.

In some embodiments, the method comprises a library of different collections of particles.

In some embodiments, the library comprises a collection of 2 to 500 distinct particles.

In some embodiments, the library comprises at least 60 distinct collections of particles.

In some embodiments, each particle comprises an MHC peptide.

In some embodiments, the MHC peptide is a mammalian MHC peptide.

In some embodiments, the MHC peptide is a human MHC peptide.

In some embodiments, the MHC peptide is a class I HLA peptide.

In some embodiments, the HLA peptide comprises an HLA-A, HLA-B or HLA-C peptide.

In some embodiments, the HLA peptide comprises HLA-A01: 01, HLA-A02: 01, HLA-A03: 01, HLA-A24: 02, HLA-A30: 02, HLA-A31: 01, HLA-A32: 01, HLA-A33: 01, HLA-A68: 01, HLA-A11: 01, HLA-A23: 01, HLA-A30: 01, HLA-A33: 03, HLA-A25: 01, HLA-A26: 01, HLA-A29: 02, HLA-A68: 02, HLA-B07: 02, HLA-B14: 02, HLA-B18: 01, HLA-B02: 27, HLA-B01: 44, HLA-B44: 44, HLA-A33: 01, HLA-A25: 01, HLA-A26: 01, HLA-A29: 02, HLA-A68: 02, HLA-B07: 46, HLA-B50: 01, HLA-B57: 01, HLA-B58: 01, HLA-B08: 01, HLA-B15: 03, HLA-B35: 01, HLA-B40: 02, HLA-B42: 01, HLA-B44: 03, HLA-B51: 01, HLA-B53: 01, HLA-B13: 02, HLA-B15: 07, HLA-B27: 05, HLA-B35: 03, HLA-B37: 01, HLA-B38: 01, HLA-B41: 02, HLA-B44: 05, HLA-B49: 01, HLA-B01: 55, HLA-C02, HLA-B05: 01, HLA-C02, HLA-B55: 01, HLA-C55, HLA-B55: 01, HLA-C55, HLA-B33: 01, HLA-B55, HLA-C, HLA-5, HLA-B35, HLA-C, HLA-C07: 01, HLA-C01: 02, HLA-C04: 01, HLA-C06: 02, HLA-C07: 02, HLA-C16: 01, HLA-C03: 03, HLA-C07: 04, HLA-C08: 01, HLA-C08: 02, HLA-C12: 03, HLA-C14: 02, HLA-C15: 02, or HLA-C17: 01.

In some embodiments, the HLA peptide comprises a Y84A or Y84C mutation.

In some embodiments, each particle comprises an HLAI peptide and a β 2M peptide.

In some embodiments, the β 2M peptide is a mammalian β 2M peptide.

In some embodiments, the β 2M peptide is a human β 2M peptide.

In some embodiments, the β 2M peptide comprises a mutation that allows or increases binding to a thiol dye.

In some embodiments, the mutation is S88C.

In some embodiments, each particle comprises a polypeptide comprising in the amino to carboxy terminal direction (i) an antigenic peptide, (ii) a β 2M peptide, and (iii) an MHC peptide.

In some embodiments, the polypeptide further comprises a first universal target peptide preceding the antigenic peptide, and a second universal target peptide different from the first universal target peptide between the antigenic peptide and the β 2M peptide.

In some embodiments, each particle comprises a polypeptide comprising, in the amino to carboxy terminal direction, (i) a first universal target peptide, (ii) an antigenic peptide, (iii) a second universal target peptide different from the first universal target peptide, (iv) a β 2M peptide, and (v) an MHC peptide.

In some embodiments, the antigenic peptide is 7-15 amino acids, 7-10,8-9,7,8,9,10,11,12,13,14, or 15 amino acids in length.

In some embodiments, the polypeptide comprising the unique antigenic peptide is biotinylated.

In some embodiments, each particle in the different set of particles comprises a streptavidin core and at least one copy of a unique antigenic peptide.

In some embodiments, the particle comprises one, two, three, or four copies of the unique antigenic peptide.

In one aspect, provided herein is a library comprising two or more distinct sets of particles, each set of particles comprising a unique antigenic peptide and a defined barcode operably associated with the identity of the antigenic peptide, wherein each set comprises a first particle comprising a first identifying label and a second particle comprising a second identifying label different from the first identifying label.

In some embodiments, the identifying label is a fluorophore.

In some embodiments, the fluorophore is APC or PE.

In one aspect, provided herein is a particle comprising a tetrameric solid support bound to a unique barcode and three or fewer attached polypeptide molecules comprising in the amino to carboxy terminal direction (i) an antigenic peptide, (ii) a β 2M peptide, and (iii) an MHC peptide, wherein the barcode is operably associated with the identity of the antigenic peptide.

In some embodiments, the polypeptide further comprises a first universal target peptide preceding the antigenic peptide, and a second universal target peptide different from the first universal target peptide between the antigenic peptide and the β 2M peptide.

In some embodiments, the solid support is a streptavidin core.

In some embodiments, the polypeptide molecule is biotinylated.

In some embodiments, the polypeptide molecule binds to the streptavidin core through a biotin-streptavidin interaction.

In some embodiments, the particle further comprises an identifying label.

In some embodiments, the identifying label is a fluorophore.

In some embodiments, the fluorophore is APC or PE.

In one aspect, provided herein is a library comprising particles, wherein the library comprises two or more different particles, wherein each different particle comprises a unique antigenic peptide.

In one aspect, provided herein is a method of monitoring a subject immune repertoire, comprising: providing two or more distinct sets of particles, each distinct set of particles comprising a unique antigenic peptide and at least one defined barcode operably associated with an antigenic peptide identity, and each set comprising a first particle comprising a first identifying label and a second particle comprising a second identifying label different from the first identifying label; providing a sample known or suspected of comprising one or more T cells, wherein the sample is obtained from a subject over time; contacting the sample with two or more collections of particles, wherein the contacting comprises providing conditions sufficient for binding of a single T cell to a unique antigen of at least one collection of particles; isolating one or more T cells associated with the first and second signature; performing an assay to identify one or more barcodes that bind to the isolated T cells; determining a ratio of barcodes bound to isolated T cells, wherein the ratio is calculated by identifying a first copy number from the primary barcode and a second copy number of a different barcode from step (e) and dividing the first copy number by the second copy number; identifying antigen specificity of the T cells based on the ratio; and monitoring the subject for changes in the antigen-specific T cells identified by the method.

In some embodiments, the first and second identifying labels are fluorophores.

In some embodiments, the fluorophore is APC or PE.

In one aspect, provided herein is a method of monitoring an immune repertoire in a subject, comprising obtaining or having obtained a dataset comprising data associated with one or more barcodes detectably binding directly or indirectly to T cells, wherein each of the one or more barcodes is operably associated with a unique antigenic peptide; determining or having determined a ratio of barcodes that bind to T cells that identify T cell antigen specificity, wherein the ratio is calculated by identifying a first copy number of the primary barcode from step (a) and a second copy number of the different barcodes and dividing the first copy number by the second copy number; identifying the sequence of a unique antigenic peptide that binds to antigen-specific T cells; and monitoring the subject for changes in the antigen-specific T cells identified by the method.

In some embodiments, the data set includes one or more barcodes and one or more barcode copy numbers.

In some embodiments, the unique antigenic peptides are the same for each different set of particles.

In some embodiments, monitoring a change in T cells comprises administering to the subject a soluble, labeled antigen-specific TCR.

In some embodiments, the immunotherapy is administered to the subject based on the identified change in the antigen-specific T cells.

In some embodiments, the immunotherapy is a checkpoint inhibitor.

In some embodiments, the checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody. In some embodiments, the anti-PD-1 antibody is selected from the group consisting of pembrolizumab, nivolumab, and cimiciprimab (cemipimab). In some embodiments, the anti-PD-LI antibody is selected from the group consisting of atelizumab (atezolizumab), avelumab, and dolvuzumab (durvalumab). In some embodiments, the anti-CTLA 4 antibody is ipilimumab (ipilimumab). In some embodiments, the checkpoint inhibitor is an anti-TIGIT antibody. In some embodiments, the anti-TIGIT antibody is selected from the group comprising AB154(Arcus), tiragolumab (genentech), BMS-986297(BMS), MK-7684(Merck), and etiglimab (oncomed).

In one aspect, provided herein is a method of identifying an antigen, comprising providing two or more distinct sets of particles, each distinct set of particles comprising a unique antigenic peptide and at least one defined barcode operably associated with an antigenic peptide identity, and each set comprising a first particle comprising a first identifying label and a second particle comprising a second identifying label different from the first identifying label; providing a sample known or suspected to comprise one or more T cells; contacting the sample with two or more collections of particles, wherein the contacting comprises providing conditions sufficient for binding of a single T cell to a unique antigen of at least one collection of particles; isolating one or more T cells associated with the first and second signature; performing an assay to identify one or more barcodes that bind to the isolated T cells; determining a ratio of barcodes bound to isolated T cells, wherein the ratio is calculated by identifying a first copy number of the primary barcode and a second copy number of the different barcodes from step (e) and dividing the first copy number by the second copy number; and identifying the sequence of the unique antigenic peptide that binds to the antigen-specific T cell.

In some embodiments, the first and second identifying labels are fluorophores.

In some embodiments, the fluorophore is APC or PE.

In one aspect, provided herein is a method of identifying an antigen, comprising obtaining or having obtained a data set comprising data associated with one or more barcodes that detectably bind directly or indirectly to T cells, wherein each of the one or more barcodes is operably associated with a unique antigenic peptide; determining or having determined a ratio of barcodes that bind to T cells, wherein the ratio is calculated by identifying a first copy number of the primary barcode and a second copy number of the different barcodes from step (a) and dividing the first copy number by the second copy number; and identifying the sequence of the unique antigenic peptide that binds to the antigen-specific T cell.

In some embodiments, step (a) comprises obtaining a T cell-based sample and assaying it to obtain a data set.

In some embodiments, the data set includes one or more barcodes and one or more barcode copy numbers.

In some embodiments, the unique antigenic peptides are the same for each different set of particles.

In one aspect, provided herein is a method of identifying HLA and peptide complexes, comprising providing two or more distinct sets of particles, each distinct set of particles comprising a unique antigenic peptide and at least one defined barcode operably associated with an antigenic peptide identity, and each set comprising a first particle comprising a first identifying label and a second particle comprising a second identifying label different from the first identifying label; providing a sample known or suspected to comprise one or more T cells; contacting the sample with two or more collections of particles, wherein the contacting comprises providing conditions sufficient for binding of a single T cell to a unique antigen of at least one collection of particles; isolating one or more T cells associated with the first and second signature; performing an assay to identify one or more barcodes that bind to the isolated T cells; determining a ratio of barcodes bound to isolated T cells, wherein the ratio is calculated by identifying a first copy number of the primary barcode and a second copy number of the different barcodes from step (e) and dividing the first copy number by the second copy number; and identifying HLA and peptide complexes that bind to the antigen-specific T cells.

In some embodiments, the first and second identifying labels are fluorophores.

In some embodiments, the fluorophore is APC or PE.

In one aspect, provided herein is a method of identifying HLA and peptide complexes, comprising obtaining or having obtained a data set comprising data associated with one or more barcodes that detectably bind directly or indirectly to T cells, wherein each of the one or more barcodes is operably associated with a unique antigenic peptide; determining or having determined a ratio of barcodes that bind to T cells, wherein the ratio is calculated by identifying a first copy number of the primary barcode from step (a) and a second copy number of the different barcodes and dividing the first copy number by the second copy number; and identifying HLA and peptide complexes that bind to the antigen-specific T cells.

In some embodiments, step (a) comprises obtaining a T cell-based sample and assaying it to obtain a data set.

In some embodiments, the data set includes one or more barcodes and one or more barcode copy numbers.

In some embodiments, the unique antigenic peptides are the same for each different set of particles.

In one aspect, provided herein is a method of identifying a subject for treatment with immunotherapy, comprising providing two or more distinct sets of particles, each distinct set of particles comprising a unique antigenic peptide and at least one defined barcode operably associated with an antigenic peptide identity, and each set comprising a first particle comprising a first identifying label and a second particle comprising a second identifying label different from the first identifying label; providing a sample known or suspected to comprise one or more T cells; contacting the sample with two or more collections of particles, wherein the contacting comprises providing conditions sufficient for binding of a single T cell to a unique antigen of at least one collection of particles; isolating one or more T cells associated with the first and second signature; performing an assay to identify one or more barcodes that bind to the isolated T cells; determining a ratio of barcodes bound to isolated T cells, wherein the ratio is calculated by identifying a first copy number of the primary barcode from step (e) and a second copy number of the different barcodes and dividing the first copy number by the second copy number.

In some embodiments, the first and second identifying labels are fluorophores.

In some embodiments, the fluorophore is APC or PE.

In one aspect, provided herein is a method of identifying a subject for treatment with immunotherapy, comprising obtaining or having obtained a data set comprising data associated with one or more barcodes that detectably bind directly or indirectly to T cells, wherein each of the one or more barcodes is operably associated with a unique antigenic peptide; and determining or having determined a ratio of barcodes that bind to T cells, wherein the ratio is calculated by identifying a first copy number of the primary barcode from step (a) and a second copy number of the different barcodes and dividing the first copy number by the second copy number.

In some embodiments, step (a) comprises obtaining a T cell-based sample and assaying it to obtain a data set.

In some embodiments, the data set includes one or more barcodes and one or more barcode copy numbers.

In some embodiments, the unique antigenic peptides are the same for each different set of particles.

In some embodiments, the immunotherapy comprises a T cell vaccine, a dendritic cell vaccine, a nucleic acid vaccine, a peptide vaccine, a viral vaccine, a soluble TCR, a TCR-drug conjugate, an antibody, or an antibody-drug conjugate.

In some embodiments, the antibody comprises a monoclonal antibody.

In one aspect, provided herein is a method of identifying unique TCR sequences, comprising providing two or more distinct sets of particles, each distinct set of particles comprising a unique antigenic peptide and at least one defined barcode operably associated with an antigenic peptide identity, and each set comprising a first particle comprising a first identifying tag and a second particle comprising a second identifying tag different from the first identifying tag; providing a sample known or suspected to comprise one or more T cells; contacting the sample with two or more collections of particles, wherein the contacting comprises providing conditions sufficient for binding of a single T cell to a unique antigen of at least one collection of particles; isolating one or more T cells associated with the first and second signature; performing an assay to identify one or more barcodes that bind to the isolated T cells; determining a ratio of barcodes bound to isolated T cells, wherein the ratio is calculated by identifying a first copy number of the primary barcode from step (e) and a second copy number of the different barcodes and dividing the first copy number by the second copy number; and identifying unique TCR sequences.

In some embodiments, the first and second identifying labels are fluorophores.

In some embodiments, the fluorophore is APC or PE.

In one aspect, provided herein is a method of identifying a unique TCR sequence, comprising obtaining or having obtained a data set comprising data associated with one or more barcodes that detectably bind directly or indirectly to T cells, wherein each of the one or more barcodes is operably associated with a unique antigenic peptide; determining or having determined a ratio of barcodes that bind to T cells, wherein the ratio is calculated by identifying a first copy number of the primary barcode from step (a) and a second copy number of the different barcodes and dividing the first copy number by the second copy number; and identifying unique TCR sequences.

In some embodiments, step (a) comprises obtaining a T cell-based sample and assaying it to obtain a data set.

In some embodiments, the data set includes one or more barcode sequences and one or more barcode copy numbers.

In some embodiments, the unique antigenic peptides are the same for each different set of particles.

In some embodiments, the method further comprises making a soluble TCR polypeptide comprising the identified unique TCR sequence.

In some embodiments, the soluble TCR polypeptide is linked to a label or drug.

In some embodiments, the method is repeated to identify at least two unique TCR sequences.

In some embodiments, the method further comprises making a library comprising at least two unique TCR sequences.

In one aspect, provided herein is a method of treating cancer in a subject, comprising obtaining or having obtained a data set comprising data associated with one or more barcodes detectably binding directly or indirectly to T cells, wherein each of the one or more barcodes is operably associated with a unique antigenic peptide; determining or having determined a ratio of barcodes that bind to T cells that identify T cell antigen specificity, wherein the ratio is calculated by identifying a first copy number of the primary barcode from step (a) and a second copy number of the different barcodes and dividing the first copy number by the second copy number; identifying TCR sequences of at least one or both T cells and generating engineered T cells comprising at least one or both TCR sequences; and administering the engineered T cells to the subject.

In some embodiments, the method further comprises administering immunotherapy.

In some embodiments, the immunotherapy is a checkpoint inhibitor.

In some embodiments, the checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody.

In some embodiments, the T cells are autologous.

In some embodiments, the engineered T cells are autologous.

In some embodiments, the unique antigenic peptide is presented by HLA class I on the cell surface of the cancer of the subject.

In certain embodiments, the presently disclosed subject matter provides methods for treating T cells. In certain embodiments, the method comprises: (a) contacting the sample with a plurality of different sets of particles; (b) isolating the one or more T cells bound to the particle; (c) identifying barcodes of particles bound to the isolated T cells; and (d) determining a ratio for each barcode. In certain embodiments, each particle comprises a unique antigenic peptide, an operably associated barcode, and at least one identifying label. In certain embodiments, the sample comprises T cells. In certain embodiments, contacting comprises providing conditions suitable for binding of a single T cell to the unique antigenic peptide of the at least one collection of particles.

In certain embodiments, the ratio is calculated by identifying the copy number of the first barcode and the copy number of the second barcode and dividing the copy number of the first barcode by the copy number of the second barcode. In certain embodiments, the unique antigenic peptides are the same for each different set of particles. In certain embodiments, each distinct set of particles comprises at least one or more barcodes, wherein each barcode is associated with the identity of an antigenic peptide. In certain embodiments, the ratio of each barcode corresponds to the antigen specificity of the isolated T cells. In certain embodiments, the isolated T cells are identified as antigen-specific T cells if the ratio of the first barcodes is above a threshold. In certain embodiments, the threshold is at least or greater than 2,3,4,5,6,7,8,9,10,2-5,3-6,4-7,5-8,5-10,7-10, or greater than 10.

In certain embodiments, identifying the barcode comprises a nucleotide-based assay. In certain embodiments, the nucleotide-based assay is a PCR, RT-PCR, sequencing, or hybridization assay. In certain embodiments, the sequence of each barcode is determined based on a nucleotide-based assay. In certain embodiments, the copy number of each barcode is determined based on a nucleotide-based assay. In certain embodiments, the nucleotide-based assay determines (a) the sequence of each barcode and/or (b) the copy number of each barcode.

In certain embodiments, the method further comprises obtaining a T Cell Receptor (TCR) CDR sequence. In certain embodiments, the method further comprises obtaining a TCR gene sequence. In certain embodiments, the TCR gene sequence is a TCR α or TCR β chain sequence.

In certain embodiments, the method comprises identifying antigen specificity of the T cell. In certain embodiments, the antigen specificity of a T cell includes the sequence of the antigenic peptide and the TCR sequence of the bound T cell.

In certain embodiments, the at least one identifying label is the same in each distinct set of particles. In certain embodiments, the method comprises at least two different identifying marks. In certain embodiments, the at least one identifying label is a fluorophore. In certain embodiments, the fluorophore is selected from the group consisting of Allophycocyanin (APC) and Phycoerythrin (PE). In certain embodiments, the at least two different signature markers are fluorophores, wherein the fluorophores are selected from the group consisting of Allophycocyanin (APC) and Phycoerythrin (PE).

In certain embodiments, the antigenic peptide is selected from the group consisting of: tumor antigens, neoantigens, tumor neoantigens, viral antigens, bacterial antigens, phospho-antigens, and microbial antigens. In certain embodiments, the neoantigen is identified from tumor sequencing data of the subject. In certain embodiments, a computer predictive algorithm is used to determine neoantigens. In certain embodiments, the prediction algorithm further comprises an MHC binding algorithm to predict binding between the neoantigen and the MHC peptide.

In certain embodiments, the sample is selected from a blood sample, a bone marrow sample, a tissue sample, a tumor sample, or a Peripheral Blood Mononuclear Cell (PBMC) sample. In certain embodiments, wherein the T cell is a human T cell. In certain embodiments, the T cell is CD8⁺T cells.

In certain embodiments, the method comprises a library of different collections of particles. In certain embodiments, the library comprises a collection of 2 to 500 distinct particles. In certain embodiments, each particle comprises an MHC peptide. In certain embodiments, the MHC peptide is a human MHC peptide. In certain embodiments, the MHC peptide is a class I HLA peptide. In certain embodiments, the HLA peptide comprises HLA-A, HLA-B or HLA-C peptide. In certain embodiments, the HLA peptide comprises HLA-A01: 01, HLA-A02: 01, HLA-A03: 01, HLA-A24: 02, HLA-A30: 02, HLA-A31: 01, HLA-A32: 01, HLA-A33: 01, HLA-A68: 01, HLA-A11: 01, HLA-A23: 01, HLA-A30: 01, HLA-A33: 03, HLA-A25: 01, HLA-A26: 01, HLA-A29: 02, HLA-A68: 02, HLA-B07: 02, HLA-B14: 02, HLA-B18: 01, HLA-B02: 27, HLA-B01: 44, HLA-B44: 44, HLA-A33: 01, HLA-A25: 01, HLA-A26: 01, HLA-A29: 02, HLA-A68: 02, HLA-B07: 46, HLA-B50: 01, HLA-B57: 01, HLA-B58: 01, HLA-B08: 01, HLA-B15: 03, HLA-B35: 01, HLA-B40: 02, HLA-B42: 01, HLA-B44: 03, HLA-B51: 01, HLA-B53: 01, HLA-B13: 02, HLA-B15: 07, HLA-B27: 05, HLA-B35: 03, HLA-B37: 01, HLA-B38: 01, HLA-B41: 02, HLA-B44: 05, HLA-B49: 01, HLA-B01: 55, HLA-C02, HLA-B05: 01, HLA-C02, HLA-B55: 01, HLA-C55, HLA-B55: 01, HLA-C55, HLA-B33: 01, HLA-B55, HLA-C, HLA-5, HLA-B35, HLA-C, HLA-C07: 01, HLA-C01: 02, HLA-C04: 01, HLA-C06: 02, HLA-C07: 02, HLA-C16: 01, HLA-C03: 03, HLA-C07: 04, HLA-C08: 01, HLA-C08: 02, HLA-C12: 03, HLA-C14: 02, HLA-C15: 02 or HLA-C17: 01.

In certain embodiments, each particle comprises an HLA peptide and a β 2M peptide. In certain embodiments, the β 2M peptide is a human β 2M peptide. In certain embodiments, the β 2M peptide comprises a mutation. In certain embodiments, the mutation is S88C.

In certain embodiments, each particle comprises a polypeptide comprising in the amino to carboxy terminal direction (i) an antigenic peptide, (ii) a β 2M peptide, and (iii) an MHC peptide. In certain embodiments, the antigenic peptide is 7-15 amino acids, 7-10,8-9,7,8,9,10,11,12,13,14, or 15 amino acids in length. In certain embodiments, the polypeptide is biotinylated. In certain embodiments, the particles are selected from the group consisting of: magnetic beads, agarose beads, styrene polymer particles, and dextran polymer particles. In certain embodiments, wherein the particle is coated with streptavidin.

In certain embodiments, the presently disclosed subject matter provides methods for monitoring an immune repertoire in a subject. In certain embodiments, the method comprises monitoring the subject for changes in antigen-specific T cells. In certain embodiments, the method comprises administering immunotherapy to the subject. In certain embodiments, the immunotherapy is an adoptive cell transfer or checkpoint inhibitor. In certain embodiments, any of the methods disclosed herein are used to monitor an immune repertoire in a subject.

In certain embodiments, the presently disclosed subject matter provides methods for identifying at least one TCR sequence. In certain embodiments, at least one TCR sequence is a TCR α sequence, a TCR β sequence, or a combination thereof. In certain embodiments, the method further comprises making a soluble TCR polypeptide. In certain embodiments, any of the methods disclosed herein are used to identify at least one TCR sequence.

In certain embodiments, the presently disclosed subject matter provides a library of particles. In certain embodiments, the library comprises at least two collections of particles. In certain embodiments, each set of particles comprises an antigenic peptide, a barcode operably associated with the identity of the antigenic peptide, and at least one identifying label. In certain embodiments, the at least one identifying label is the same in each set of particles. In certain embodiments, there are at least two different identifying labels in each different set of particles. In certain embodiments, the at least one identifying label is a fluorophore. In certain embodiments, the fluorophore is selected from the group consisting of Allophycocyanin (APC) and Phycoerythrin (PE). In certain embodiments, the at least two different signature markers are fluorophores, wherein the fluorophores are selected from the group consisting of Allophycocyanin (APC) and Phycoerythrin (PE).

In certain embodiments, the presently disclosed subject matter further provides particles. In certain embodiments, the particle comprises at least one polypeptide, a barcode, and an identifying label. In certain embodiments, the polypeptide comprises an antigenic peptide, a β 2M peptide, and an MHC peptide. In certain embodiments, the barcode is operably associated with the identity of the antigenic peptide. In certain embodiments, the particles are selected from the group consisting of: magnetic beads, agarose beads, styrene polymer particles, and dextran polymer particles. In certain embodiments, the identifying label is a fluorophore. In certain embodiments, the particles are coated with streptavidin. In certain embodiments, the polypeptide is labeled.

In certain embodiments, the presently disclosed subject matter further discloses methods of treating cancer in a subject. In certain embodiments, the method comprises: (a) preparing a plurality of particles, each particle comprising a plurality of labeled polypeptides; (b) contacting a plurality of particles with a plurality of T cells from the subject under conditions suitable for specific binding of the T cells to the particle antigen; (c) isolating the T cells bound to the particles and identifying the TCR gene sequence of the isolated T cells; (d) preparing a polynucleotide comprising homology arms and at least one TCR gene sequence; (e) recombining the polynucleotide into an endogenous locus of a subject T cell; (f) culturing the modified T cells to produce a population of T cells; and (g) administering a therapeutically effective amount of the modified T cell to the subject, thereby treating the cancer. In certain embodiments, the polypeptide comprises an antigenic peptide, a β 2M sequence, an HLA sequence, and a detectable label. In certain embodiments, the TCR gene sequences are patient-specific. In certain embodiments, the TCR gene sequences are located between the homology arms.

In certain embodiments, the presently disclosed subject matter further discloses methods of modifying cells. In certain embodiments, the method comprises: (a) introducing a Homologous Recombination (HR) template nucleic acid sequence into a cell; and (b) recombining the HR template nucleic acid into the endogenous locus of the cell. In certain embodiments, the HR template nucleic acid comprises: (a) first and second homology arms homologous to first and second endogenous sequences of the cell; (b) a T Cell Receptor (TCR) gene sequence obtained by any of the methods disclosed herein; and (c) a first 2A coding sequence located upstream of the TCR gene sequence and a second 2A coding sequence located downstream of the TCR gene sequence, wherein the first and second 2A coding sequences encode the same amino acid sequence that is codon-diverged from one another. In certain embodiments, the first and second endogenous sequences are homologous to the first and second homology arms of the HR template nucleic acid. In certain embodiments, the TCR gene sequence is located between the first and second HR arms. In certain embodiments, the 2A-coding sequence is a P2A-coding sequence. In certain embodiments, the HR template further comprises a sequence encoding a flexible linker. In certain embodiments, the sequence encoding the flexible linker is located immediately upstream of the 2A coding sequence. In certain embodiments, the flexible linker has a Gly Ser Gly amino acid sequence. In certain embodiments, the HR template further comprises a sequence encoding a protease cleavage sequence. In certain embodiments, the protease cleavage sequence is a furin sequence. In certain embodiments, the protease cleavage sequence is a TEV sequence. In certain embodiments, the protease cleavage sequence is upstream of the second 2A coding sequence.

In certain embodiments, the presently disclosed subject matter further discloses compositions comprising the modified cells. In certain embodiments, the modified cell comprises an exogenous nucleic acid sequence integrated into an endogenous locus. In certain embodiments, the exogenous nucleic acid sequence comprises: (a) a TCR gene sequence; and (b) a first 2A coding sequence located upstream of the TCR gene sequence and a second 2A coding sequence located downstream of the TCR gene sequence. In certain embodiments, the TCR gene sequences are identified by any of the methods disclosed herein. In certain embodiments, the first and second 2A coding sequences encode the same amino acid sequence that is codon divergent from each other. In certain embodiments, the 2A-coding sequence is a P2A-coding sequence. In certain embodiments, the exogenous nucleic acid sequence further comprises a sequence encoding a flexible linker. In certain embodiments, the sequence encoding the flexible linker is located immediately upstream of the 2A coding sequence. In certain embodiments, the flexible linker has a Gly Ser Gly amino acid sequence. In certain embodiments, the exogenous nucleic acid further comprises a sequence encoding a protease cleavage sequence. In certain embodiments, the protease cleavage sequence is a furin sequence. In certain embodiments, the protease cleavage sequence is a TEV sequence. In certain embodiments, the protease cleavage sequence is upstream of the second 2A coding sequence.

Brief Description of Drawings

These and other features, aspects, and advantages of the disclosed compositions and methods will become better understood with regard to the following description and accompanying drawings where:

fig. 1 shows the design of an exemplary comact minigene. SS refers to an optional signal sequence; US1 refers to a first universal target site; NeoE refers to a neoepitope, i.e., an antigenic peptide sequence site; US2 refers to a second universal target site; l1 refers to an optional first linker sequence; beta2m refers to the Beta-2-microglobulin domain sequence; l2 refers to an optional second linker sequence; MHC heavy chain refers to an MHC heavy chain allele; l3 refers to an optional third linker sequence; and a purification cluster refers to an optional purification cluster having a biotinylation sequence, a protease cleavage site, and an affinity tag sequence. Although fig. 1 shows His6(SEQ ID NO:34) (6-His tag (SEQ ID NO:34)) as the affinity tag, any other suitable affinity tag may be used, including but not limited to histidine tags of different lengths (poly-His tag), HAT tags, FLAG tags (or FLAG epitopes), epitopes specific for any antibody used for purification, galactose binding protein tags, fluorescent tags, GST tags, HA tags, Halo tags, MBP tags, Myc tags, poly-Asp tags, poly-Phe tags, protein C, streptavidin/biotin tags, Strep-tags, protein G, or any other protein purification tag capable of purifying paccomt polypeptides.

In addition, while FIG. 1 shows the use of nickel resin (see "Ni" in the figure) to purify His 6-tagged (SEQ ID NO:34) comPACT polypeptides, other His6(SEQ ID NO:34) affinity resins have been used. Specifically, zinc resin has been used to successfully purify comPACT polypeptide with a His6 tag (SEQ ID NO:34) from solution. Cobalt and calcium resins are two additional exemplary His6(SEQ ID NO:34) affinity resins that can be used.

Figure 2 shows a schematic of an exemplary modular off-the-shelf platform for rapid assembly of libraries of antigenic peptide ligands complexed to selected MHC alleles. FIG. 2 discloses SEQ ID NO 9, 11 and 13, respectively, in order of appearance.

FIG. 3 is a schematic of an exemplary restriction digestion cloning reaction replacing a virtual insert in an MHC template with a selected new epitope sequence. The virtual insert (underlined, bold; top sequence in the figure) contains four stop codons in different frames and unique restriction sites for disrupting uncleaved or religated templates. Restriction sites on either side of the insert are shown in boxes. The bottom sequence set shows the NeoE insert after the virtual insert has been cleaved and ligated to the correct NeoE sequence. FIG. 3 discloses SEQ ID NO 270-274, respectively, in order of appearance.

FIG. 4 is a schematic of an exemplary restriction digestion cloning reaction for insertion of selected novel epitope sequences in MHC templates. The new epitope sequences (underlined, bold) were synthesized as primers, flanked by two different restriction sites (boxed). A universal primer with the reverse complement of the 3' restriction site was used in the PCR reaction to form a double-stranded primer dimer of the new epitope sequence. Restriction digestion of both the neoepitope and the MCH template vector allows ligation reactions to insert selected neoepitope sequences into MHC template sequences. The ligation reaction was transformed into E.coli, and the plasmid prepared from the transformed E.coli was used for mammalian producer cell transfection reaction. FIG. 4 discloses SEQ ID NO 275-276, 271-274 and 277, respectively, in order of appearance.

Figure 5 is a diagram of an exemplary alternative form of restriction digestion cloning reaction to insert selected novel epitope sequences in MHC templates. Two complementary NeoE-encoding primers were synthesized with partial 5 'and 3' restriction sites. These primers were annealed and mimicked the restriction digested overhangs. The precut vector (with the overhang retaining mainly the 5' phosphate) was then ligated to the annealed NeoE insert and the ligation product was transformed into e. FIG. 5 discloses SEQ ID NO 278-279, 271-274 and 277, respectively, in order of appearance.

Figure 6 is a diagram of an exemplary PCR-based method of inserting selected novel epitope sequences in MHC templates. Synthesizing two complementary NeoE coding primers, wherein the forward primer has a 3' sequence aiming at a second universal site in the MHC template; and the reverse primer has a 3' sequence to the complement of the first universal site in the MHC template. These primers were mixed with a 5' fragment of the MHC template with the first universal sequence site and a second fragment of the MHC template with the second universal site and the rest of the comPACT minigene. The first PCR amplification cycle produced two nucleotide fragments, one encoding the first universal site region with the downstream neo-epitope, the other encoding the neo-epitope and then the rest of the comPACT gene. The two fragments, which overlap at the unique new epitope sequence, are then assembled and the complete assembly is amplified and cleaned up for transfection.

Fig. 7 shows total protein expression in 30mL mammalian cells transfected with comPACT gene (Neo12) during 7 days time and western blot using NTA-HRP reagent to detect His-tag.

FIG. 8 shows a Ni-NTA affinity chromatography purified gel of Neo12 comPACT protein. Pre represents crude lysate, FT represents flow-through, W represents wash, and E represents eluted.

Figure 9 shows a size exclusion chromatogram of purified Neo12 protein. The major peak is Neo12 protein and the minor peak is ATP added during the biotinylation step.

Figure 10 shows a purification experiment similar to that shown in figure 8, using 0.7 cell culture volume.

Fig. 11 shows crude and purified proteins of eight different NeoE copact proteins, each with a different antigenic sequence. FIG. 11 discloses SEQ ID NO 11 and 13, respectively, in order of appearance.

Fig. 12 shows size exclusion chromatograms of the eight NeoE copact proteins of fig. 11.

Fig. 13A shows NeoE copact protein (in particular Neo12 copact protein) produced using the PCR assembly method (linear amplicon) described in fig. 6, compared to NeoE copact protein (in particular Neo12 copact protein) produced from plasmids. FIG. 13B shows a DNA gel of linear amplicons generated by the PCR assembly method. Each lane contains a comPACT minigene (in particular the Neo12comPACT minigene) with a different new epitope sequence.

Fig. 14 shows a streptavidin bead pull-down assay to test for complete biotinylation of comapt protein. FIG. 14 discloses "(His 6)" as SEQ ID NO: 34.

Fig. 15 shows biotinylation of different comact proteins in crude cell lysates, visualized by western blot using streptavidin-HRP. FIG. 15 discloses "His 6" as SEQ ID NO: 34. FIG. 15 also discloses SEQ ID NOs 280, 280-281 and 281, respectively, in order of appearance.

FIGS. 16A, 16B and 16C show the production and purification of BirA enzyme (FIGS. 16B and 16C) and TEV protease (FIG. 16A) in E.coli.

FIG. 17 shows biotinylation of comPACT protein using BirA (lane 2) and cleavage of His6 tag using TEV protease (SEQ ID NO:34) (lane 3).

Figure 18 shows cell sorting of cells transduced with BirA and V5 based on V5 expression.

Fig. 19 shows antigen-specific capture of T cells using multimerized comact protein.

Fig. 20 shows comPACT NTAmer production using S88C β 2M comPACT protein.

Figure 21 shows the coupling of Cy5 to S88C comPACT protein monomer.

Fig. 22A shows an exemplary diagram of a cloning strategy for making comPACT polynucleotides. Fig. 22B provides sequence validation statistics obtained from 824 individual comact polynucleotides.

Fig. 23 provides an exemplary illustration of a workflow for making comPACT polynucleotides and proteins.

Fig. 24A and 24B show the percentage of patients covered by the highest HLA allele in the united states relative to the comPACT HLA library size.

Fig. 25A shows a representative selection of comPACT protein monodispersity of comPACT proteins. Fig. 25B shows the comPACT protein yields for a representative selection of comPACT proteins. Fig. 25C shows comPACT protein expression for a representative selection of comPACT proteins.

Figure 26 provides a schematic of the imPACT signal to noise ratio neo-antigen T cell isolation process.

FIG. 27A provides a graphical representation of non-specific barcode signal intensity for signal and noise identification. FIG. 27B provides a graphical representation of specific barcode signal intensity for identifying signal and noise.

Fig. 28 provides an illustration of single and double bar codes.

Fig. 29A provides a schematic of a bistetramer staining process. Figure 29B provides data showing imPACT tetramer staining of MART5 antigen-specific T cells. Figure 29C provides data showing imPACT tetramer staining of neo12 antigen specific T cells.

Figure 30 provides an example of isolated neoantigen T cell sensitivity by neoID signal to noise ratio.

Figure 31 provides an example of the specificity of the imPACT neo-antigen separation process (i.e., the imPACT separation technique).

Figure 32A provides FACS plots of gene-edited specific T cells (filled squares) exhibiting a NeoID signal to noise ratio greater than 10. Non-specific T cells (circles) displayed S/N < 10. Fig. 32B shows the quantification of the signal-to-noise ratio and mean values for specific and non-specific T cells.

Figure 33A provides an example of imPACT analysis of PBMC samples and validation of TCR isolated by imPACT using a single barcode approach. FIG. 33A discloses SEQ ID NO 282-284 in appearance order, respectively. Figure 33B provides an example of imPACT analysis of PBMC samples and validation of TCR isolated by imPACT using a single barcode method.

Figure 34A provides FACS plots of double-stained T cells for ipact analysis of PBMC samples using a single barcode method. FIG. 34A discloses SEQ ID NO 285-292, respectively, in order of appearance. Fig. 34B provides FACS plots of CD45RA and CD95 stained T cells after double tetramer staining. FIG. 34B discloses SEQ ID NO 285-292, respectively, in order of appearance. FIG. 34C provides a table summarizing the TRA (SEQ ID NO:293-300 in appearance order), TRB (SEQ ID NO:285-292 in appearance order), gene and novel antigenic peptide (SEQ ID NO:204, 203, 205, 203 and 206-207 in appearance order) sequences of T cells isolated after imPACT analysis. Figure 34D provides a summary of the isolated neoantigen-specific T cells. Figure 34E provides a summary of the number of isolated lymphocytes for each TCR identified using the imPACT method.

Figure 35A provides an example of a validation screen for the ipact analysis of the PBMC samples used in figure 34. FIG. 35A discloses SEQ ID NOs 207, 301, 206 and 302, respectively, in order of appearance. Fig. 35B provides an example of a validation screen for the ipact analysis of the PBMC samples used in fig. 34.

Figure 36 provides a graphical representation of mutant targeted T cells of the PBMC sample used in figure 34.

Fig. 37A provides FACS plots of double-stained T cells for imPACT analysis of PBMC samples using the double barcode method. Fig. 37B provides FACS plots of CD45RA and CD95 stained T cells after double tetramer staining. FIG. 37B discloses SEQ ID NO 303-305 in appearance order, respectively. Fig. 37C provides a table summarizing TRA, TRB, genes and neoantigenic peptide sequences of T cells isolated after imPACT analysis. In order of appearance, FIG. 37C discloses all that the "top1. NeoAg" sequences are SEQ ID NOS: 306,208 and 208, respectively, the "top2. NeoAg" sequences are SEQ ID NOS: 307,306,208,208,208,208,208,208 and 208, the "tra. CDR 3" sequences are SEQ ID NOS: 308-310,310-312, 312-312 and 312, the "trb. CDR 3" sequences are SEQ ID NOS: 313-314,304, 303,305 and 305, and the "peptide tumor" sequences are SEQ ID NOS: 306,306,208,208,208,208,208,208 and 208. Fig. 37D provides an example of validation screening of imPACT analysis using comPACT9 dextramer.

Figure 38A provides a summary of neoantigen-specific TCRs isolated from patient samples. Fig. 38B provides a table summarizing the HAL types, cancers, target numbers and TCR numbers found in TILs.

Fig. 39A shows antigen specificity of HCMV and EBV T cells. Figure 39B shows the number of TCR hits.

FIG. 40A shows a comparison of tetramer, trimer and dextramer separation methods using F5T cells. FIG. 40B shows a comparison of tetramer, trimer and dextramer separation methods using neo 12T cells.

FIG. 41A shows a comparison of trimer and dextramer separation methods using PBMC samples and neo 12T cells, CMV T cells, and M1WT cells. FIG. 41B shows the average barcode signal to noise ratio when using trimers or dextramers.

Fig. 42A shows the change over time of neoantigen-specific T cells in peripheral blood of PACT157 in patients. FIG. 42A discloses SEQ ID NO 315-317, respectively, in order of appearance. Fig. 42B shows the change over time of neoantigen-specific T cells in peripheral blood of patient PACT 132. FIG. 42B discloses SEQ ID NO 318-. Fig. 42C shows the change over time of neoantigen-specific T cells in peripheral blood of patient PACT 131. FIG. 42C discloses SEQ ID NO 320-323 in order of appearance, respectively.

Figure 43 shows phenotypic characterization of neoantigen-specific T cells from one patient.

Figure 44A shows functional characterization of TCR clones isolated against PIK3CA neoantigen target. Figure 44B shows functional characterization of TCR clones isolated against PIK3CA neoantigen target.

Fig. 45 provides a summary of the number of neoantigen-specific T cells per CD 8T cells in each T cell sample collected during anti-PD-1 antibody treatment of patient PACT 135. FIG. 45 discloses "KTYFKPFHPK" as SEQ ID NO:256 and "YFKPFHPKF" as SEQ ID NO: 227.

Figure 46 shows the strong T cell gene editing efficiency of 14 neo TCRs in both CD4 and CD 8T cells.

FIG. 47 shows that neo TCR is internalized when co-cultured with a neo TCR T cell cognate (cognate) comPACT-dextramer and melanoma matched cell line M489 with and without IFN γ pre-incubation.

FIG. 48 shows that neoTCR T cells derived from patient PACT135 express the activation marker 4-1 BB.

Fig. 49 shows that neoTCR T cells derived from patient PACT135 express the activation marker OX 40.

Figure 50 provides a graph of the percentage of tumor cells confluent after co-culture with all neoTCR T cells identified from PACT 135.

Figure 51A provides a separate graph of the percentage of tumor cells confluent after co-culture with each neoTCR T cell. Figure 51B provides a separate graph of the percentage of tumor cells confluent after co-culture with each neoTCR T cell.

Figure 52 shows IFN γ, IL2, and TNF α secretion by TCR 218T cells after co-culture with M489 cells.

Figure 53 shows IFN γ, IL2, and TNF α secretion by TCR221 and TCR 227T cells after co-culture with M489 cells.

Figure 54 shows IFN γ and TNF α secretion by TCR 222T cells after co-culture with M489 cells.

Figure 55 shows IFN γ secretion from TCR219, TCR220, TCR223, TCR224, TCR225, TCR228, TCR229, TCR232, TCR240, TCR 241T cells following coculture with M489 cells, with or without IFN γ pretreatment.

Fig. 56 provides a summary of the number of neoantigen-specific T cells isolated from patient PACT 035.

FIG. 57A shows HLA-A2 expression in KV1858 cell lines. FIG. 57B shows HLA-A2 expression in KV1832 cell line. FIG. 57C shows HLA-A2 expression in SW620 cell line. Fig. 57D shows GFP expression in transfected SW620 cells.

Figure 58 shows Nur77 expression in TCR089 neoTCR T cells co-cultured with SW620 cells homozygous for the COX6C-R20Q mutation.

Figure 59A shows that T cells expressing neoTCR killed SW620 homozygous tumor cells. Fig. 59B shows that wild-type SW620 cells were not killed.

Figure 60 shows that TCR089 killed SW620 cells homozygous for the COX6C-R20Q mutation, but not wild-type SW620 cells.

Figure 61 shows IFN γ secretion in TCR 089T cells after coculture with SW620 cells homozygous for the COX6C-R20Q mutation.

Fig. 62 shows a method of the imPACT separation technique: NeoE-specific TCRs were isolated from patients treated with checkpoint inhibitors, sequenced, tumor antigens identified, neoepitopes selected using algorithms to screen using comapt polypeptides and imPACT isolation techniques, and neoepitope-specific T cells captured.

FIG. 63 shows a patient sample from a patient who did not respond to anti-PD-1 treatment, the patient's neoE-HLA complex breakdown and the resulting identified TCR. FIG. 63 discloses SEQ ID NO 324.

FIG. 64 shows neoTCR-T cells killing autologous melanoma tumor cells.

FIG. 65A shows the ability of neoTCR-T cells to kill autologous tumor cells. FIG. 65B shows that neoTCR-T cells express activation markers when co-cultured with autologous tumor cells. FIG. 65C shows that neoTCR-T cells secrete interferon gamma when co-cultured with autologous tumor cells.

Figure 66A shows neoTCR T cell therapy eradicates tumors implanted in mice. Figure 66B shows the number of human CD8T cells/mL present in mouse blood at day 4 post neoTCR T cell infusion and at day 35 post infusion.

Fig. 67A and 67B illustrate neoantigen-specific TCR construct designs for integration of a neoantigen-specific TCR construct (neoTCR) into the TCR α locus. Figure 67A illustrates the target TCR α locus (endogenous TRAC, top panel) and its CRISPR Cas9 target site (horizontal stripes, cleavage site designated by arrows), as well as the circular plasmid Homologous Recombination (HR) template with the polynucleotide encoding neoTCR (bottom panel), which was located between the left and right homology arms ("LHA" and "RHA", respectively) prior to integration. RNP: CRISPR/Cas9 complex. Figure 67B illustrates the translation and processing of the integrated neoTCR in the TCR α locus (top panel), the transcribed and spliced neoTCR mRNA (middle panel), and the expressed neoTCR (bottom panel).

Detailed Description

Definition of

Unless otherwise indicated, the terms used in the claims and the specification are defined as follows.

As used herein, the term "antigen-specific T cells" refers to cells that are distinguished from each other by their T Cell Receptors (TCRs) that confer their antigen specificity.

Embodiments of the compositions and methods disclosed herein include recombinant antigen-MHC complexes capable of pairing with cognate T cells. As used herein, "antigen-MHC," "antigen-MHC complex," "recombinant antigen-MHC complex," "peptide MHC," and "p/MHC" are used interchangeably to refer to the major histocompatibility complex with a peptide in the antigen-binding groove.

As used herein, "antigen" includes any antigen, including patient-specific antigens.

"antigenic peptide" and "antigenic peptide" as well as "neo-epitope" and "NeoE" are used interchangeably to refer to a peptide derived from an antigen identified on a cell of interest (e.g., an antigen expressed by a tumor cell if it is a tumor cell), which is incorporated into a comPACT polypeptide using the molecular biology techniques described herein. Furthermore, as explicitly stated in the examples, the terms "neoantigen sequence" and "neoantigen insert" may have the same meaning as "antigenic peptide" and "neo-epitope" and "NeoE". The term also refers to peptides or peptide fragments that are capable of binding to MHC molecules.

"antigen-MHC complex" and "antigen-MHC" and "recombinant antigen-MHC complex" and "peptide MHC" and p/MHC "and" neoantigen-MHC complex "are used interchangeably and refer to a ternary complex consisting of HLA/MHC heavy chain, β 2M chain and antigen peptide.

The "anti-CTLA 4 antibody" antibody attaches to CTLA-4 and prevents its work. This may enhance the body's immune response to cancer cells. Including ipilimumab. Including AB154(Arcus), tiragolumab (Genentech/Roche), BMS-986297(BMS), MK-7684(Merck), and etiglimab (Oncomed). In addition to anti-CTLA 4 antibodies, CTLA4 inhibitors (both large and small molecules) can be used in combination with any neoTCR product.

anti-PD-1 antibodies "and" antibodies that bind to PD-1 "and" anti-PD-1 therapy "refer to antibodies that bind to and are capable of binding to PD-1 with sufficient affinity such that the antibodies are useful as diagnostic and/or therapeutic agents targeting PD-1. In certain embodiments, an antibody that binds or is capable of binding to PD-1 is capable of blocking the interaction of PD-1 and PD-L1 and enhancing an immune response against cancer cells. anti-PD-1 antibodies include, but are not limited to, pembrolizumab, nivolumab, and cimiralizumab. In addition to anti-PD 1 antibodies, PD1 inhibitors (both large and small molecules) can be used in combination with any neoTCR product.

"anti-PD-L1 antibody" and "antibody that binds to PD-L1" and "anti-PD-L1 therapy" refer to an antibody that binds to and is capable of binding PD-L1 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent targeting PD-L1. In certain embodiments, an antibody that binds or is capable of binding PD-L1 is capable of blocking the interaction of PD-1 and PD-L1 and enhancing the immune response against cancer cells. anti-PD-L1 antibodies include, but are not limited to, atelizumab (atezolizumab), avelumab, and dolacizumab (durvalumab). In addition to the anti-PD-L1 antibody, PD-L1 inhibitors (both large and small) can be used in combination with any neoTCR product.

"attachment moiety" refers to any chemical or biological moiety that can be used to attach a polynucleotide or polypeptide to a chemical or biological substrate. As used herein, an attachment moiety is used to attach a polynucleotide or polypeptide to a particle.

"checkpoint inhibitors" refer to the class of drugs that block certain proteins produced by some types of immune system cells (e.g., T cells) and some cancer cells. These proteins help to control the immune response and may prevent T cells from killing cancer cells. When these proteins are blocked, the "brakes" of the immune system are released and the T cells are better able to kill the cancer cells. Examples of checkpoint proteins found on T cells or cancer cells include PD-1/PD-L1 and CTLA-4/B7-1/B7-2. Some immune checkpoint inhibitors are useful for treating cancer.

"barcode" and "barcode sequence" and "nucleotide barcode" and "barcoded polynucleotide" and "neoID barcode" are used interchangeably and refer to a nucleotide sequence used to label and identify a particular peptide.

"barcoded particles" refers to particles to which barcodes are attached.

"beta-2-microglobulin", "beta 2M" are used interchangeably and have the same meaning.

"comPACT" and "comPACT construct" are used interchangeably and refer to a polynucleotide or polypeptide, including neo-antigen and MCH complexes, based on the context of how the term is used. The comPACT may also comprise a signal sequence, universal target sites, linkers and purification clusters. Fig. 1 shows a non-limiting representation of comact.

The "comPACT library" and the "comPACT-neoID library" are used interchangeably and represent one or more comPACTs.

"comPACT minigene" or "comPACT polynucleotide" or "comPACT gene" or "comPACT polynucleotide molecule" are used interchangeably and mean a nucleic acid sequence encoding a comPACT protein.

By "comPACT protein" or "comPACT polypeptide molecule" is meant an MHC molecule expressed as a single polypeptide fusion of a universal target sequence, an antigenic peptide, a second universal target sequence, β 2-microglobulin, and an MHC class I heavy chain, comprising the α 1, α 2, and α 3 domains forming an MHC display moiety. The comPACT polypeptides described herein may also optionally include linker sequences between any or all of the individual components of the comPACT polypeptide. An example of placement of an optional linker sequence in a comPACT polypeptide is shown in the comPACT minigene of fig. 1.

An "effective amount" is an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

An "epitope" or "epitope tag" refers to an affinity tag in which a peptide sequence is genetically engineered into a polypeptide and in which an antibody can bind to the peptide sequence. Epitope tags include, but are not limited to, the V5 tag, the Myc tag, the HA tag, the Spot tag, the NE tag, and all other epitopes that can be used as affinity tags. Epitope tags can be formed from a contiguous sequence of amino acids (linear epitopes) or comprise non-contiguous amino acids (conformational epitopes), for example, in spatial proximity due to folding of the antigen. "linker" refers to any amino acid sequence (or nucleic acid sequence encoding such amino acid sequence) used to link components in a fusion protein. When applied to comPACT proteins (fusion proteins), linkers can be used, for example, to link NeoE to β 2M or β 2M to an MHC heavy chain or to link an MHC heavy chain to a purification cluster.

Both "host cell" and "producer cell" refer to a cell into which an exogenous nucleic acid has been introduced, including progeny of such a cell. Host cells include primary transformed cells and progeny derived therefrom, regardless of passage number. Progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Included herein are mutant progeny that have the same function or biological activity as screened or selected in the originally transformed cell.

An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain aspects, the individual or subject is a human.

An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is extrachromosomal or at a chromosomal location different from its natural chromosomal location.

By "MHC complex" is meant a complex comprising β 2-microglobulin and an MHC heavy chain. The MHC complex may be a polypeptide or a polynucleotide encoding such a polypeptide. MHC complexes are contained in all comPACT proteins, and polynucleotides encoding such β 2-microglobulin and MHC heavy chains are contained in all comPACT minigenes. Fig. 1 and 2 show two examples of inclusion of MHC complexes in comPACT minigenes. For example, fig. 11 shows a western blot of comPACT protein comprising MHC class I heavy chain complexes.

By "MHC display portion" is meant an MHC class I heavy chain comprising the α 1, α 2 and α 3 domains.

"MHC" refers to the major histocompatibility complex, which is a group of genes that encode cell surface proteins essential for the adaptive immune system or for recognition of foreign molecules. The main function of MHC molecules is to bind foreign antigens, including antigens present on endogenous cells that are harmful to an organism (e.g., a human), and to display them on the cell surface for recognition by appropriate T cells. Three subgroups of the MHC family are class I, class II and class III.

"MHC class I" refers to a subgroup of the MHC family comprising the subunit of Beta-2-microglobulin.

"neoantigen" refers to an antigen having at least one alteration that renders the presentation of the neoantigen or neoantigen different from its corresponding wild-type antigen, e.g., a mutation in the polypeptide sequence, a difference being a post-translational modification or a difference in the level of expression. "neoantigen" and "tumor neoantigen" refer to a specific antigen on a cell that can be used as a recognition target for killing. When applied to cancer and tumors, the neoantigen is an antigen specific to the tumor or cancer. When applied to pathogens and pathogen-infected cells, the neoantigens are antigens that are specific to the pathogen or pathogen-infected cell. By "tumor neoantigen" is meant a neoantigen derived from a tumor or cancer, e.g., from a patient's tumor.

"neoTCR product" and "neoTCR T cell therapy" and "neoTCR T cell" are used interchangeably and both refer to genetically engineered T cells expressing TCRs that recognize neoepitopes that have been identified and designed using copack polypeptides and polynucleotides and imPACT isolation techniques.

"Neo 12" and "Neo 12 protein" refer to exemplary Neo epitopes.

"NTAmer" refers to a complex comprising comPACT polypeptide.

By "off-the-shelf" is meant, in terms of the design of the comPACT polynucleotides and comPACT polypeptides prepared therefrom, the comPACT minigenes comprising Beta-2-microglobulin, the MHC heavy chain allele, and the location of the inserted neo-epitope in such constructs. In certain embodiments, the construct is in 5 'to 3' order 1) neo-epitope, 2) Beta-2-microglobulin, and 3) MHC heavy chain allele. In certain embodiments, a signal sequence, a universal target site (e.g., a restriction enzyme site), a flexible linker, and a purification cluster are also incorporated into the construct. In certain embodiments, the construct with the additional elements is the construct disclosed in figure 2.

By "operatively associated" is meant that, in terms of the construction of particles, each particle constructed using a given comact (in which a specific neoantigen is expressed) is associated with one or more barcodes unique to that particle. In this way, downstream sequencing determines which barcodes bind to specific cells, which can be used to determine which comPACT (and which neoantigen) is responsible for the binding.

By "particle", "collection of particles", "pair of particles" and "collection of distinct particles", it is meant, with the term "particle", the core of a comact comprising a matrix capable of being specifically sorted or separated and to which components of the comact (as well as additional polypeptides, polynucleotides and chemicals) may be attached. In certain embodiments, "collection of particles" refers to a plurality of particles.

The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a formulation that is present in a form that allows for the biological activity of the active ingredient contained therein to be effective, and that does not contain additional ingredients that have unacceptable toxicity to the subject to whom the pharmaceutical composition is administered.

By "pharmaceutically acceptable carrier" is meant an ingredient of a pharmaceutical composition or formulation that is non-toxic to a subject, other than the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

As used herein, "polynucleotide" or "nucleic acid" are used interchangeably and include any compound and/or substance that contains a nucleotide polymer. Each nucleotide consists of a base, in particular a purine or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (a), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or ribose) and a phosphate group. Typically, a nucleic acid molecule is described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The base sequence is usually represented as 5 'to 3'. By polynucleotide is meant any DNA (including but not limited to cDNA, ssDNA and dsDNA) and any RNA (including but not limited to ssRNA, dsRNA and mRNA), and also synthetic forms of DNA and RNA and mixed polymers comprising two or more of these molecules. Those skilled in the art will understand which form is referred to, e.g., based on the context in which the polynucleotide is used. The polynucleotide may be linear or circular. In addition, the term polynucleotide includes both sense and antisense strands, as well as single-and double-stranded forms. The polynucleotide may comprise naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derivatized sugar or phosphate backbone linkages or chemically modified residues. Polynucleotides encompass DNA and RNA molecules suitable as vectors for direct expression of the polypeptides of the invention in vitro and/or in vivo.

By "proliferative disorder" is meant excessive cellular proliferation of one or more subpopulations of cells in a multicellular organism, resulting in injury (i.e., discomfort or decreased life expectancy) to the multicellular organism. Cell proliferative disorders can occur in different types of animals and humans. As used herein, proliferative disorders include neoplastic disorders.

"protein" and "polypeptide" are used interchangeably herein.

By "purification cluster" is meant an optional portion of comPACT that includes gene coding elements that allow purification of comPACT.

"Signal sequence" refers to a short peptide present at the N-terminus of a newly synthesized protein, which is destined to be oriented in the secretory pathway. Signal sequences may be included in the comPACT design and production.

As used herein, "treatment" (and grammatical variations thereof such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the disease in the individual being treated, and may be performed for prophylaxis or in the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of disease, alleviating symptoms, alleviating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and/or improving prognosis. In some aspects, the antibodies of the invention are used to delay the progression of a disease or slow the progression of a disease.

"Universal target site", "universal target sequence" and "universal sequence" are used interchangeably and refer to a polynucleotide sequence that can be cleaved by a restriction endonuclease or a primer binding site that can be used for binding of primers and amplification of a desired sequence.

"vector," "expression vector," and "expression construct" are used interchangeably and refer to a discrete element used to introduce heterologous DNA into a cell for its expression or replication. As used herein, a vector can be engineered and used to express in vivo or in vitro the polypeptide gene product encoded by the coding sequence inserted into the vector.

"young" or "young" when associated with T cells refers to memory stem cells (T)_MSC) And central memory cells (T)_CM)。These cells have T cell proliferation upon specific activation and are capable of multiple cell divisions. They also have the ability to implant after re-infusion, rapidly differentiate into effector T cells and target and kill tumor cells after exposure to their cognate antigen, and continue to monitor and control cancer.

As used herein, the terms "barcoded T cells," "paired T cells," "T cell-binding nanoparticles," and "T cell-paired antigen MHC complex" refer to complexes of T cells having a T cell receptor that binds to an antigenic peptide presented by an MHC molecule on a barcoded NP-antigen-MHC complex (i.e., a particle-comact complex).

As used herein, "antibody" or "antibodies" are used in the broadest sense and include a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab '-SH, F (ab') 2; a diabody; a linear antibody; single chain antibody molecules (e.g., scFv and scFab); a single domain antibody (dAb); and multispecific antibodies formed from antibody fragments.

The term "in vivo" refers to a process that occurs in a living organism (including a cell).

As used herein, the term "mammal" includes humans and non-humans, and includes, but is not limited to, humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

The term "percent sequence identity," in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to the skilled artisan) or by visual inspection. Depending on the application, "percent sequence identity" may be present over a region of the sequences being compared, e.g., over a functional domain, or alternatively, over the entire length of the two sequences being compared.

For sequence comparison, one sequence is typically used as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the established program parameters.

Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, adv.appl.Math.2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.mol.biol.48:443(1970), by the similarity search method of Pearson & Lipman, Proc.Nat' l.Acad.Sci.USA 85:2444(1988), by computerized implementation of these algorithms (GAP, BESTFIT, FASTA and TFASTA, in the Wisconsin Genetics software package, Genetics Computer Group,575Science Dr., Madison, Wis.), or by visual inspection (see Ausubel et al, infra).

One example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al, J.mol.biol.215: 403-. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov /).

It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

Other explanatory convention

Ranges recited herein are to be understood as shorthand for all values within the range, including the endpoints recited. For example, a range of 1 to 50 is understood to include any number or fraction thereof, combination of numbers or fractions thereof, or subranges from (including any number of fractions from) the group consisting of 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49, and 50.

Introduction to the design reside in

T cell-mediated immunity is characterized by the activation of antigen-specific cytotoxic T cells that are capable of inducing the death of cells displaying antigen in the Major Histocompatibility Complex (MHC) on their surface. These cells displaying antigen-loaded MHC complexes include virus-infected cells, cells with intracellular bacteria, cells with internalized or phagocytized extracellular protein sources, and cancer cells displaying tumor antigens.

Natural MHC class I heavy chains comprise about 350 amino acids; native β 2-microglobulin comprises about 100 amino acids, and class I antigenic peptides typically have a length of about 7 to about 15 amino acids. The class I heavy chains are encoded by genes of the major histocompatibility complex and are designated HLA-A, -B, and-C in humans and H-2K, D and L in mice. Class I heavy chains and β 2-microglobulin are encoded on different chromosomes, respectively. Antigenic peptides are typically processed by cells from protein sources, such as viruses, bacteria, or cancer cells. Different variants of the polypeptides encoded by the human HLA-A, -B and-CMHC genes and the murine H-2K, D and LMHC genes have been identified.

Embodiments of the methods disclosed herein relate to methods of making single molecules in which a selected neoantigen is linked to an MHC complex comprising β 2-microglobulin (β 2M) and an MHC heavy chain. Different MHC heavy chains can be attached to β 2M molecules, forming different numbers of MHC templates. The method disclosed herein for inserting neoantigens into MHC templates by restriction digestion or PCR-based assembly by using universal target sequences flanking the neo-epitope insertion sites (also referred to as neoantigen insertion sites) leads to the ability to construct libraries of different neoantigen-MHC complexes in a high-throughput manner, which can be personalized for a specific patient. These complexes, referred to as "comPACT proteins," can then be attached to, for example, particles, barcode particles or surfaces for the isolation and identification of patient-specific T cell populations targeting patient-specific neo-antigens. Methods of attaching antigen-MHC complexes and uses of such complexes are disclosed in PCT/US2018/21611 filed on 3, 8, 2018, which is incorporated herein by reference in its entirety.

Nucleotide and peptide compositions

MHC complexes

Briefly, as used herein, a comPACT polypeptide refers to an MHC molecule expressed as a single fusion polypeptide of a universal target sequence, an antigenic peptide, a second universal target sequence, β 2-microglobulin, and an MHC class I heavy chain, comprising α 1, α 2, and α 3 domains forming an MHC display moiety. The comPACT polypeptides described herein may also optionally include linker sequences between any or all of the individual components of the comPACT polypeptide. An example of placement of an optional linker sequence in a comPACT polypeptide is shown in the comPACT minigene of fig. 1. The MHC display portion may comprise a recombinant MHC molecule. The design and manufacture of individual comPACT polypeptides and comPACT polypeptide molecular libraries is described in international application PCT/US2019/025415, filed 2019, 4, month 2, which is incorporated herein by reference in its entirety. In certain embodiments, the comPACT polypeptide may comprise a disulfide trap, as described in U.S. publication No. 2009/0117153 and U.S. publication No. 2008/0219947; each of which is incorporated herein by reference. The antigen-MHC complex formed by the comPACT proteins results in the display of antigens such that they can be recognized by the cognate TCR molecules. In some embodiments, the MHC complex may be an MHC class I (MHC I) complex paired with a CD8 positive (CD8+) T "killer" cell. In some embodiments, the MHC complex may be an MHC class II (MHC II) complex paired with a CD4 positive (CD4+) T cell.

In some embodiments, the MHC class I heavy chain sequence of comPACT may include a single amino acid substitution, addition, and/or deletion, such as substituting Tyr-84 with a non-aromatic amino acid other than proline. In these embodiments, the amino acid substitution can be any amino acid encoded by the standard genetic code, such as leucine, isoleucine, valine, serine, threonine, alanine, histidine, glutamine, asparagine, lysine, aspartic acid, glutamic acid, cysteine, arginine, serine, or glycine, or can be a modified or unusual amino acid. In one embodiment, the MHC class I heavy chain sequence of comPACT comprises a substitution of tyrosine 84 to alanine. In another embodiment, the MHC class I heavy chain sequence of comPACT comprises a Tyrosine-84 to cysteine substitution.

Beta 2-microglobulin (beta 2M) may comprise a recombinant beta 2M molecule. In some embodiments, the β 2M sequence may include single amino acid substitutions, additions and/or deletions as described above. In one embodiment, the substitution comprises a substitution of serine-88 to cysteine. In one embodiment, the substitution may be a substitution of any naturally occurring non-cysteine amino acid of β 2M to cysteine, wherein the substitution does not negatively affect the function of β 2M within the comPACT polypeptide, and the substitution allows conjugation of a thiol-reactive moiety. Such substitutions can be achieved, for example, by cysteine screening of the protein using mutagenesis techniques known to those skilled in the art. Such thiol-reactive moieties may be used to detect β 2M or whole comact polypeptides. In certain embodiments, the thiol-reactive moiety is a thiol-reactive dye (fluorophore) conjugate, which allows measurement of kinetic parameters of TCR-comact binding using comPACT (see, e.g., example 8). In certain embodiments, the thiol-reactive moiety is a dye (fluorophore) comprising a thiol-reactive crosslinker reactive group, including, but not limited to, maleimide, iodoacetamide or derivatives thereof, haloacetyl, pyridyl disulfide, and all other thiol-reactive conjugation partners (see, e.g., Haughland, 2003, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc.; Brinkley,1992, Bioconjugate Chem.3: 2; Garman,1997, Non-Radioactive Labelling: A Practical laboratory, Academic Press, London; Means (1990) Bioconjugate Chem.1: 2; Hermanan, G.in Bioconjugate Chem (1996) Academic Press, Sanego, pp.40-643, 671).

Universal sequence

Antigenic peptides are typically flanked by universal sequences or portions thereof. These sequences allow for a rapid, high-throughput method for replacing or inserting antigenic peptide-encoding nucleotides in polynucleotide MHC templates. The universal sequence may comprise restriction sites for restriction digestion based cloning. Exemplary restriction sites include, but are not limited to, Nco1, BamHI, BlpI, BspEI, BstBI, Xbal, HindIII, EcoRI, ApaI, NotI, any restriction site not present in the β 2M, MHC heavy chain, NeoE, the purification cluster of the signal sequence (if present) or the fusion of any of its components (including optional linker sequences), and any combination thereof. Alternatively, the universal sequence may be a primer binding site. Universal primer sequences known in the art may be used in the compositions and methods disclosed herein, or the sequences may be different from the previously described universal primer sequences and may be designed to promote specific binding/amplification and eliminate non-specific binding/amplification. The length of the universal sequence may be between 4-50, between 4-15, between 15-40, between 15-35, between 15-30, between 20-40, between 25-40, or between 30-40 nucleotides. The universal sequence can be at least 4, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 nucleotides in length. In some embodiments, the universal target sequence is 4-8 nucleotides in length. In other embodiments, the universal target sequence is between 9-25 nucleotides in length. In other embodiments, the universal target sequence is between 25-35 nucleotides in length. In other embodiments, the universal target sequence is at least about 15 nucleotides in length. In certain aspects, one or more universal target sequences are not present in the manipulated genetic material, e.g., in order to reduce or eliminate off-target effects and/or increase specificity.

Joint

In various embodiments, the comPACT may comprise a first flexible linker interposed between the antigenic peptide segment and the β 2-microglobulin segment. Such a linker may extend from the carboxy terminus of the antigenic peptide segment and link the carboxy terminus of the antigenic peptide segment to the amino terminus of the β 2-microglobulin segment. In a non-limiting example, when comPACT is expressed, the linked peptide ligands can fold into the binding groove, producing a functional comPACT protein. In various embodiments, the linker is at least about 10 amino acids and up to about 15 amino acids. In various embodiments, the linker is between 4 and 32 amino acids.

In various embodiments, the comPACT may comprise a second flexible linker interposed between the β 2-microglobulin segment and the MHC heavy chain segment. Such linkers may extend from the carboxy terminus of the β 2-microglobulin segment and link the carboxy terminus of the β 2-microglobulin segment to the amino terminus of the MHC heavy chain segment. In a non-limiting example, when comPACT is expressed, β 2-microglobulin and MHC heavy chain can fold into the binding groove, producing molecules that can play a role in promoting T cell expansion. In various embodiments, the linker may comprise at least about 15 amino acids, up to about 20 amino acids. In various embodiments, the linker is at least about 10 amino acids and up to about 15 amino acids. In various embodiments, the linker is between 4 and 32 amino acids.

In various embodiments, the comPACT may comprise a third flexible linker interposed between the MHC heavy chain segment and the purification cluster. Such linkers can extend from the carboxy terminus of the MHC heavy chain segment and attach it to the amino terminus of the purification cluster. In various embodiments, the linker is at least about 10 amino acids and up to about 15 amino acids. In various embodiments, the linker is between 4 and 32 amino acids. In some embodiments, the linker is only 2 or 3 amino acids.

In certain embodiments, the same linker may be used for the first and second linkers, and optionally the third linker (if present). In certain embodiments, the same linker is used for the first, second and third linkers. In certain embodiments, all three linkers are the (G4S)4 linker (SEQ ID NO: 19). In certain embodiments, all three linkers are (G3S) n linkers (SEQ ID NO: 201). In certain embodiments, all three linkers are (GSGGS) n linkers (SEQ ID NO: 11).

In certain embodiments, all three linkers are (GCGGS) n linkers (SEQ ID NO: 13).

In certain embodiments, a different sequence is used for each of the first and second linkers, and optionally the third linker (if present).

In certain embodiments, two of the first, second, and optional third linkers are the same and one is different.

Any suitable flexible linker sequence known in the art may be used. Suitable linker sequences include, but are not limited to, those comprising GGGGS (G)₄S)(SEQ ID NO:9),GGGS(G₃S) (SEQ ID NO:201), GSGGS (SEQ ID NO:11) or GCGGS (SEQ ID NO:13) sequence motifs. In certain embodiments, a cleavable linker can be used for any of the first, second, and third linkers. In certain embodiments, a cleavable linker is used only for the first, second, or third linker. In certain embodiments, a cleavable linker is used for only the first linker. In certain embodiments, a cleavable linker is used for only the second linker. In certain embodiments, a cleavable linker is used for the third linker only.

In certain embodiments, the linkers (first, second, and/or third) may be selected from the group consisting of less rigid or flexible linkers.

Signal sequence

In various embodiments, the comPACT polynucleotides and polypeptides comprise a signal sequence and a signal peptide. In one embodiment, the signal sequence is a signal sequence from human growth hormone (hGH). Additional signal sequences may also be used, including but not limited to signal sequences from β 2M, or any other eukaryotic or prokaryotic signal sequence known in the art. Any signal sequence that directs the comPACT protein to the secretory pathway (for secreting comPACT from the cell) may be used.

In certain embodiments, the signal sequence comprises the amino acid sequence of SEQ ID NO 2. In certain embodiments, the signal sequence comprises the nucleic acid sequence of SEQ ID NO 1.

The signal sequence may be between 70 and 80 nucleotides in length. The signal sequence may be between 40-90,40-60,45-70,50-80,60-90,55-70,60-80 or 70-80 nucleotides in length. The signal peptide may be between 10-30,10-20,15-30 or 20-30 amino acids in length.

Promoters

The comPACT polynucleotide composition may further comprise a promoter for transcribing the encoded polynucleotide into an mRNA transcript that is translatable by the host cell. The source of the promoter may be prokaryotic, viral, or eukaryotic (such as, but not limited to, mammalian). Any suitable promoter for gene transcription in a cell may be used. In certain embodiments, eukaryotic promoters may be used. In certain embodiments, the type of eukaryotic promoter is a constitutive promoter, an inducible promoter, or a specific promoter. In certain embodiments, the eukaryotic promoter is an EF1a, Cytomegalovirus (CMV), CAG, PGK, RE, U6, or UAS promoter. In certain embodiments, prokaryotic promoters may be used. In certain embodiments, the type of prokaryotic promoter is a constitutive promoter, a constitutive promoter that requires the presence of a particular polymerase (e.g., T7 or Sp6 RNA polymerase), a promoter that is constitutive in the absence of a repressor and inducible in the presence of an inducing agent (e.g., without limitation, a lac promoter that is constitutive in the absence of a lac repressor but inducible by IPTG or lactose), an inducible promoter, a repressible promoter, or a regulated promoter. In certain embodiments, the prokaryotic promoter is a T7, Sp6, lac, araBad, trp, or Ptac promoter. In certain embodiments, viral promoters may be used. In certain embodiments, the type of viral promoter is an AAV promoter or an SV40 promoter.

In some embodiments, the comPACT polynucleotide comprises SV40 or any viral promoter. In certain embodiments, a strong viral promoter may be beneficial depending on the cell line and agent.

In some embodiments, the comPACT polynucleotide comprises a CMV promoter.

Affinity tag

The comPACT polynucleotide composition may further comprise at least one sequence encoding an affinity tag or an epitope tag. In some embodiments, the comPACT polynucleotide comprises at least two affinity tags or epitope tag sequences. Any suitable affinity tag or epitope tag may be used in the comPACT polynucleotide or polypeptide. Such epitope tags include, but are not limited to, AviTag (or any avidin/streptavidin tag), strep tag, polyhistidine (His6) -tag (SEQ ID NO:34), FLAG-tag, HA-tag, and Myc-tag. The sequence in the polynucleotide comPACT gene is translated into a peptide in the comPACT polypeptide. These epitope tags can be used for affinity chromatography purification or quantification of the expressed comPACT polypeptide. For example, the His6 tag (SEQ ID NO:34) can be used to purify comPACT protein by HA-tag binding affinity chromatography. In certain embodiments, metal ion resins can be used to purify HA-tagged proteins. In certain embodiments, Ni2+ (nickel) resin, Co2+ (cobalt) resin, Cu2+ (copper) resin, Ca2+ (calcium) resin, Zn2+ (zinc) resin, or any combination thereof, may be used to purify HA-tagged proteins. In certain embodiments, Ni2+ resin is used to purify HA-tagged comPACT protein. In certain embodiments, a mixture of Ni2+ and Zn2+ resins is used to purify HA-tagged comPACT protein. In certain embodiments, the resin is an immobilized metal affinity chromatography resin (IMAC). In certain embodiments, the metal ion is coupled to the resin matrix by a chelating ligand. In certain embodiments, the metal ions are coupled to the resin matrix with nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA).

In addition, AviTag encodes a known biotinylation site that is recognized by the BirA enzyme. The inclusion of this peptide sequence in the protein allows biotinylation of the sequence by enzymatic modification by BirA. Thus, a comPACT polypeptide comprising an AviTag (or any avidin/streptavidin tag) sequence and a His6 tag (SEQ ID NO:34) can be biotinylated, purified via the His6 tag (SEQ ID NO:34) by metal affinity chromatography (e.g., Ni-NTA affinity chromatography or any other metal affinity resin described herein), and the purity or quantity of the purified protein assessed by biotin visualization using streptavidin or other avidin reagents. In some embodiments, the comPACT polynucleotide comprises an AviTag (or any avidin/streptavidin tag) sequence. In some embodiments, the comPACT polypeptide comprises an AviTag (or any avidin/streptavidin) epitope. In some embodiments, the comPACT polynucleotide comprises a His6 sequence (SEQ ID NO: 34). In some embodiments, the comPACT polypeptide comprises the His6 epitope (SEQ ID NO: 34). In some embodiments, the comPACT polynucleotide comprises an AviTag (or any avidin/streptavidin) sequence and a His6 sequence (SEQ ID NO: 34). In some embodiments, the comPACT polypeptide comprises an AviTag (or any avidin/streptavidin) epitope and a His6 epitope (SEQ ID NO: 34).

Protease cleavage site

The comPACT polynucleotide composition may further comprise a sequence encoding a protease cleavage site in the purification cluster. The cleavage site may be encoded between the first and second affinity tag sequences and allow cleavage of the second affinity tag from the comPACT protein once the comPACT has been expressed and subjected to one round of purification. Any suitable protease cleavage site known in the art may be used, including but not limited to those recognized by TEV, thrombin, factor Xa, enteropeptidase, and rhinovirus 3C protease, among others. In one embodiment, the protease cleavage site nucleotide sequence encodes a TEV cleavage site. In another embodiment, the comPACT polypeptide comprises a TEV protease cleavage site.

PolyA tail

The comPACT polynucleotide composition may further comprise a polyadenylation (polyA) tail. Eukaryotic (including mammalian) or prokaryotic polyA sequence motifs may be used. This sequence may be included when the comPACT polynucleotide is assembled by PCR for direct transfection into a host cell (e.g., not in the case of an expression construct or vector). Any suitable polyA tail and sequence motif may be used in comapt polynucleotides, including but not limited to polyA tails of SV40, hGH, bGG, and rbGlob sequences. Such sequences include the sequence motif AAUAA. In one embodiment, the comPACT polynucleotide comprises a BHG polyA tail sequence.

Antigen sequences

The length of the antigen sequence (i.e., the sequence of the neoantigen to which the neo-epitope portion of the comPACT polypeptide is designed to bind) may be between 20-60, between 20-30, between 25-35, between 20-45, between 30-45, between 40-60, or between 45-60 nucleotides. The antigenic peptide may be or be derived from an exogenous antigen, an endogenous antigen (including heterologous, autologous and homologous antigens) or an autoantigen. The antigenic peptide can be or be derived from an antigen that originates from an exogenous antigen and then subsequently becomes an endogenous antigen (e.g., an intracellular virus). The antigenic peptide may be or be derived from a tumor antigen, a neoantigen, a tumor neoantigen, a viral antigen, a bacterial antigen, a phospho antigen or a microbial antigen. In one embodiment, the antigenic peptide is a neoantigen. The antigenic peptide may be selected from patient data and may comprise one or more somatic mutations.

In order to make an inclusive comact library with multiple new epitopes and, in turn, multiple comact polypeptides, the antigen sequences need to be predicted and identified. Prediction of antigenic peptides may include prediction algorithms and they may be designed to predict binding of antigenic peptides or neoantigens to MHC alleles. Prediction of antigenic peptides is discussed further below.

In some embodiments, the nucleotide sequence encoding the antigenic peptide is between 20-60, between 20-30, between 25-35, between 20-45, between 30-45, between 40-60, or between 45-60 nucleotides in length. In other embodiments, the nucleotide sequence encoding the antigenic peptide is between 20-30 nucleotides in length. In some embodiments, the antigenic peptide is 7-15 amino acids, 7-10,8-9,7,8,9,10,11,12,13,14, or 15 amino acids in length.

Biotinylation of the compound

The comPACT protein described herein may be further biotinylated by any suitable method. One such method utilizes BirA biotin-protein ligase and is commercially available. A specific amino acid sequence, designated the AviTag sequence (GLNDIFEAQKIEWHE (SEQ ID NO:30)), is encoded in the protein of interest. BirA ligase, d-biotin and ATP are added to a reaction mixture containing the target protein. BirA covalently links biotin to lysine in the AviTag sequence, thus biotinylating the target protein. The new biotinylated protein can then be purified and used for downstream applications. Other methods known in the art for biotinylating proteins may also be used. For clarity, any suitable avidin/streptavidin sequence may be used in the comPACT protein preparation.

Expression constructs and vectors

The comPACT polynucleotide molecules may be inserted into expression constructs or expression vectors, such as for plasmids (to increase the number of expression constructs or expression vectors encoding comPACT polynucleotides for protein production) and protein production. The expression construct or expression vector may be a eukaryotic, prokaryotic, or viral expression vector. Any suitable expression construct or expression vector known in the art may be used, including bacterial expression plasmids, such as e.coli or bacillus subtilis plasmids; eukaryotic expression vectors, such as mammalian expression vectors or yeast expression vectors; or a viral vector, such as an adenoviral expression vector, a lentiviral expression vector, a vaccinia expression vector, or a baculovirus expression vector. Mammalian expression constructs or expression vectors can be used (e.g., transfected) with cultured mammalian cell lines, such as Chinese Hamster Ovary (CHO), J558, NSO, SP2-O, HEK293, HECK293T, Expi293, HeLa or any derivative or modified cell line of CHO, HEK293, Expi293 or HeLa cell lines, and any other suitable mammalian cell line. Mammalian expression constructs or expression vectors can be used in primary mammalian cell lines, such as immune or tumor cells obtained or collected (e.g., from a human) directly from an organism (e.g., a human), frozen, and then thawed as needed. In addition to mammalian expression vectors and expression constructs, eukaryotic expression vectors and expression constructs may be used (e.g., transfected) as appropriate in insect cell lines such as Sf9 or Sf12 (or any derivative or modification thereof) or yeast cell lines such as Pichia pastoris (or any derivative or modification thereof). In addition, the expression construct or expression vector may comprise a nucleotide barcode. The nucleotide barcode of each expression construct or vector may be unique. In some embodiments, the nucleotide sequences encoding the signal sequence, beta-2-microglobulin, and MHC allele may be ligated into an expression construct or expression vector having a non-coding or mimetic antigen insertion sequence. The non-coding antigen insert can then be removed by appropriate cloning techniques (e.g., restriction digestion) and the desired antigen sequence (in this case, for clarity, the antigen sequence refers to the neoantigen sequence) can be inserted by ligation or any other appropriate cloning technique.

In some aspects, provided herein are comPACT libraries comprising two or more comPACT polypeptides. Such libraries are created by encoding two or more comact polypeptides in an expression construct or expression vector. In certain embodiments, each expression construct or expression vector comprises a single comact polynucleotide. In certain embodiments, the number of expression constructs or expression vectors (each expression construct or expression vector may be the same expression construct or expression vector) is the same as the number of different comPACT polynucleotides. In certain embodiments, the comPACT polynucleotide is inserted into an expression construct or expression vector using the same or different universal target sites. In other aspects, provided herein are MHC libraries comprising two or more MHC. Such libraries are created by encoding two or more MHC polypeptides in an expression construct or vector. In other aspects, provided herein are HLA libraries comprising two or more HLA's. Such libraries are created by encoding two or more HLA polypeptides in an expression construct or expression vector.

Host cell

In another aspect, provided herein is a host cell comprising a polynucleotide molecule or expression construct described herein. The host cell may be any suitable host cell known in the art, including but not limited to bacterial cells such as e.coli or b.subtilis, or eukaryotic host cells such as Chinese Hamster Ovary (CHO), J558, NSO, SP2-O, HEK293, HEK293T, Expi293, HeLa, insect cell lines (e.g. Sf9 or Sf12), yeast cells such as pichia pastoris, other suitable eukaryotic or prokaryotic cell lines selected scientifically reasonable based on the construct and vector, or any derivative or modification of any such cell line. The host cell can also stably express the biotinylation enzyme BirA. The host cell may be a primary cell or an immortalized cell line.

In some embodiments, the polynucleotide is integrated into the genome of the cell. In some embodiments, the polynucleotide is extrachromosomal. In some embodiments, the host cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is selected from the group consisting of a stem cell, a tumor cell, an immortalized cell, and a fetal cell. In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the cell is an escherichia coli cell. In some embodiments, the cell expresses a BirA protein or a fragment thereof.

In certain embodiments, any of the expression constructs or expression vectors described herein can be inserted into a host cell (e.g., by transfection, transformation, or similar process based on the host cell type) for polypeptide production. In certain embodiments, expression constructs or expression vectors encoding a comPACT polypeptide library, MHC or HLA as described above may be inserted into host cells (e.g., by transfection, transformation, or similar processes based on host cell type) for polypeptide production and purification. In certain embodiments, expression constructs or expression vectors encoding a comPACT polypeptide library, as described below, may be inserted into host cells (e.g., by transfection, transformation, or similar processes based on host cell type) for polypeptide production and purification.

Libraries

In certain embodiments, the library comprises two or more different comPACT polynucleotide molecules. In certain embodiments, the library comprises two or more different polypeptide molecules. In certain embodiments, the library comprises two or more different comact polypeptide molecules attached to the particle.

In certain embodiments, any of 1) a comPACT polynucleotide library, 2) a comPACT polypeptide library or 3) a comPACT polypeptide attached to a particle library comprises more than two respective (respecive) molecules in such respective libraries. In certain embodiments, any of 1) a comPACT polynucleotide library, 2) a comPACT polypeptide library, or 3) a comPACT polypeptide attached to a particle library, has no upper limit on the number of respective molecules in such respective libraries, and in turn comprises as many respective comPACT polypeptides, comPACT polypeptides attached to particles, or comPACT polynucleotides as possible. In certain embodiments, the upper limit is determined by the number of tumor neoantigens detected. In certain embodiments, the upper limit is determined by the number of potential neoepitopes identified based on the detected tumor neoantigen. In certain embodiments, the upper limit is determined by an algorithm.

The library may comprise 2 to 1000 comatt polypeptides, comatt polypeptides attached to particles, or comatt polynucleotides. In some embodiments, the library comprises 2-900,2-800,2-700,2-600,2-500,2-480,2-400,2-300,2-200,2-100,2-50,2-66,2-48,2-30,2-20,2-19,10-1000,10-900,10-800,10-700,10-600,10-500,10-480,10-400,10-300,10-200,10-100,10-50,10-66,10-48,10-30,10-20,20-1000,20-900,20-800,20-700,20-600,20-500,20-480,20-400,20-300,20-200,20-100,20-50,20-50,20-66,20-48,20-30,30-1000,30-900,30-800,30-700,30-600,30-500,30-480,30-400,30-300,30-200,30-100,30-50,30-50,30-66,30-48,30-40,40-1000,40-900,40-800,40-700,40-600,40-500,40-480,40-400,40-300,40-200,40-100,40-60,40-50,40-66,40-48,50-1000,50-900,50-800,50-700,50-600,50-500,50-480,50-400,50-300,50-200,50-100,50-60,50-66,60-1000,60-900,60-800,60-700,60-600,60-500,60-480,60-400,60-300,60-200,60-100,70-1000,70-900,70-800,70-700,70-600,70-500,70-480,70-400,70-300,70-200,70-100,70-80,70-90,80-1000,80-900,80-800,80-700,80-600,80-500,80-480,80-400,80-300,80-200,80-100 comPACT polypeptides, A comPACT polypeptide or a comPACT polynucleotide attached to the particle. In some embodiments, the library comprises between 2-19, 48-480, 48-66, 66-480, 220-240, 40-60, 48-66, 50-70, or 60-80 comPACT polypeptides, comPACT polypeptides attached to particles, or comPACT polynucleotides. In some embodiments, the library comprises at least 2,5,10,15,20,25,30,35,40,45,48,50,55,60,65,66,70,75,80,85,90,100,110,120,130,140,150,160,170,180,190,200,225,250,275,300,325,350,375,400,425,450,475,500,525,550,600,562,650,675,700,725,750,775,800,825,850,875,900,925,950,975, or 1000 paccomt polypeptides, paccomt polypeptides attached to the particles, or comPACT polynucleotides. In some embodiments, the library comprises 2,10,15,20,24,48,66,100,200,300,400,500,600,700,800,900 or 1000 comact polypeptides, comact polypeptides attached to particles, or comact polynucleotides. In some embodiments, two or more comPACT polypeptides, particle-attached comPACT polypeptides, or comPACT polynucleotides in the library have different novel epitope sequences and different MHC sequences.

In certain embodiments, the library comprises two or more comPACT polynucleotides, wherein each comPACT polynucleotide in the library comprises a neoepitope sequence corresponding to a neoantigen detected from the patient sample and an MHC heavy chain sequence.

In some embodiments, the library comprises greater than or equal to two different polynucleotide molecules, wherein each different polynucleotide molecule comprises (i) a first universal sequence, (ii) a nucleotide sequence encoding an antigenic peptide, wherein the nucleotide sequence is not identical for each of the greater than or equal to two polynucleotide molecules, (iii) a second universal target sequence, (iv) a β 2M sequence, and (v) an MHC allele sequence. In some embodiments, the MHC allele sequence is different for each of greater than or equal to two polynucleotide molecules.

In one embodiment, the library comprises HLA-A01: 01, HLA-A02: 01, HLA-A03: 01, HLA-A24: 02, HLA-A30: 02, HLA-A31: 01, HLA-A32: 01, HLA-A33: 01, HLA-A68: 01, HLA-A11: 01, HLA-A23: 01, HLA-A30: 01, HLA-A33: 03, HLA-A25: 01, HLA-A26: 01, HLA-A29: 02, HLA-A68: 02, HLA-B07: 02, HLA-B14: 02, HLA-B18: 01, HLA-B27: 02, HLA-A01: 39: 44, HLA-B46, HLA-B50: 01, HLA-B57: 01, HLA-B58: 01, HLA-B08: 01, HLA-B15: 03, HLA-B35: 01, HLA-B40: 02, HLA-B42: 01, HLA-B44: 03, HLA-B51: 01, HLA-B53: 01, HLA-B13: 02, HLA-B15: 07, HLA-B27: 05, HLA-B35: 03, HLA-B37: 01, HLA-B38: 01, HLA-B41: 02, HLA-B44: 05, HLA-B49: 01, HLA-B01: 55, HLA-C02, HLA-B05: 01, HLA-C02, HLA-B55: 01, HLA-C55, HLA-B55: 01, HLA-C55, HLA-B33: 01, HLA-B55, HLA-C, HLA-5, HLA-B35, HLA-C, HLA-C07: 01, HLA-C01: 02, HLA-C04: 01, HLA-C06: 02, HLA-C07: 02, HLA-C16: 01, HLA-C03: 03, HLA-C07: 04, HLA-C08: 01, HLA-C08: 02, HLA-C12: 03, HLA-C14: 02, HLA-C15: 02, and HLA-C17: 01 alleles. In one embodiment, the library comprises at least HLA-A01: 01, HLA-A02: 01, HLA-A03: 01, HLA-A24: 02, HLA-A30: 02, HLA-A31: 01, HLA-A32: 01, HLA-A33: 01, HLA-A68: 01, HLA-A11: 01, HLA-A23: 01, HLA-A30: 01, HLA-A33: 03, HLA-A25: 01, HLA-A26: 01, HLA-A29: 02, HLA-A68: 02, HLA-B07: 02, HLA-B14: 02, HLA-B18: 01, HLA-B02, HLA-B01: 39: 44, HLA-B46, HLA-B50: 01, HLA-B57: 01, HLA-B58: 01, HLA-B08: 01, HLA-B15: 03, HLA-B35: 01, HLA-B40: 02, HLA-B42: 01, HLA-B44: 03, HLA-B51: 01, HLA-B53: 01, HLA-B13: 02, HLA-B15: 07, HLA-B27: 05, HLA-B35: 03, HLA-B37: 01, HLA-B38: 01, HLA-B41: 02, HLA-B44: 05, HLA-B49: 01, HLA-B01: 55, HLA-C02, HLA-B05: 01, HLA-C02, HLA-B55: 01, HLA-C55, HLA-B55: 01, HLA-C55, HLA-B33: 01, HLA-B55, HLA-C, HLA-5, HLA-B35, HLA-C, HLA-C07: 01, HLA-C01: 02, HLA-C04: 01, HLA-C06: 02, HLA-C07: 02, HLA-C16: 01, HLA-C03: 03, HLA-C07: 04, HLA-C08: 01, HLA-C08: 02, HLA-C12: 03, HLA-C14: 02, HLA-C15: 02 and HLA-C17: 01 alleles.

In certain embodiments, the HLA library comprises HLA-A01: 01, HLA-A02: 01, HLA-A03: 01, HLA-A11: 01, HLA-A23: 01, HLA-A24: 02, HLA-A25: 01, HLA-A26: 01, HLA-A29: 02, HLA-A30: 01, HLA-A30: 02, HLA-A31: 01, HLA-A32: 01, HLA-A33: 03, HLA-A68: 01, HLA-A68: 02, HLA-B07: 02, HLA-B08: 01, HLA-B13: 02, HLA-B01: 15, HLA-B15: 15, HLA-A15: 15, HLA-B27: 02, HLA-B27: 05, HLA-B35: 01, HLA-B35: 03, HLA-B37: 01, HLA-B38: 01, HLA-B39: 01, HLA-B40: 02, HLA-B41: 02, HLA-B42: 01, HLA-B44: 02, HLA-B44: 03, HLA-B44: 05, HLA-B46: 01, HLA-B49: 01, HLA-B50: 01, HLA-B51: 01, HLA-B52: 01, HLA-B53: 01, HLA-B55: 01, HLA-B01: 57, HLA-B03: 03, HLA-C02, HLA-B02: 58, HLA-C03: 04, HLA-C04: 01, HLA-C05: 01, HLA-C06: 02, HLA-C07: 01, HLA-C07: 02, HLA-C07: 04, HLA-C08: 01, HLA-C08: 02, HLA-C12: 03, HLA-C14: 02, HLA-C15: 02, HLA-C16: 01, and HLA-C17: 01. In certain embodiments, the HLA library is composed of HLA-A01: 01, HLA-A02: 01, HLA-A03: 01, HLA-A11: 01, HLA-A23: 01, HLA-A24: 02, HLA-A25: 01, HLA-A26: 01, HLA-A29: 02, HLA-A30: 01, HLA-A30: 02, HLA-A31: 01, HLA-A32: 01, HLA-A33: 03, HLA-A68: 01, HLA-A68: 02, HLA-B07: 02, HLA-B08: 01, HLA-B13: 02, HLA-B01: 15, HLA-B15: 15, HLA-A3: 15, HLA-B27: 02, HLA-B27: 05, HLA-B35: 01, HLA-B35: 03, HLA-B37: 01, HLA-B38: 01, HLA-B39: 01, HLA-B40: 02, HLA-B41: 02, HLA-B42: 01, HLA-B44: 02, HLA-B44: 03, HLA-B44: 05, HLA-B46: 01, HLA-B49: 01, HLA-B50: 01, HLA-B51: 01, HLA-B52: 01, HLA-B53: 01, HLA-B55: 01, HLA-B01: 57, HLA-B03: 03, HLA-C02, HLA-B02: 58, HLA-C03: 04, HLA-C04: 01, HLA-C05: 01, HLA-C06: 02, HLA-C07: 01, HLA-C07: 02, HLA-C07: 04, HLA-C08: 01, HLA-C08: 02, HLA-C12: 03, HLA-C14: 02, HLA-C15: 02, HLA-C16: 01, and HLA-C17: 01. In certain embodiments, the HLA library comprises at least 50%, 60%, 70%, 80%, or 90% or more of the following HLA alleles: HLA-A01: 01, HLA-A02: 01, HLA-A03: 01, HLA-A11: 01, HLA-A23: 01, HLA-A24: 02, HLA-A25: 01, HLA-A26: 01, HLA-A29: 02, HLA-A30: 01, HLA-A30: 02, HLA-A31: 01, HLA-A32: 01, HLA-A33: 03, HLA-A68: 01, HLA-A68: 02, HLA-B07: 02, HLA-B08: 01, HLA-B13: 02, HLA-B14: 02, HLA-B01: 15, HLA-B03: 15, HLA-B15: 15, HLA-A03: 15, HLA-B15: 15, HLA-A33: 01, HLA-B33: 03, HLA-A29: 15, HLA-B15: 15, HLA-B27: 05, HLA-B35: 01, HLA-B35: 03, HLA-B37: 01, HLA-B38: 01, HLA-B39: 01, HLA-B40: 02, HLA-B41: 02, HLA-B42: 01, HLA-B44: 02, HLA-B44: 03, HLA-B44: 05, HLA-B46: 01, HLA-B49: 01, HLA-B50: 01, HLA-B51: 01, HLA-B52: 01, HLA-B53: 01, HLA-B55: 01, HLA-B57: 01, HLA-B01: 58, HLA-B03: 03, HLA-C03, HLA-B03: 03, HLA-B55: 01, HLA-B02: 03, HLA-C03, HLA-B55: 01, HLA-B55: 57:01, HLA-B55: 58, HLA-B02: 58, HLA-C03, HLA-C04: 01, HLA-C05: 01, HLA-C06: 02, HLA-C07: 01, HLA-C07: 02, HLA-C07: 04, HLA-C08: 01, HLA-C08: 02, HLA-C12: 03, HLA-C14: 02, HLA-C15: 02, HLA-C16: 01, HLA-C17: 01.

In some embodiments, the library comprises greater than or equal to two different polypeptide molecules, wherein the antigenic peptide is not identical for each of the greater than or equal to two polypeptide molecules, and wherein each different polypeptide is attached to a particle. In some embodiments, the library further comprises a uniquely defined barcode sequence operably associated with the identity of each different polypeptide.

Embodiments include barcoded polynucleotides comprising defined barcode sequences. Barcoded polynucleotides may be polynucleotides that provide unique antigen-specific sequences for identification after T cell isolation. Thus, each unique comact is attached to a particle with a uniquely defined barcode sequence. This allows for efficient association between a given antigen and a given barcode, wherein the barcode is unique to the pair.

The barcoded polynucleotides may be ssDNA or dsDNA. The polynucleotide comprising the barcode may be modified at its 5' end to comprise an attachment moiety for attachment to a particle. For example, a polynucleotide comprising a barcode sequence is conjugated to a biotin molecule to bind to a streptavidin core attached to a particle (e.g., dextran). However, any suitable attachment moiety may be used to attach the polynucleotide to the particle. Suitable pairs of attachment portions are known in the art, as described herein and understood by those skilled in the art. Non-limiting examples of attachment moieties include thiols, maleimides, adamantanes, cyclodextrins, amines, carboxyls, azides, and alkynes.

Granules

As used herein, "nanoparticle" or alternatively referred to as "particle" refers to a substrate that is capable of being specifically sorted or separated and to which other entities may be attached. In certain embodiments, the "entity" attached to the particle is comPACT and a barcode. In certain embodiments, in addition to the comPACT and barcode, additional entities (e.g., fluorophores or other imaging agents) may be attached to the particles. In certain embodiments, in addition to comPACT and barcodes, additional proteins may be attached to the particles. For example, additional proteins may be attached to the particles to promote T cell binding or increase the stability of comPACT. In certain embodiments, the comPACT protein, barcode, imaging agent, and additional protein may be attached to the particle. In certain embodiments, a plurality of comPACT proteins are attached to the particle.

In some embodiments, the particles are magnetic, e.g., for separation using a magnet. In some embodiments, the magnetic particles comprise magnetic iron oxide. Examples of magnetic particles include, but are not limited to, dynabead (thermo fisher). In some embodiments, the particles are polystyrene particles, e.g., for separation by gravity. In other embodiments, the particles may be surfaces, beads, or polymers. Examples of beads include, but are not limited to, agarose (agarose) beads and sepharose (sepharose) beads. In particular embodiments, the particles may be fluorescent or attached directly or indirectly to a fluorophore.

According to certain embodiments, the particle is modified with an attachment moiety for attaching additional molecules. The modification of the particle includes an attachment moiety that can pair (e.g., covalently bind) with a corresponding homologous (e.g., complementary) attachment moiety attached to the polynucleotide. Any suitable pair of attachment moieties may be used to modify the particles and polynucleotide detection tags for attachment. Non-limiting examples of attachment moiety pairs include streptavidin/biotin systems, thiol groups (e.g., cysteine) and cysteine-reactive moieties (e.g., maleimide, adamantane, and cyclodextrin), amino and carboxyl groups, and azido and alkynyl groups. In some embodiments, the attachment portion may comprise a cut portion. In other embodiments, the attachment moiety that binds to a complementary cognate attachment moiety may be reversible, such as a reducible thiol group. In exemplary embodiments, the modified particle is a streptavidin-coated magnetic nanoparticle, such as a 1 μm particle (e.g., Dynabeads MyOne streptavidin T1 bead from ThermoFisher Scientific), and the polynucleotide can be biotinylated to attach to the modified particle.

The particles may be dextran, such as biotinylated dextran or streptavidin coated dextran. Modified dextrans are described in further detail in Bethune et al, BioTechniques62: 123-. Biotinylated comaps may be attached to streptavidin-coated dextran.

The comPACT proteins may also assemble into tetramers, including 1,2, 3, or 4 biotinylated comPACT proteins bound to a streptavidin core. The tetramer may also contain a fluorophore, such as Phycoerythrin (PE) or Allophycocyanin (APC) bound to a streptavidin core. MHC class I and class II tetramers are well known in the art. MHC class I tetramers are further described in detail in Burrow SR et al, J Immunol December 1,2000,165(11) 6229-.

The comPACT proteins can also assemble into multimers. In some embodiments, the comPACT protein multimer may be a dimer, trimer, tetramer, pentamer, hexamer, or higher order multimer. In some embodiments, the multimer may comprise at least two or more comact proteins. In some embodiments, the multimer may comprise at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 comapt proteins.

Particle collections and libraries

Different sets of particles are also contemplated, each different set of particles comprising a unique antigenic peptide (referred to herein as comact) and at least one defined barcode operably associated with the identity of the antigenic peptide. The collection of particles comprises at least two particles, each individual particle comprising a unique antigenic peptide and at least one defined barcode operably associated with the identity of the antigenic peptide. In some embodiments, the distinct set of particles comprises at least two particles. In some embodiments, the distinct set of particles comprises at least three particles. In some embodiments, the distinct set of particles comprises at least four particles. In some embodiments, the unique antigenic peptide (referred to as comact, as used herein) comprises a comact polynucleotide molecule or polypeptide molecule.

Libraries of different particle collections are also contemplated. The library of different particle sets may comprise from 2 to 1000 particle sets. In some embodiments, the library comprises 2-900,2-800,2-700,2-600,2-500,2-480,2-400,2-300,2-200,2-100,2-50,2-66,2-48,2-30,2-20,2-19,10-1000,10-900,10-800,10-700,10-600,10-500,10-480,10-400,10-300,10-200,10-100,10-50,10-66,10-48,10-30,10-20,20-1000,20-900,20-800,20-700,20-600,20-500,20-480,20-400,20-300,20-200,20-100,20-50,20-50,20-66,20-48,20-30,30-1000,30-900,30-800,30-700,30-600,30-500,30-480,30-400,30-300,30-200,30-100,30-50,30-50,30-66,30-48,30-40,40-1000,40-900,40-800,40-700,40-600,40-500,40-480,40-400,40-300,40-200,40-100,40-60,40-50,40-66,40-48,50-1000,50-900,50-800,50-700,50-600,50-500,50-480,50-400,50-300,50-200,50-100,50-60,50-66,60-1000,60-900,60-800,60-700,60-600,60-500,60-480,60-400,60-300,60-200,60-100,70-1000,70-900,70-800,70-700,70-600,70-500,70-480,70-400,70-300,70-200,70-100,70-80,70-90,80-1000,80-900,80-800,80-700,80-600,80-500,80-480,80-400,80-300,80-200,80-100, 150, 200, 250, 300, 350, 400, 450 or 450, 250, 300, 350, 400, 450, or 450 Between 500 particles. In some embodiments, the library comprises a collection of between 2-19, 48-480, 48-66, 66-480, 220-240, 40-60, 48-66, 50-70, or 60-80 particles. In some embodiments, the library comprises at least 2,5,10,15,20,25,30,35,40,45,48,50,55,60,65,66,70,75,80,85,90,100,110,120,130,140,150,160,170,180,190,200,225,250,275,300,325,350,375,400,425,450,475,500,525,550,600,562,650,675,700,725,750,775,800,825,850,875,900,925,950,975, or 1000 collections of particles. In some embodiments, the library comprises a collection of 2,10,15,20,24,48,66,100,200,300,400,500,600,700,800,900 or 1000 particles.

In certain embodiments, a library of a collection of particles of the present disclosure may comprise one, two, three, four, five or more collections of particles. In certain embodiments, each collection of particles can include one, two, three, four, five or more types of particles. In certain embodiments, the collection of particles can include a single type of particle. In certain embodiments, a collection of particles can include multiple types of particles. For example, but not by way of limitation, a collection of particles may include particles bound to the same barcode or particles bound to different barcodes, or a combination thereof. In certain embodiments, the collection of particles may comprise particles bound to a comPACT polypeptide. In certain embodiments, the collection of particles may comprise particles bound to the same comact polypeptide, a different comact polypeptide, or a combination thereof.

Dextramer and tetramer

The comPACT polypeptide may be attached to dextran, such as biotinylated dextran or streptavidin-coated dextran. Modified dextrans are described in further detail in Bethune et al, BioTechniques 62: 123-. The biotinylated copat polypeptide may be attached to streptavidin-coated dextran. In certain embodiments, the dextran is coated with streptavidin. In certain embodiments, streptavidin is covalently conjugated to dextran. In certain embodiments, streptavidin is non-covalently coupled to biotin-dextran.

comPACT can also assemble into tetramers, including 1,2, 3, or 4 biotinylated comPACT proteins bound to a streptavidin core. The tetramer may also contain a fluorophore, such as Phycoerythrin (PE) or Allophycocyanin (APC) bound to a streptavidin core. In certain embodiments, the fluorophore is selected from the group consisting of PerCP, Cy3, Cy5, and Alexa 488. In certain embodiments, the fluorophore is a quantum dot (a non-limiting example is Qdot 800). In certain embodiments, any fluorophore with a high extinction coefficient may be used. MHC class I and class II tetramers are well known in the art. MHC class I tetramers are further described in detail in Burrow SR et al, JImmunol December 1,2000,165(11) 6229-.

Method for preparing comPACT polypeptide

Antigen prediction

To manufacture a comact, one of the initial steps may include identifying a tumor-specific antigen (e.g., a neoantigen) of the patient. The compositions produced by this method can then be used in T cell-mediated immune processes, for example, for patient-specific cancer immunotherapy. To identify a putative neoantigen (tumor or pathogen) in a patient, tumor, viral or bacterial sequencing data (including whole genome, whole exome or transcriptome sequencing data) can be analyzed using a computer predictive algorithm program to identify one or more mutations corresponding to the putatively expressed neoantigen. In addition, Human Leukocyte Antigen (HLA) typing can be determined from tumor or blood samples from patients, and this HLA information can be used in prediction algorithms for MHC binding along with putative novel antigen peptide sequences that have been identified (see Fritsch et al, 2014, Cancer Immunol Res.,2:522-529, which is incorporated herein by reference in its entirety). HLA common in the human population, e.g. HLA-a 02,24,01 in caucasians; HLA-B35, 44, 51; DRB1 x 11,13, 07; HLA-a 02,03,30 in african brazilians; HLA-B35, 15, 44; DRB1 x 13,11, 03; and HLA-a 24,02,26 in asian; HLA-B40, 51, 52; DRB1 × 04,15, 09. Specific pairing of HLA alleles can also be used. Common alleles found in the human population are further described in Bardi et al (Rev Bras Hematol promoter.2012; 34(1): 25-30.)

Additional examples of methods for identifying neoantigens include combining sequencing with mass spectrometry and MHC presentation prediction (e.g., U.S. publication No. 2017/0199961), and combining sequencing with MHC binding affinity prediction (e.g., issued U.S. patent No. 9,115,402). In addition, methods that can be used to identify the presence or absence of neoantigen-specific T cells in a patient sample can be used in combination with the methods described herein, e.g., as described in U.S. publication No. 2017/0003288 and PCT/US17/59598, which are all incorporated herein by reference. These analyses produced a ranked list of patient candidate neoantigenic peptides (which can be readily synthesized using conventional methods) for screening for cognate antigen-specific T cells.

Restricted digestive assembly

In general, preparation of the comPACT polynucleotides may be accomplished by the procedures disclosed herein and recognized recombinant DNA techniques, such as preparation of plasmid DNA, cleavage of the DNA with restriction enzymes, DNA ligation, transformation or transfection of the host, culture of the host, and isolation and purification of the expressed fusion complex. Such procedures are generally known and disclosed in standard references, such as Sambrook et al, supra.

In some aspects, DNA encoding MHC class I heavy chains can be obtained from a suitable cell line, such as a human lymphoblast cell. In various configurations, the gene or cDNA encoding the class I heavy chain may be amplified by Polymerase Chain Reaction (PCR) or other means known in the art. In some aspects, the PCR product may also include sequences encoding linkers, and/or one or more restriction enzyme sites for ligating such sequences.

In some embodiments, the vector encoding the comPACT polynucleotide may be prepared by ligating sequences encoding MHC class I heavy chains and β 2-microglobulin to sequences encoding antigenic peptides.

The DNA encoding the antigenic peptide may be obtained by isolating the DNA from a natural source or by known synthetic methods such as the phosphotriester method. See, e.g., Oligonucleotide Synthesis, IRL Press (m.gait, ed., 1984). Synthetic oligonucleotides can also be prepared using a commercially available automated oligonucleotide synthesizer. The DNA sequence encoding the universal target sequence discussed herein may be inserted between the sequence encoding the signal sequence and the sequence encoding the antigenic peptide, and a second universal target sequence may be inserted between the sequence encoding the antigenic peptide segment and the sequence encoding the β 2-microglobulin segment. In some embodiments, the segments may be joined using a ligase.

PCR assembly

In some aspects, comPACT may be assembled by Polymerase Chain Reaction (PCR) amplification. Similar to the restriction digestion method, DNA encoding MHC heavy chain and β 2-microglobulin may be obtained from a suitable source. The second DNA segment encoding the selected signal sequence may also be obtained from a suitable source. Two fragments of DNA may have different universal target sequences such that primers of one universal sequence do not anneal to a second universal sequence. Two sequences encoding the selected antigenic peptides can be synthesized; a forward primer having an antigen sequence at its 5 'end and a sequence complementary to the sequence of the universal primer on the MHC DNA fragment at its 3' end; a reverse primer having at its 5 'end the reverse complement of the selected antigen sequence and at its 3' end the reverse complement of the universal primer from the signal sequence fragment. PCR reactions with primers for all four DNA fragments and 5 'signal sequence fragments and 3' MHC allele fragments will result in amplification of two DNA fragments, one with 3 'signal sequence and 5' antigen sequence and the other with 3 'antigen sequence and 3' MHC allele. Further cycles of PCR amplification will anneal overlapping antigenic peptide sequences and produce a single full-length DNA fragment. In some embodiments, the signal peptide fragment further comprises a promoter sequence. In some embodiments, the MHC fragment further comprises a purification cluster and/or a polyA tail.

Transfection, transduction, and genetic modification of host cells

The comPACT polynucleotide may be inserted into the host cell by known appropriate methods including, but not limited to, transfection, transduction, electroporation, lipofection, sonoporation, mechanical disruption or viral vectors. Exemplary transfection reagents include, but are not limited to, FectroPro, Expifeacylamine, Lipofectamine, Polyethyleneimine (PEI), Fugene, or any other transfection reagent that provides optimal transfection efficiency based on the cell type, transfection system, transfection type, transfection conditions, and construct to be transfected. In some examples, Expifectamine is used to transfect mammalian cells with comPACT polynucleotides. In some examples, polyethyleneimine is used to transfect mammalian cells with comPACT polynucleotides. In some examples, FectrorPro is used to transfect mammalian cells with comPACT polynucleotides.

The comPACT polynucleotide may be transiently or stably expressed in the host cell. In some embodiments, the comPACT polynucleotide is integrated into the host genome. In other embodiments, the comPACT polynucleotide is maintained extrachromosomally. Any suitable gene editing technique known in the art may also be used to modify the host cell with the comPACT polynucleotide, including CRISPR/Cas9, zinc finger nucleases, or TALEN nucleases.

Expression of

Various strategies may be employed to express comPACT polypeptides. For example, the comPACT polynucleotide can be incorporated into a suitable vector by known methods, e.g., by using restriction enzymes and ligases (see, e.g., Sambrook et al, supra). The vector may be selected based on factors associated with the cloning protocol. For example, the vector may be compatible with the host in use and have a suitable replicon. Suitable host cells include eukaryotic and prokaryotic cells, and may be cells that are readily transformed and exhibit rapid growth in culture. Examples of host cells include prokaryotes such as Escherichia coli and Bacillus subtilis, and eukaryotes such as animal cells and yeast, such as mammalian cells and human cells. Non-limiting examples of mammalian cells that can be used as hosts to express comPACT include J558, NSO, SP2-O, 293T, Expi293, and CHO (as well as any derivatives or modifications of any J558, NSO, SP2-O, 293T, Expi293, and CHO cell lines). Other examples of possible hosts include insect cells, such as Sf9, which can be grown using conventional culture conditions. See Sambrook et al, supra. In various embodiments, cells expressing comPACT polypeptides may be identified using known methods. For example, expression of comPACT polypeptide can be determined by ELISA, FACS or Western blot. In certain embodiments, expression of the comPACT polypeptide may be determined by ELISA, FACS or Western blot using antibody probes directed to the MHC heavy chain portion of comPACT or antibodies directed to an affinity tag, such as His6(SEQ ID NO:34), or streptavidin reagent (if comPACT has been biotinylated).

In some aspects, comPACT is expressed in mammalian cells. The benefits of expressing proteins in mammalian cells, rather than in E.coli cells, are manifold. Proteins expressed in E.coli cells must be carefully purified from Lipopolysaccharide (LPS). Expression of the protein in mammalian cells does not result in contamination of the purified protein with LPS. In addition, mammalian cells are more likely to fold mammalian proteins correctly because mammalian cells produce proteins with the correct post-translational modifications required for correct folding, including the correct formation of disulfide bonds. In addition, mammalian cells provide the correct chaperones to assist protein folding in the endoplasmic reticulum or golgi apparatus. This results in an increased purification of the homogeneously well folded protein compared to proteins expressed in E.coli cells.

In some aspects, comPACT is expressed in prokaryotic cells. In certain embodiments, the prokaryotic cell has been genetically modified to post-translationally modify the comPACT. In certain embodiments, the comPACT expressed in the prokaryotic cells is substantially free of LPS or no detectable LPS as measured using LPS detection methods known in the art.

comPACT may be substantially free of LPS. The comPACT may be LPS free, e.g., the comPACT may have no detectable LPS as measured using LPS detection methods known in the art. comPACT may be glycosylated. comPACT may have one or more post-translational modifications. comPACT may be modified by expression in eukaryotic cells or, in particular embodiments, in mammalian cells, e.g., by one or more post-translational modifications such as glycosylation. The comPACT may include one or more post-translational modifications. comPACT may (1) be substantially free of LPS or free of LPS, and (2) be glycosylated.

Exemplary comPACT workflow

Fig. 23 shows an exemplary schematic of the assembly and expression of comPACT protein. Sense and antisense oligonucleotides encoding the desired neoantigenic peptide sequences are synthesized and annealed to form double-stranded oligonucleotides with overhangs at the 5 'and 3' ends, which can then be ligated into plasmids containing the β 2M gene and MHC alleles. The intact comPACT oligo can be amplified into a double-stranded amplicon and transfected into cells for protein expression and optionally biotinylation. comPACT protein can be assessed by SDS-PAGE. The comPACT polynucleotide can then be selected for amplified plasmid production in E.coli. Protein-producing cells are transfected with the selected plasmid, and comPACT is purified from the producing cells for functional assays.

Purification (chromatography)

The expressed comPACT polypeptide can be isolated and purified by known methods. For example, comPACT containing a His6 affinity tag (SEQ ID NO:34) can be purified by affinity chromatography on a metal affinity chromatography column (e.g., a Ni-NTA column or any other metal affinity resin column described herein such as Co2+, Ca2+, Zn2+, Cu2+ resin or any combination thereof (including Ni2+)) and by well known and published procedures. In addition, comPACT containing human HLA sequences can be purified by well known and published procedures by affinity chromatography on monoclonal antibody-Sepharose columns.

Method for isolating antigen-specific T cells

The comPACT libraries described herein have been used to isolate antigen-specific T cells, and can be used to isolate any cells presenting a novel antigen. A schematic diagram of a T cell isolation process according to one embodiment is shown in figure 26. This process may also be referred to herein as an 'imPACT' or 'imPACT separation technique' approach.

The steps and components of the imPACT separation technique method include, but are not limited to, steps (1) - (5) illustrated in fig. 26:

(1) creating a comPACT element library for patient-specific neo-antigen T cell isolation

(2) Adding unique DNA oligonucleotides, neoID or barcodes to comPACT element library

(3) Each individual comapt polypeptide and its corresponding NeoID barcode, DNA oligonucleotide, NeoID or barcode bind to two separate fluorescent streptavidin proteins (phycoerythrin (PE) and Allophycocyanin (APC) in the example provided in fig. 26)

(4) This assembly process resulted in two paired barcoded fluorescent tetramers per comPACT polypeptide and barcode element

(5) A tetramer library assembled with all comact polypeptides and NeoID barcodes, DNA oligonucleotides, NeoID or barcodes targeting each patient's predicted neoantigen candidate was pooled for isolation of neoantigen-specific T cells from the peripheral blood of the subjects.

The use of the comPACT library to identify and characterize neoantigen-specific T cells is also shown in panels 6-8 of FIG. 26. The comPACT-neoID library was incubated with patient samples (6) and then Fluorescence Activated Cell Sorting (FACS) (7). A fixed number of T cells designed to express the tool neoTCR can be added to the patient sample as an internal positive control to calibrate each assay. Double fluorescently labeled (PE and APC) tetramer bound neoantigen-specific T cells, as well as internal positive control cells and potentially non-specific T cells, were sorted as single cells into individual wells in the plate for subsequent RT-PCR analysis, including barcode and neo-TCR sequencing (8).

Bar code signal to noise ratio (S/N) analysis

True positive neoantigen-specific ditag T cells can be resolved from the identified false positive T cells by flow cytometry through sequence analysis of the neoID barcodes bound to the isolated T cells. The presence of multiple copies of the same neoID barcode results in a high proportion of specific neoID barcode species compared to non-specifically bound barcodes. This results in a higher signal-to-noise barcode ratio (S/N). Non-specific T cells bind a relatively equal number of different tetramer species, resulting in a lower proportion of different neoID barcodes. Schematic representations of non-specific binding to specific T cells are shown in fig. 27A (non-specific) and fig. 27B (specific). The numbers represent different neoID barcodes. In fig. 27A, the ratio of the unique DNA copy number of the most predominant neoID divided by the second most predominant neoID was 1, indicating cells that were non-specifically bound by comPACT elements. In fig. 27B, the ratio of the unique DNA copy number of the most predominant neoID divided by the second most predominant neoID was 5, indicating that this T cell was bound by the primary comPACT element and represents a true positive neoantigen-specific CD 8T cell. This can be further confirmed by functional characterization using neoTCR engineered T cells cloned from single cells.

S/N1 and S/N2 analysis

In some embodiments, one TCR can recognize two different neoantigens. In this case, even if the T cells are specific, the S/N ratio may be less than 10. In this case, two different S/N calculations may be used, namely S/N1 and S/N2. S/N1 is the highest signal divided by the next highest signal, and S/N2 is the highest signal from one mutation divided by the highest signal from a different mutation. In the S/N2 analysis, the highest signal from different mutations may not be the next highest signal in the sample.

In an illustrative example, 8 different TCRs can be identified in one sample. Of these 6 may have an S/N1 ratio greater than 10 and may be identified as specific neoantigen T cells. For the other 2T cells, the S/N1 ratio may be below 10. However, S/N2 may be higher than 10. Cloning of these 2 TCRs indicated that they could recognize two different neoantigens sharing the same mutation, explaining the reason for the low S/N1 ratio. In some embodiments, the S/N2 assay can be used to call (call) non-specificity from a particular cell when there are multiple new antigens derived from the same mutation.

In some embodiments, a higher S/N ratio indicates a higher TCR binding specificity.

Threshold value

In some embodiments, an isolated T cell is identified as the antigen-specific T cell if the barcode signal to noise ratio S/N1 or S/N2 is above a threshold.

In some embodiments, the threshold is at least or greater than 2,5,10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100,110,120,130,140,150,160,170,180,190,200,250,300,350,400,450,500,550,600,650,700,750,800,850,900,950, or 1000. In some embodiments, the threshold is at least or greater than 2,5,10, or 20. In some embodiments, the ratio corresponds to the specificity of the isolated antigen-specific T cells. In some embodiments, the S/N ratio is 10 or greater.

Marking

As used herein, "identification marker" or "identification marker" refers to a molecule or compound used to mark a collection of particles. In some embodiments, the identifying label is a fluorophore. In some embodiments, the identifying label is a metal, lanthanide, quantum dot, radioisotope, nanoparticle, or dye. Any suitable fluorophore can be used, including, but not limited to, Allophycocyanin (APC), Phycoerythrin (PE), Fluorescein (FITC), rhodamine, texas red, DAPI, C2, Cy3, Cy5, Cy7, AlexaFluor fluorophore, BODIPY fluorophore, DyLight fluorophore, FluoProbes fluorophore, or any combination thereof.

Bar code

As used herein, "barcode" or "barcodes" refers to a nucleotide sequence used to tag and identify a particular peptide, including but not limited to antigenic peptides. In certain embodiments, the barcode is selected from the group consisting of a 3-mer, a 4-mer, a 5-mer, a 6-mer, a 7-mer, an 8-mer, a 9-mer, a 10-mer, an 11-mer, a 12-mer, a 13-mer, a 14-mer, a 15-mer, a 16-mer, a 17-mer, an 18-mer, a 19-mer, and a 20-mer. In certain embodiments, the barcode is an 8 mer.

In some embodiments, the different particle pairs comprise a unique antigenic peptide and a defined barcode operably associated with the identity of the antigenic peptide.

In some embodiments, the first particle comprises a first barcode and the second particle comprises a second barcode different from the second barcode, wherein the first and second barcodes are associated with the identity of the antigen.

In some embodiments, the pair of particles comprises a third particle comprising a third barcode different from the first and second barcodes, wherein the first, second and third barcodes are associated with the identity of the antigen.

Exemplary barcodes and barcode structures are provided in table a below. The barcode may also be referred to as "neoID".

In some embodiments, the barcode signal to noise ratio is based on at least one barcode. In such embodiments, each paired particle comprises the same antigen, a different label, and at least one barcode, wherein the at least one barcode is associated with a neo-antigen. In some embodiments, the conjugate particle has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 barcodes.

In some cases, aggregates of particles with fluorophore labeling can result in high average fluorescence intensity of individual fluorophores staining cells during separation. This is not due to the specific binding of the fluorescent particles, but due to the non-specific binding of the comPACT particle aggregates, resulting in high neoantigen barcodes S/N, since there may be a large number of identical barcodes that are non-specifically bound to T cells. A dual barcode system can be used to solve this problem.

In some embodiments, each particle pair of a comPACT library element comprises at least two barcodes. The dual barcodes conjugated two different DNA barcodes of each antigen separately to each comPACT tetramer. FIG. 28 shows a graph comparing single and double barcodes for one antigen. In the top panel, the same neoantigen is associated with two different particles, two different fluorophores, and a single barcode, labeled "1". In the bottom panel, the same antigen is associated with two different particles, two different fluorophores, and two different barcodes, labeled "1" and "2". This results in increased recognition of false positives with high signal-to-noise ratio caused by tetramer aggregation. The signal-to-noise ratio of each DNA barcode assigned to each fluorescent particle and the same antigen can be analyzed separately.

In some embodiments, each particle pair of a comPACT library element comprises at least two barcodes. Double-barcoding two different DNA barcodes of each antigenic peptide were conjugated separately to each comPACT tetramer. FIG. 28 shows a graph comparing single and double barcodes for an antigenic peptide. In the top panel, the same antigenic peptide is associated with two different particles, two different fluorophores, and a single barcode, labeled "1". In the bottom panel, the same antigenic peptide is associated with two different particles, two different fluorophores, and two different barcodes, labeled "1" and "2". This results in increased recognition of false positives with high signal-to-noise ratio caused by tetramer aggregation. The signal-to-noise ratio of each DNA barcode assigned to each fluorescent particle and the same antigenic peptide can be analyzed separately.

Cell sample

The imPACT method (i.e., imPACT isolation techniques) may be used to isolate immune cells, such as T cells and B cells, from any suitable patient-derived sample comprising immune cells, including, but not limited to, blood, plasma, Peripheral Blood Mononuclear Cell (PBMC) samples, bone marrow, tumor-infiltrating lymphocyte (TIL) samples, tissues, solid tumors, hematological cancers, and liquid tumors, or any combination thereof. For example, both CD4+ and CD8+ T cells can be labeled and sorted from PBMCs or TILS using anti-CD 4 and anti-CD 8 fluorescent antibodies, and live cell populations of CD4+ and CD8+ single positive cells sorted using Fluorescence Activated Cell Sorting (FACS) to isolate only CD4+ or CD8+ cells. In some embodiments, T cells positive for both CD4 and CD8 may be isolated using anti-CD 3 fluorescent antibody followed by FACS. In addition, the imPACT method can also be used for antibody discovery of B cells. The person skilled in the art is able to determine the type of immune cells to be isolated with respect to the type of comPACT protein used. In some embodiments, the sample is a blood sample. In some embodiments, the sample is a PBMC sample. In some embodiments, the sample is a solid tumor sample. In some embodiments, the sample is a hematological tumor sample. In some embodiments, the sample is a bone marrow sample. In some embodiments, the sample is a tumor sample comprising tumor infiltrating lymphocytes. The T cells may be CD8+ T cells or CD4+ T cells. In some embodiments, the T cell is a CD8+ T cell. In some embodiments, the T cell is a CD4+ T cell. In some embodiments, the T cell is a human T cell. In some embodiments, the T cell is a human CD8+ T cell.

T cell isolation

In another aspect, provided herein is a method of isolating antigen-specific T cells, the method comprising the steps of: (a) providing a polypeptide comprising in an amino-terminal to carboxy-terminal direction, (i) a first universal target peptide, (ii) an antigenic peptide, (iii) a second universal target peptide different from the first universal target peptide, (iv) a β 2M peptide, and (v) an MHC peptide, wherein the polypeptide is linked to one particle; (b) providing a sample known or suspected to comprise one or more T cells; (c) contacting the polypeptide with the sample, wherein the contacting comprises providing conditions sufficient to allow binding of a single T cell to the polypeptide attached to the particle, and (d) isolating the single T cell associated with the particle.

Isolating and identifying patient-derived and antigen-specific T cells using comPACT as described herein may include incubating comPACT protein with patient-derived T cells or a sample containing patient-derived T cells. In some embodiments, the library comprising at least two compacts may be incubated with patient-derived T cells. T cells can be prepared using standard methods starting from tissues such as blood, lymph nodes or tumors.

Incubation of comPACT or comPACT libraries with T cell suspensions allows complete and complete exposure of the particle-bound antigenic peptides to various T cell receptors. The method may comprise shaking or rotating the cells. In some embodiments, the comPACT is associated with a particle.

After comPACT or comPACT libraries (both bound to the granulocytes) and T cell incubation, the bound comPACT-T cell complexes are selectively isolated or selectively collected. T cells may bind to many identical copies of the same comact library element (i.e., a single comact polypeptide and a particle-associated comact polypeptide) and may be isolated based on these interactions. For example, if comPACT or comPACT associated with a particle contains a fluorophore, or is attached to a particle with a fluorophore, fluorescence-associated cell sorting (FACS), including single cell sorting, may be used to selectively isolate T cells. If comPACT or particles associated therewith are attached to the magnetic particles, application of a magnet to the suspension can separate the particles complexed with the antigen-paired T cells and remove the unpaired T cells. Alternatively, if the particles associated with comPACT are polystyrene particles, the unpaired T cells may be separated by gravity (e.g., centrifugation). After removing unpaired T cells, in some embodiments, the isolated binding particles are washed at least once to remove any non-specifically bound T cells.

comPACT-bound T cells can also be separated by FACS into separate collection containers, e.g., multi-well plates. The separate collection vessel may be a single cell reaction vessel. For example, components for downstream processing and analysis may be added to each single-cell reaction vessel. comPACT-bound T cells can be isolated by FACS into large volume collection containers (e.g., each isolated T cell is collected in the same container).

The comPACT-bound T cells can also be isolated individually in droplets using droplet-generating microfluidic devices (i.e., "droplet generators"). Droplet generation devices for encapsulating individual cells are known to those skilled in the art, for example, as described in U.S. publication No. 2006/0079583, U.S. publication No. 2006/0079584, U.S. publication No. 2010/0021984, U.S. publication No. 2015/0376609, U.S. publication No. 2009/0235990, and U.S. publication No. 2004/0180346.

After the comPACT-bound T cells are isolated into single-cell reaction vessels (e.g., in single wells or droplets), the comPACT-bound T cell nucleic acids may be further processed for downstream analysis. Specifically, expressed TCR α and TCR β mRNA transcripts may first be converted to cDNA by reverse transcription, and the amplified cDNA used in Next Generation Sequencing (NGS) methods known to those skilled in the art, including but not limited to sequencing by synthesis techniques (e.g., Illumina or any other NGS sequencer).

Method for identifying antigen specificity of T cells

In certain embodiments, the presently disclosed subject matter provides methods for identifying antigen specificity of T cells. In certain embodiments, the T cell isolation methods described herein provide information about the antigen specificity of the isolated T cells. For example, but not limited to, information about antigen specificity may be obtained by nucleic acid analysis of isolated T cells. In certain embodiments, the nucleic acid of an isolated T cell can be analyzed to determine the sequence of T cell receptor gene sequences (e.g., TCR α and TCR β sequences). In certain embodiments, information about the antigen specificity of the isolated T cells can be used for downstream applications. Non-limiting examples of downstream applications include immunohistochemical library analysis, manufacturing methods, and clinical follow-up of patients receiving immunotherapy. In certain embodiments, information about the antigen specificity of isolated T cells can be used to prepare reagents and compositions for making cells useful for adoptive cell transfer therapy.

In a non-limiting embodiment, monitoring of the immune repertoire is performed. In certain embodiments, monitoring of the immune repertoire is performed before, during, or after treatment. In certain embodiments, the treatment is immunotherapy. Non-limiting examples of immunotherapy include administration of vaccines, oncolytic viruses, antibodies, T cells expressing chimeric antigen receptors, T cells expressing recombinant T cell receptors, tumor infiltrating lymphocytes.

Method of treatment

In certain embodiments, the presently disclosed subject matter provides methods of treatment, including but not limited to inducing and/or increasing an immune response in a subject in need thereof. In certain embodiments of the present disclosure, the methods of treatment disclosed herein involve the isolation and/or administration of cells. For example, but not by way of limitation, in certain embodiments, the cells employed in the methods described herein can be obtained from a subject. In certain embodiments, the cell is a tumor cell, a non-cancer cell, a T cell, or any combination thereof. In certain embodiments, nucleic acids can be extracted from cells as described herein. In certain embodiments, the nucleic acid of the cell can be sequenced as outlined herein. In certain embodiments, the information obtained from the subject, e.g., nucleic acid sequence information, provides information about antigen-specific T cells. In certain embodiments, the information relates to the identity (e.g., amino acid sequence) of the antigenic peptide. In certain embodiments, the antigenic peptide is a tumor neoantigen. In certain embodiments, the information relates to the identity of the MHC sequence.

In certain embodiments, the methods described herein relate to the treatment of cancer. In certain embodiments, the cancer is a solid cancer. Non-limiting examples of tumors that can be treated by the methods described herein include, for example, carcinomas, lymphomas, sarcomas, blastomas, and leukemias. Non-limiting specific examples include, for example, breast cancer, pancreatic cancer, liver cancer, lung cancer, prostate cancer, colon cancer, kidney cancer, bladder cancer, head and neck cancer, thyroid cancer, soft tissue sarcoma, ovarian cancer, primary or metastatic melanoma, squamous cell carcinoma, basal cell carcinoma, brain cancers of all histopathological types, angiosarcoma (angiosarcoma), angiosarcoma (hemangiosarcoma), osteosarcoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma (angiosarcoma), endotheliosarcoma, lymphangiosarcoma, lymphangiolicosarcoma, synovioma, testicular cancer, uterine cancer, cervical cancer, gastrointestinal cancer, mesothelioma, cancers associated with viral infections (such as, but not limited to, Human Papillomavirus (HPV) -associated tumors (e.g., cervical cancer, vaginal cancer, vulvar cancer, head and neck cancer, anal cancer, and penile cancer)).

In certain embodiments, a comPACT minigene comprising a candidate antigen-peptide is produced according to the methods disclosed herein. In certain embodiments, a comPACT polypeptide comprising an antigenic peptide is produced.

In certain embodiments, particles comprising a comPACT polypeptide are produced according to the methods disclosed herein. In certain embodiments, at least one collection of particles comprising a comPACT polypeptide is produced according to the methods disclosed herein.

In certain embodiments of the methods of treatment disclosed herein, T cells are isolated by binding to particles. In certain embodiments, T cells isolated in this manner are obtained from a subject. In certain embodiments, the T cell is CD8⁺T cells.

In certain embodiments, the isolated T cells are sorted and analyzed for their genome. In certain embodiments, TCR sequences of the isolated T cells are obtained. In certain embodiments, the TCR gene sequences or portions thereof are inserted into a homologous recombination template. In certain embodiments, the homologous recombination template comprises the features described in international patent application No. PCT/US2018/058230, the contents of which are incorporated herein by reference in their entirety.

In certain embodiments, the T cell is modified by inserting a homologous recombination template comprising the TCR gene sequence of the isolated T cell, or a portion thereof.

In certain embodiments, the modified T cells are adoptively transferred to the patient. Adoptive Cell Transfer (ACT) is a potent form of immunotherapy and involves the transfer of immune cells with anti-tumor activity into cancer patients. Lymphocytes for adoptive transfer can be derived from the blood or from the stroma of resected tumors, although other sources of such cells are known in the art. In certain embodiments, the lymphocytes used in ACT may be administered in a single dose. Such administration may be by injection, for example, intravenous injection. In certain embodiments, the lymphocytes may be administered in multiple doses. The administration may be once, twice, three times, four times, five times, six times or more than six times per year. Administration may be monthly, biweekly, weekly, or every other day. The cytotoxic lymphocytes may continue to be administered as long as desired. In certain embodiments, the methods described herein can be used to determine an immune repertoire of a subject. In certain embodiments, the immune repertoire is analyzed before, during, and/or after treatment. In certain embodiments, the treatment is a cancer treatment. In certain embodiments, the cancer treatment is immunotherapy. In certain embodiments, immunotherapy comprises administering an antibody. In certain embodiments, the immunotherapy comprises adoptive cell transfer of T cells. In certain embodiments, the T cell comprises a recombinant TCR or a chimeric antigen receptor. In certain embodiments, the immune repertoire provides information to provide targeted therapy.

Method for modifying cells

In certain embodiments, the presently disclosed subject matter provides methods for modifying a cell. For example, but not by way of limitation, modified cells can be obtained using the methods and compositions described herein.

In certain embodiments, the presently disclosed subject matter provides a method of modifying a cell by introducing and recombining a Homologous Recombination (HR) template nucleic acid sequence into an endogenous locus of the cell. In certain embodiments, the cells are modified using non-viral methods. In certain embodiments, the HR template nucleic acid sequence is circular. In certain embodiments, the HR template nucleic acid sequence is linear. In certain embodiments, the HR template nucleic acid sequence comprises first and second homology arms. In certain embodiments, the homology arms can be from about 300 bases to about 2,000 bases. For example, each homology arm can be 1,000 bases. In certain embodiments, the homology arms can be homologous to first and second endogenous sequences of the cell. In certain embodiments, the endogenous locus is a TCR locus. For example, the first and second endogenous sequences are within a TCR α locus or a TCR β locus. In certain embodiments, the HR template comprises a TCR gene sequence. In a non-limiting embodiment, the TCR gene sequence is a patient-specific TCR gene sequence. In non-limiting embodiments, TCR gene sequences are identified and obtained using the methods described herein. For example, methods for identifying T cell antigen specificity can be used to obtain the sequence of a TCR gene from a patient, and the TCR sequence can be incorporated into an HR template. In certain embodiments, the HR template comprises a TCR α gene sequence and a TCR β gene sequence. Additional information on HR template nucleic acids and methods of modifying cells thereof may be found in International patent application No. PCT/US2018/058230, the contents of which are incorporated herein by reference.

In certain embodiments, a construct comprising a gene of interest can be inserted into an endogenous locus using non-viral gene editing methods. In certain embodiments, this may be achieved by using a homologous repair template comprising the coding sequence of the gene of interest flanked by the left and right HR arms. In certain embodiments, the target gene is sandwiched between the 2A peptide, a protease cleavage site located upstream of the 2A peptide to remove the 2A tag from the upstream translated target gene, and a signal sequence, in addition to the HR arm; wherein the gene of the target expression cassette is transcribed into single messenger RNA upon integration into the genome. In certain embodiments, during translation of the target gene messenger RNA, the flanking region is disconnected from the target gene by self-cleaving the 2A peptide, and the protease cleavage site is cleaved to remove the upstream 2A sequence from the translated target gene. In certain embodiments, in addition to the 2A and protease cleavage sites, a glycine-serine-glycine (GSG) linker may be inserted before each 2A peptide to further enhance the separation of the gene of interest from other elements in the expression cassette. In certain embodiments, the P2A peptides are used because they are superior to other 2A peptides due to their efficient cleavage. In certain embodiments, two (2) P2A peptides and codon divergence (codon divergence) are used to express the target gene without introducing any exogenous epitopes from the remaining amino acids at either end of the target gene from the P2A peptide.

In certain embodiments and as described in PCT/US/2018/058230, the neoTCR is integrated into the TCR α locus of the T cell. In certain embodiments, a homologous repair template is used that contains the neoTCR coding sequence flanked by the left and right HR arms. In certain embodiments, the endogenous TCR β locus is disrupted, resulting in expression of only the TCR sequences encoded by the neoTCR construct. In certain embodiments, a general strategy is applied using a cycle HR template. In some embodiments, the general strategy is applied using a linear template.

In certain embodiments, the target TCR α locus (ca) is displayed along with the plasmid HR template, and the resulting edited sequence and downstream mRNA/protein products are shown in fig. 67A and 67B. In certain embodiments, additional elements in the neoTCR cassette include: 2A ═ P2A ribosome skip element; a furin cleavage site upstream of F ═ 2A, which removes the 2A tag from the upstream TCR β protein; HGH is a human growth hormone signal sequence. The HR template of the neoTCR expression cassette includes two flanking homology arms for direct insertion of TCR α guide RNA into the TCR α genomic locus targeted by the CRISPR Cas9 nuclease RNP. In certain embodiments, the homology arms (LHA and RHA) flank the neoE-specific TCR sequence of the neoTCR expression cassette. In certain embodiments, the protease cleavage site is any suitable protease cleavage site known to those of skill in the art that can be used. In certain embodiments, any signal sequence known to those skilled in the art may be selected based on the desired transport (trafficking) and use.

In certain embodiments, once integrated into the genome, the neoTCR expression cassette is transcribed as a single messenger RNA from the endogenous TCR α promoter, which still includes a portion of the endogenous TCR α polypeptide from the T cells of the individual. In certain embodiments, during translation of the ribosomal polypeptide of a single neoTCR messenger RNA, the neoTCR sequence is disconnected from the endogenous CRISPR-disrupted TCR alpha polypeptide by self-cleavage at the P2A peptide. In certain embodiments, the encoded neoTCR α and neoTCR β polypeptides are also cleaved from each other by cleavage of a second self-cleaving P2A sequence motif contained in the endogenous cellular human furin and neoTCR expression cassettes (fig. 67B). In certain embodiments, the neoTCR α and neoTCR β polypeptides are targeted to the endoplasmic reticulum by a signal leader sequence (e.g., derived from human growth hormone, HGH), respectively, for multimeric assembly and trafficking of the neoTCR protein complex to the T cell surface. In certain embodiments, the inclusion of a furin cleavage site facilitates removal of the 2A sequence from the upstream TCR β chain to reduce potential interference with TCR β function. In certain embodiments, the inclusion of a gly-ser-gly linker (not shown) before each 2A further enhances the separation of the three polypeptides.

In certain embodiments, the three repeated protein sequences are codon-diverged within the HR template to promote genome stability. In certain embodiments, within the TCR cassette, the two P2A codon diverged relative to each other, and the two HGH signal sequences codon diverged relative to each other, to promote stability of the introduced new TCR cassette sequences within the genome of the ex vivo engineered T cells. In certain embodiments, the 5' end of the reintroduced TRAC exon 1 (fig. 67A and 67B, vertical bars) reduces the likelihood of loss of the entire cassette over time by removing two direct repeats of the intervening sequence.

The presently disclosed subject matter further provides compositions comprising cells modified by the methods disclosed herein.

Exemplary embodiments

A. In certain non-limiting embodiments, the presently disclosed subject matter provides a method comprising: (a) contacting the sample with a plurality of different sets of particles, wherein each particle comprises a unique antigenic peptide, an operably associated barcode, and at least one identifying label, wherein the sample comprises T cells, and wherein contacting comprises providing conditions suitable for binding of a single T cell to the unique antigenic peptide of at least one set of particles; (b) isolating the one or more T cells bound to the particle; (c) identifying barcodes of particles bound to the isolated T cells; and (d) determining a ratio for each barcode.

A1. The method of the foregoing a, wherein the ratio is calculated by identifying the copy number of the first barcode and the copy number of the second barcode and dividing the copy number of the first barcode by the copy number of the second barcode.

A2. The method of the foregoing a or a1, wherein the unique antigenic peptide is the same for each different set of particles.

A3. The method of any one of the preceding a-a2, wherein each distinct set of particles comprises at least one or more barcodes, wherein each barcode is associated with the identity of the antigenic peptide.

A4. The method of any one of the preceding a-a3, wherein the ratio of each barcode corresponds to the antigen specificity of the isolated T cells.

A5. The method of any one of the preceding a-a4, wherein the isolated T cell is identified as an antigen-specific T cell if the ratio of the first barcodes is above a threshold value.

A6. The method of the foregoing a5, wherein the threshold is at least or greater than 2,3,4,5,6,7,8,9,10,2-5,3-6,4-7,5-8,5-10,7-10, or greater than 10.

A7. The method of any one of the preceding a-a6, wherein identifying the barcode comprises a nucleotide-based assay.

A8. The method of the foregoing A7, wherein the nucleotide-based assay is a PCR, RT-PCR, sequencing, or hybridization assay.

A9. The method of the foregoing A7 or A8, wherein the nucleotide-based assay determines (a) the sequence per barcode and/or (b) the copy number per barcode.

A10. The method of any one of the preceding a-a9, further comprising obtaining a T Cell Receptor (TCR) CDR sequence.

A11. The method of any one of the preceding a-a10, further comprising obtaining a TCR gene sequence.

A12. The method of A11, wherein the TCR sequence is a TCR α or TCR β chain sequence.

A13. The method of any one of the preceding a-a12, for use in identifying antigen specificity of T cells.

A14. The method of the foregoing a13, wherein the antigen specificity of the T cell comprises the sequence of the antigenic peptide and the TCR sequence of the bound T cell.

A15. The method of any one of the preceding claims a-a14, wherein the at least one identifying mark is the same in each distinct set of particles.

A16. The method of any one of the preceding claims a-a15, comprising at least two different identifying labels.

A17. The method of any one of the preceding a-a16, wherein the at least one identifying label is a fluorophore.

A18. The method of the foregoing a17, wherein the fluorophore is selected from the group consisting of Allophycocyanin (APC) and Phycoerythrin (PE).

A19. The method of the foregoing a18, wherein the at least two different signature labels are fluorophores, wherein the fluorophores are selected from the group consisting of Allophycocyanin (APC) and Phycoerythrin (PE).

A20. The method of any one of the preceding a-a19, wherein the antigenic peptide is selected from the group consisting of: tumor antigens, neoantigens, tumor neoantigens, viral antigens, bacterial antigens, phospho-antigens, and microbial antigens.

A21. The method of the foregoing a20, wherein the neoantigen is identified from tumor sequencing data of the subject.

A22. The method of the foregoing A21, wherein a computer predictive algorithm is used to determine the neoantigen.

A23. The method of the foregoing a22, wherein the prediction algorithm further comprises an MHC binding algorithm to predict binding between the neoantigen and the MHC peptide.

A24. The method of any one of the preceding a-a23, wherein the sample is selected from a blood sample, a bone marrow sample, a tissue sample, a tumor sample, or a Peripheral Blood Mononuclear Cell (PBMC) sample.

A25. The method of any one of the preceding claims A-A24, wherein the T cell is a human T cell.

A26. The method of the foregoing A25, wherein the T cells are CD8+ T cells.

A27. The method of any preceding claim a-a26, wherein the method comprises a library of different collections of particles.

A28. The method of the foregoing a27, wherein the library comprises a collection of 2 to 500 distinct particles.

A29. The method of any one of the preceding a-a28, wherein each particle comprises an MHC peptide.

A30. The method of the foregoing A29, wherein the MHC peptide is a human MHC peptide.

A31. The method of the foregoing A29, wherein the MHC peptide is a class I HLA peptide.

A32. The method of A29, wherein the HLA peptide comprises HLA-A, HLA-B or HLA-C peptide.

A33. The method according to A32, wherein the HLA peptide comprises HLA-A01: 01, HLA-A02: 01, HLA-A03: 01, HLA-A24: 02, HLA-A30: 02, HLA-A31: 01, HLA-A32: 01, HLA-A33: 01, HLA-A68: 01, HLA-A11: 01, HLA-A23: 01, HLA-A30: 01, HLA-A33: 03, HLA-A25: 01, HLA-A26: 01, HLA-A29: 02, HLA-A68: 02, HLA-B07: 02, HLA-B14: 02, HLA-B18: 01, HLA-B02: 27:02, HLA-B01: 44, HLA-B44: 44, HLA-A46, HLA-B50: 01, HLA-B57: 01, HLA-B58: 01, HLA-B08: 01, HLA-B15: 03, HLA-B35: 01, HLA-B40: 02, HLA-B42: 01, HLA-B44: 03, HLA-B51: 01, HLA-B53: 01, HLA-B13: 02, HLA-B15: 07, HLA-B27: 05, HLA-B35: 03, HLA-B37: 01, HLA-B38: 01, HLA-B41: 02, HLA-B44: 05, HLA-B49: 01, HLA-B01: 55, HLA-C02, HLA-B05: 01, HLA-C02, HLA-B55: 01, HLA-C55, HLA-B55: 01, HLA-C55, HLA-B33: 01, HLA-B55, HLA-C, HLA-5, HLA-B35, HLA-C, HLA-C07: 01, HLA-C01: 02, HLA-C04: 01, HLA-C06: 02, HLA-C07: 02, HLA-C16: 01, HLA-C03: 03, HLA-C07: 04, HLA-C08: 01, HLA-C08: 02, HLA-C12: 03, HLA-C14: 02, HLA-C15: 02 or HLA-C17: 01.

A34. The method of any one of the preceding a-a33, wherein each particle comprises an HLA peptide and a β 2M peptide.

A35. The method of the foregoing A34, wherein the β 2M peptide is a human β 2M peptide.

A36. The method of the foregoing a35, wherein the β 2M peptide comprises a mutation.

A37. The method of the foregoing A36, wherein the mutation is S88C.

A38. The method of any one of the preceding a-a37, wherein each particle comprises a polypeptide comprising in the amino to carboxy terminal direction (i) an antigenic peptide, (ii) a β 2M peptide, and (iii) an MHC peptide.

A39. The method of any one of the preceding a-a38, wherein the antigenic peptide is 7-15 amino acids, 7-10,8-9,7,8,9,10,11,12,13,14 or 15 amino acids in length.

A40. The method of the foregoing A38 or A39, wherein the polypeptide is biotinylated.

A41. The method of any one of the preceding a-a40, wherein the particles are selected from the group consisting of: magnetic beads, agarose beads, styrene polymer particles, and dextran polymer particles.

A42. The method of any one of the preceding a-a41, wherein the particles are coated with streptavidin.

A43. The method of any one of the preceding a-a42, for use in monitoring an immune repertoire in a subject.

A44. The method of the foregoing a43, further comprising monitoring the change in antigen-specific T cells in the subject.

A45. The method of the foregoing a43 or a44, comprising administering immunotherapy to the subject.

A46. The method of the foregoing a45, wherein the immunotherapy is adoptive cell transfer or checkpoint inhibitor.

A47. The method of any one of the preceding a-a46, for use in identifying at least one TCR sequence.

A48. The method of a47, wherein the at least one TCR sequence is a TCR α sequence, a TCR β sequence, or a combination thereof.

A49. The method of A47 or A48, further comprising making a soluble TCR polypeptide.

B. In certain non-limiting embodiments, the presently disclosed subject matter provides a library comprising at least two sets of particles, each set of particles comprising an antigenic peptide, a barcode operably associated with the identity of the antigenic peptide, and at least one identifying label.

B1. The library of the foregoing B, wherein the at least one identifying label is the same in each set of particles.

B2. The library of the aforementioned B or B1, comprising at least two different identifying labels in each different set of particles.

B3. The library of any one of the preceding B-B2, wherein the at least one identifying label is a fluorophore.

B4. The library of the foregoing B3, wherein the fluorophore is selected from the group consisting of Allophycocyanin (APC) and Phycoerythrin (PE).

B5. The library of the foregoing B2, wherein the at least two different signature tags are fluorophores, wherein the fluorophores are selected from the group consisting of Allophycocyanin (APC) and Phycoerythrin (PE).

C. In certain non-limiting embodiments, the presently disclosed subject matter provides a particle comprising at least one polypeptide, a barcode, and an identifying label, wherein the polypeptide comprises an antigenic peptide, a β 2M peptide, and an MHC peptide, and wherein the barcode is operably associated with the identity of the antigenic peptide.

C1. Particles of the foregoing C selected from the group consisting of: magnetic beads, agarose beads, styrene polymer particles, and dextran (dextran) polymer particles.

C2. The particle of the aforementioned C or C1, wherein the identifying label is a fluorophore.

C3. The particle of any one of the preceding C-C2, coated with streptavidin.

C4. The particle of any one of the preceding C-C3, wherein the polypeptide is labeled.

D. In certain non-limiting embodiments, the presently disclosed subject matter provides a method of treating cancer in a subject, comprising: (a) preparing a plurality of particles, each particle comprising a plurality of labeled polypeptides, wherein the polypeptides comprise an antigenic peptide, a β 2M sequence, an HLA sequence, and a detectable label; (b) contacting a plurality of particles with a plurality of T cells from the subject under conditions suitable for specific binding of the T cells to the particle antigen; (c) isolating the T cells bound to the particles and identifying the TCR gene sequence of the isolated T cells; (d) preparing a polynucleotide comprising homology arms and at least one TCR gene sequence, wherein the TCR gene sequence is located between the homology arms; (e) recombining the polynucleotide into an endogenous locus of a subject T cell; (f) culturing the modified T cells of step (ii) to produce a population of T cells; and (g) administering a therapeutically effective amount of the modified T cell to the subject, thereby treating the cancer.

E. In certain non-limiting embodiments, the presently disclosed subject matter provides a method of modifying a cell comprising: (a) introducing into a cell a Homologous Recombination (HR) template nucleic acid sequence, wherein the HR template nucleic acid sequence comprises (i) first and second homology arms homologous to first and second endogenous sequences of the cell, (ii) a T Cell Receptor (TCR) gene sequence obtained by a method according to any of a-a49, wherein the TCR gene sequence is located between the first and second HR arms, and (iii) a first 2A coding sequence located upstream of the TCR gene sequence and a second 2A coding sequence located downstream of the TCR gene sequence, wherein the first and second 2A coding sequences encode identical amino acid sequences that are codon divergent from each other; and (b) recombining the HR template nucleic acid into an endogenous locus of the cell, the locus comprising first and second endogenous sequences homologous to the first and second homology arms of the HR template nucleic acid.

F. In certain non-limiting embodiments, the presently disclosed subject matter provides compositions comprising a modified cell, wherein the modified cell comprises an exogenous nucleic acid sequence integrated into an endogenous locus, the exogenous nucleic acid sequence comprising: (a) a TCR gene sequence identified by a method according to any one of a-a49, and (b) a first 2A coding sequence located upstream of the TCR gene sequence and a second 2A coding sequence located downstream of the TCR gene sequence, wherein the first and second 2A coding sequences encode the same amino acid sequence that is codon divergent from each other.

Examples

The following examples are merely illustrative of the presently disclosed subject matter and should not be considered limiting in any way.

EXAMPLE 1 design and cloning of comPACT minigene by restriction digestion cloning

Structure of comPACT minigene for restriction digestion:

the basic components of the comPACT minigene include a signal sequence directing secretion of the encoded protein, a universal target sequence such as a restriction site or a primer binding site, an encoded antigenic peptide (or neo-antigen, NeoE), a second universal target site, β 2m, the extracellular domain of the MHC allele and purification clusters, e.g., enzymatic modification (e.g., biotinylation) and purification of the comPACT by an affinity tag. The clusters may also contain protease cleavage sites and linker sequences between the components shown in the figures and in the examples. The minigene may also encode cysteine mutations that may act as disulfide traps. Certain comPACT minigenes prepared and disclosed herein encode cysteine mutations that act as disulfide traps. To increase the success rate of manufacturing comPACT polypeptides by stabilizing the protein, such minigenes were made to include disulfide traps. Fig. 1 shows a schematic representation of comPACT minigenes. Additional restriction sites upstream and downstream of the MHC heavy chain sequence can be used to insert other MHC alleles to construct different MHC templates and construct a pool of MHC templates (figure 2). The DNA encoding the signal sequence, the universal target sequence, the extracellular domains of the β 2m and MHC alleles are the basic MHC template.

For the restriction digestion cloning approach, each comAT DNA construct is a basic MHC template with a virtual antigen sequence insert containing a stop codon in three frames and one unique restriction site for destruction of the uncleaved or religated template (FIG. 3) and can be used as part of a ready-to-use platform for rapid assembly of an antigenic peptide library complexed with the MHC alleles. MHC alleles can also be modified or mutated (e.g., Y84A or Y84C), for example, to improve folding or increase binding of antigenic peptides to MHC proteins. In addition, the β 2m protein may also be mutated (e.g., S88C), for example, to allow binding to a thiol dye.

In this example, a comPACT minigene is shown having the following structure: a 5' NotI restriction site; the signal sequence from human growth hormone hGH, as shown in table 1; the restriction site Blp1 upstream of the antigenic peptide region and the BamHI restriction site downstream of the antigenic peptide region, as shown in Table 2; a coded linker sequence consisting essentially of glycine and serine residues (i.e., a Gly-Ser linker); beta is a₂m sequence; a second encoded Gly-Ser linker sequence with a BspI restriction site; an MHC heavy chain; a third encoded Gly-Ser linker sequence with a BstBI restriction site; and a coded purification cluster with an AviTag (or any avidin/streptavidin) sequence, a TEV cleavage site, and a tandem histidine tag.

Restriction digestion cloning and Assembly of comPACT minigenes

Three different methods of insertion of the encoded neoantigen by restriction digestion are described herein. In the first approach, as shown in FIG. 4, a primer encoding an antigenic peptide (NeoE) spans a first restriction site (BlpI in this example) at the 5 'end and a second restriction site (BamHI in this example) at the 3' end. This primer amplifies a universal reverse primer encoding a second restriction site, resulting in a primer dimer of about 70 bp.

In the second approach, a primer encoding an antigenic peptide spans the second restriction site as the 5' end and is the reverse complement of the antigenic coding sequence. The primer is primed in the reverse direction of the template DNA encoding the signal sequence. Paired with a forward primer that spans the first restriction site sequence, if a forward primer that spans a restriction site further upstream from the antigenic site is used, the reaction produces a 70bp product, or a product of about 140 bp.

In the first and second methods, the insert is typically washed on a commercial column, digested with a suitable restriction enzyme, washed again on a commercial column, and then ligated into a vector with a pre-digested MHC template. The ligation reaction was transformed into E.coli, and the plasmid prepared from the transformed E.coli was used for mammalian producer cell transfection reaction.

EXAMPLE 2 design and cloning of comPACT minigene by primer annealing

In a third variation of MHC template vector ligation, PCR and restriction digestion were bypassed by annealing two reverse complementary neo-antigen encoding primers. These primers were designed to have 5 'and 3' ends, which start and terminate with complementary sequences that mimic the overhangs produced by restriction digestion (FIG. 5). Sense and antisense primers were incubated with T4 polynucleotide kinase and ATP to phosphorylate the 5' end (FIG. 22A). When these primers anneal to each other, they form a double-stranded oligonucleotide sequence having overhang nucleotides, as if digested with restriction enzymes.

Phosphorylated neoantigen inserts (alternatively referred to as neoepitopes) are ligated into pre-cut MHC templates in a vector. The comPACT minigene has the same structure as described in example 1. The ligation products were then used for PCR amplification of linear copat amplicons, and the entire copat minigene was amplified using the backbone universal primer and sequenced. Using this method 824 comact minigenes with unique neoantigen sequences (otherwise referred to as new epitope sequences) were prepared, with more than 99% of the generated comact minigenes having the correct neoantigen sequence (otherwise referred to as new epitope sequences) (fig. 22B). The neoantigen sequence (alternatively referred to as a neoepitope sequence) cloned into the comPACT polynucleotide and expressed as a polypeptide is based on a tumor neoantigen identified from a patient sample (e.g., a tumor sample or other patient sample expressing a tumor antigen). Based on the identified neoantigens, a series of predicted neoantigen sequences (alternatively referred to as neoepitope sequences) are made for each identified neoantigen.

Next, E.coli was transformed with the ligation product plasmid and plated onto selective agar plates containing ampicillin. Single colonies were picked and grown overnight for plasmid purification and sequencing for complete gene validation. After sequencing validation, plasmid batches were archived and propagated to larger numbers.

Alternatively, T4 kinase is not used if the pre-cut MHC template carrier retains 5' phosphate at its overhang. The annealed antigen insert neoantigen sequence (also referred to as neo-epitope insert) can then be ligated to the cleaved MHC vector and the ligation product transformed into e.

Example 3 design and cloning of a ComPACT minigene by PCR Assembly

Structure of comp oct minigene for PCR assembly:

a fourth method of inserting an antigen, e.g. a neoantigen (for clarity, as used in this example, both refer to the NeoE insert in figure 6 and the neoepitope as described in example 2) may also be used. In this method, the antigen-coding sequence (for clarity, as used in this example, the antigen-coding sequence refers to the novel epitope sequence described in example 2) is inserted into an MHC template flanked by an upstream promoter and a downstream polyadenylation signal by the polymerase chain reaction to form a 2.5kb minigene. A schematic diagram of the PCR assembly reaction is shown in FIG. 6.

In this example, the comPACT minigene is shown to have the following structure: a promoter at the 5' end; a signal sequence having a first universal target sequence; an encoded antigenic peptide; having a coding linker sequence consisting essentially of glycine and serine residues (i.e., aGlySer linker); beta is a₂An M sequence; a second encoded Gly-Ser linker sequence; MHC heavy chain alleles; a third Gly-Ser linker sequence; purifying the clusters; and polyA sequences. In this exemplary method, the universal target sequences are not identical, i.e., they are different from each other.

PCR assembly of the comp PACT minigene:

in this method, two primers (<60nt) with selected antigen sequences (for clarity, antigen sequences refer to neoantigen sequences as used in this example) were synthesized. The first primer has a neoantigen sequence at the 5 'end (for clarity, as used in this example, the neoantigen sequence refers to the novel epitope sequence described in example 2), followed by a second universal target sequence segment at the 3' end. The second primer has the reverse complement of the neoantigen sequence at the 5 'end (for clarity, as used in this example, the neoantigen sequence refers to the new epitope sequence described in example 2), and the reverse complement of the first universal sequence at the 3' end. These primers are mixed with a DNA fragment encoding the promoter region, signal sequence and first universal target sequence and another DNA fragment encoding the second universal target sequence, β 2m sequence, MHC alleles, purification clusters and polyA sequence. Each antigenic peptide primer anneals to its complementary sequence and a PCR reaction is performed to amplify the neoantigenic sequence (for clarity, as used in this example, the neoantigenic sequence refers to the neoantigenic sequence described in example 2) onto a promoter fragment or an MHC allele fragment. These two newly synthesized fragments now each have a neoantigenic sequence (for clarity, as used in this example, the neoantigenic sequence refers to the new epitope sequence described in example 2). Further PCR reactions, along with primers at the 5 'end of the promoter sequence and 3' end of the polyA sequence, allowed neoantigen sequences (for clarity, as used in this example, neoantigen sequences refer to the neoepitope sequences described in example 2) to anneal to each other and initiate assembly of the full-length linear copack amplicon.

The fully assembled linear comPACT polynucleotide is then washed for direct transfection into mammalian producer cells, bypassing the steps of E.coli and plasmid production.

EXAMPLE 4 expression and purification of comPACT protein in plasmid

Expression of proteins

Neoantigen12(neo12) was ligated into the HLA-A2 template sequence and inserted into the expression plasmid (pPACT0010) by restriction digestion and ligation of NotI and BamHI restriction sites as previously described in example 1.

On day-1, Expi293 mammalian producer cells in a 30mL shake flask volume were transfected with pPACT0010 incubated with Expifeacylamine transfection reagent. Enhancers included in the expifctamine transfection kit were added on day 0. Samples were collected from cell supernatants on days 1 to 7 and secreted proteins were assessed by SDS-PAGE and total protein staining using safestain (thermofisher). Secreted comPACT protein levels increased until day 3, at which time protein secretion tended to plateau (fig. 7). Secreted comPACT protein was initially identified by its apparent molecular weight (═ 53kDa) and confirmed by western blot using NTA-HRP to detect His6 affinity tag (SEQ ID NO: 34).

Purification of proteins

The Neo12 comPACT protein collected on day 7 was purified by Ni-NTA affinity chromatography via the binding of His6 affinity tag (SEQ ID NO: 34). Samples were analyzed for total protein by SDS-PAGE and Safetain. The absence of comPACT protein in the flow-through (FT) fraction of the affinity column confirmed that the His6 tag (SEQ ID NO:34) was not cleaved during expression and purification (FIG. 8). Purification yield >400mg per liter culture volume.

The Neo12 comPACT protein was biotinylated (discussed in example 5 below) and further purified by size exclusion chromatography. A single major peak was observed, indicating that the protein was correctly folded and monomeric, with little aggregation (figure 9). The second peak is ATP, which is added for BirA catalyzed biotinylation reaction.

Although Ni-NTA chromatography is used in example 4, any HA-affinity chromatography (including but not limited to the metal affinity chromatography described herein) can be used to purify HA-tagged comact.

Optimization of production volumes and parallel production

Production of comPACTs was reduced from 30mL culture volume in shake flasks to 0.7mL in 96 deep well shake blocks. Expi293 mammalian producer cells were transfected with plasmid DNA containing the pact0010 plasmid and the secreted Neo12 comPACT protein was purified as described previously. 437mg/L of purified Neo12 comPACT protein was collected from 0.7mL pore volume compared to the yield >400mg/L of the 30mL purification experiment described previously (FIG. 10). The protein yield for the 0.7mL experiment corresponds to >300 micrograms of protein, or about 1000-fold higher than that required for a typical flow cytometry experiment.

Next, parallel expression of multiple comact constructs was evaluated. Eight different comPACT constructs with different neoantigens (neoantigens 10, 15, 64, 65, 66, 67, 80 and 83) were expressed in 30mL shake flasks as medium throughput assays (fig. 11). As previously described, each comact construct was transfected into cells, where the comPACT protein was expressed and secreted into the cell supernatant. The expressed protein was purified, concentrated and normalized as described previously. Crude supernatant samples and concentrated proteins were assayed for total protein as previously described. comPACT protein was purified by size exclusion chromatography (fig. 12). A single peak containing 2-20mg of protein was observed for each protein, which also indicates that the comPACT protein is correctly folded and monomeric.

Example 5 expression and purification of comPACT protein from Linear amplicon

In the previous examples, comPACT protein was expressed from plasmids transfected into mammalian producer cells. As an alternative method, linear amplicons of neo12comp PACT minigene (neoantigen 12 assembled into a minigene with HLA-A2 template sequence) flanked by promoter sequence and polyA sequence were transfected into 0.7mL producer cells in 96-deep well plates. As a control, the pact0010 plasmid was also transfected into individual producer cells. Total protein of proteins from both samples was expressed, purified and assayed as described previously. Both the linear amplicon and the plasmid produced similar levels of expressed protein (fig. 13A), indicating that the protein encoded by the comPACT minigene can be produced without the need for plasmid intermediates. A number of different comact minigenes with different novel epitope sequences have been generated (fig. 13B) for direct transfection of producer cells.

Additional comcats with different HLA alleles were prepared using the annealing and phosphorylation workflow described in example 2. Linear amplicons were obtained from the expression vectors using the bookmark PCR and universal primers and transfected into Expi293F cells for comPACT protein production (data not shown).

Example 6 biotinylation of comPACT protein

In vitro biotinylation of comPACT with isolated BirA enzyme

The comPACT purification cluster included a BirA recognition sequence (Avitag) for biotinylation. The purified comPACT protein was either not biotinylated (no BirA treatment) or biotinylated with commercial BirA proteins according to the manufacturer's instructions (BirA treatment). After overnight BirA enzyme treatment, samples were bound to two different types of magnetic streptavidin beads (C1 and T1) and incubated to bind biotinylated proteins to the streptavidin beads. The Supernatant (SN) and beads ("pellet", P) were separated by SDS-PAGE. Total protein of the samples was determined with Safestain and the presence of comPACT protein was determined by western blot and NTA-HRP (fig. 14). In the untreated sample, comPACT protein was found mainly in the SN fraction, confirming that it was not biotinylated. Among the biotinylated samples, comPACT protein was found in both the precipitated samples of C1 and T1 streptavidin beads, although the interaction between biotinylated protein and C1 streptavidin beads was the most complete. Biotinylated copact protein was not detected by western blotting in C1 streptavidin bead depleted supernatant, indicating that about 100% of the copact protein was biotinylated.

The comPACT protein may also be biotinylated in the clarified supernatant prior to purification. As previously described, multiple comact proteins were expressed in the producer cells. Cell culture supernatants were collected and clarified by centrifugation. The clarified supernatant was treated with commercial BirA protein, then purified by Ni-NTA affinity chromatography, and evaluated for biotinylation by western blotting, according to the manufacturer's instructions (figure 15). Biotinylation of all tested comact proteins using this method showed that biotinylation of comact proteins in the clarified cell supernatant was effective. Although Ni-NTA chromatography is used in example 6, any HA-affinity chromatography (including but not limited to the metal affinity chromatography described herein) can be used to purify HA-tagged comact.

In order to generate enough BirA for high-throughput biotinylation of comPACT proteins, the BirA protein with the His6 tag (SEQ ID NO:34) was expressed in E.coli cells. This BirA with His6 tag (SEQ ID NO:34) was purified by Ni-NTA affinity chromatography (FIG. 16B) and was used to biotinylate comPACT proteins. The second version of BirA-His6 (disclosed as "His 6" of SEQ ID NO:34) with a TEV cleavable His6 tag (SEQ ID NO:34) was also expressed and purified by Ni-NTA affinity chromatography (fig. 16C). This BirA-TEV-His6 protein ("His 6" disclosed as SEQ ID NO:34) can be purified by Ni-NTA, the His6 tag (SEQ ID NO:34) removed by TEV cleavage, and then untagged BirA was used for biotinylated copack protein. After biotinylation of the comPACT protein, the unlabeled BirA protein can be purified by Ni-NTA affinity chromatography. In addition, TEV protease was heterologously expressed in E.coli for use with BirA-TEV-His6 (disclosed as "His 6" of SEQ ID NO:34) (FIG. 16A) for biotinylated comPACT protein production. Although Ni-NTA chromatography is used in example 6, any HA-affinity chromatography (including but not limited to the metal affinity chromatography described herein) can be used to purify HA-tagged comact.

Cleavage of the His6 tag (SEQ ID NO:34) on the comPACT protein after biotinylation was also evaluated, and the results are shown in FIG. 17. The comPACT protein was treated with BirA or untreated to biotinylate it as previously described (lanes 1 and 2 of FIG. 17). A third sample of comPACT protein was treated with BirA followed by TEV to cleave the His6 tag (SEQ ID NO:34) present on the protein (lane 3). Samples were separated by SDS-PAGE and total protein was assessed by Safetain. All three samples contained equal amounts of comPACT protein. Biotinylation of comPACT protein and cleavage of His6 tag (SEQ ID NO:34) were assessed by western blotting using SA-HRP reagent for biotin signaling and NTA-HRP reagent for His6 tag (SEQ ID NO: 34). The non-biotinylated sample showed NO biotin signal, but did have a His6 signal (SEQ ID NO:34), and the biotinylated and non-cleaved samples had both signals. Biotinylated and TEV cleaved samples had only biotin signal, indicating that the His6 tag (SEQ ID NO:34) was successfully cleaved from the comPACT protein.

A third method for in vitro biotinylation of comact was the expression of BirA in Expi293 producer cells. Cells co-expressing BirA and cell surface transduction labeled Expi293 cells labeled V5 were generated. Transduced cells sorted against V5+ also expressed BirA (fig. 18). These cells can be used to produce biotinylated comact in vivo prior to purification of the comact protein.

Example 7 antigen-specific T cell staining and affinity assessment Using comPACT protein

To compare antigen-specific T cell staining using comPACT protein and conventional peptide-MHC, comPACT dextramers were prepared according to published protocols (bethoune, m.t., et al. biotechniques 62, 123-. T cells were engineered to express an A2/neo12 specific TCR and stained with HLA-A2/neo12 peptide-MHC dextramer or HLA-A2/neo12 peptide comPACT dextramer. Staining with comPACT dextramer was at least as effective as peptide-MHC dextramer staining (FIG. 19). This data indicates that comPACT dextramers can be used to sort antigen-specific T cells for TCR sequencing.

Example 8 functional T cell assay

In addition to antigen-specific capture of T cells, the modular design and ease of production of compacts facilitates their use in functional T cell assays. For example, incorporation of a mutated version of β 2m (S88C) enables comPACT to be labeled with maleimide dye conjugates, assembled into NTAmer, and used to measure kinetic parameters of TCR-comPACT binding. The S88C mutant comPACT protein was constructed and expressed at-150 mg/L. These mutated copacs exhibit similar purity and elution profiles to the non-mutated copacs (fig. 20). Other dyes such as Cy5 may also be conjugated to S88C comPACT (fig. 21).

Example 9 comPACT library production

HLA allele diversity in the American population is analyzed from an allele frequency network database (www.allelefrequencies.net) by bioinformatics to identify the optimal number of alleles included in the HLA library to achieve high coverage of subject HLA frequencies. 9736 alleles were analyzed. Figure 24A shows an analysis of the percentage of patients in which one or both alleles from each of HLA a, B, and C loci are covered by a 66 HLA allele library. The solid line indicates coverage of 1 allele, while the dashed line indicates coverage of two alleles. The 66 alleles enabled coverage of at least 4 of the 6 HLA alleles per patient in > 95% of the total population and 6/6 alleles in > 80% of the population (fig. 24B). The most common HLA-I allele is HLA-A02:01, with an prevalence of about 50% in the United states. Thus, the HLA libraries presented herein for the first time are most likely to be used to widely perform personalized NeoTCR-T cell therapy for different populations worldwide.

Next, a comPACT protein library with different neo-epitopes and selected HLA alleles was prepared. The neoepitope candidates are selected from immune epitope databases (www.iedb.org). The complete sequence of each of the 66 HLA-I alleles in the panel was obtained from the IMGT database and modified to include the Y84C mutation. All clones were sequence verified and stored in the database and reagent list. 10 new epitope peptides were selected from the IEDB database and inserted into a set of 36 HLA alleles. comPACT polypeptides of selected neo-epitopes and HLA alleles were expressed and purified by size exclusion chromatography column (Agilent Sec Bio 300) (SEC-HPLC) connected to an Agilent Infinity II HPLC system according to the manufacturer's instructions. The results are shown in FIGS. 25A-C. The comPACT polypeptide was purified as monodisperse polypeptide as assessed by SEC-HPLC by measuring the area under the curve of the monomer peak divided by the area under the entire chromatogram (fig. 25A and 25B). Most comact polypeptides were expressed at high titers (fig. 25C). The described at least one comact protein per HLA allele has been purified and characterized by HPLC, indicating that the comact platform is robust and applicable to many alleles.

Example 10 Impact T cell isolation method

Materials and methods

comPACT library preparation

Paired PE and APC tetrameric particles with three comPACT library elements and barcodes were prepared prior to the experiment. Biotinylated compPACT (1. mu.M, generated internally) and DNA barcode (1. mu.M, IDT) were mixed at a molar ratio of 3: 1. PE-streptavidin (3.33. mu.M, Life Technologies) or APC-streptavidin (6.26. mu.M, Life Technologies) was added to react with biotin in a ratio of 1: 4. After incubation, additional biotin was introduced to occupy the free streptavidin sites.

CD 8T cell staining

Cells were incubated with 40nM fluorescent comPACT tetramer for neoantigen-specific T cell staining. An Fc receptor blocking solution was then added to minimize non-specific antibody staining. The samples were also incubated with an antibody cocktail containing FITC CD4, CD14, CD19, CD20, CD40, PerCp-Cy5.5 CD8, BV711 CD45RA, BV786 CD95, and BV510 IP26(Biolegend) to identify the phenotype of the T cells.

Single cell sorting

The fluorescently labeled cells were sorted into single cells using FACSAria III (BD Biosciences). Cells were first sorted for T cell phenotype based on IP26 and CD3 staining, and then sorted for dual p-HLA binding based on APC and PE staining. comPACT positive cells were sorted into 96-well plates containing 10mM Tris and RNAse inhibitor (Promega) lysis buffer.

TCR cloning

A RT-PCR master mix (master mix) was prepared containing the following reagents: nuclease free water (Invitrogen), 5x buffer (Qiagen), 10mM dntps (Qiagen), alpha multi-primer mixture, beta multi-primer mixture, alpha antisense primer, beta antisense primer, DNA barcode sense primer, DNA barcode antisense primer (all primers ordered by IDT), onestrep RT-PCR enzyme (Qiagen), and KOD polymerase (Millipore). The RT-PCR master mix was then added to each well to initiate reverse transcription and polymerase chain amplification of the TCR and DNA barcode sequences.

Sensitivity and S/N determination

CD 8T cells expressing TCR against MART1 antigen (F5) or neoantigen neo12 were incubated with fluorescent comPACT particles with the corresponding comPACT neoantigen element (MART1 or neo 12). Cells were stained and sorted by FACS as described above (fig. 29A-C).

CD 8T cells expressing TCR against neoantigen neo12 were spiked into control PBMC samples at a ratio of 1 to 300,000. Spiked samples were incubated with 33 tetramer libraries, which included neo12 comPACT and 32 additional neoantigen comPACT elements. Single cells were sorted based on the above-described APC and PE double-labeled CD 8T cell gating strategy (fig. 30).

Specificity and S/N assays

CD 8T cells expressing TCR against neoantigen neo12 were spiked into control PBMC samples at a ratio of 1 to 300,000 or 1 to 30,000. PBMC alone were used as negative controls. The spiked samples were incubated with a library containing neo12 comatt and 28 unrelated control comatt elements. Single cells were sorted based on the above-described APC and PE double-labeled CD 8T cell gating strategy. The barcodes and the signal to noise ratio of each barcode associated with a given cell are determined.

Results

Sensitivity of the probe

Fig. 29A provides a schematic of the endogenous TCR gating strategy used to isolate PACT neoantigen CD 8T cells (upper panel) and tetramer-positive ditag T cells (lower panel). T cells expressing neoantigen F5 (fig. 29B) or Neo12 (fig. 29C) were labeled and gated according to a gating strategy. Corresponding tetramer staining produced over 99% accuracy for F5 and Neo12 compPACT Neo-antigen T cells. Repetition of this assay resulted in tetramer staining of gene-edited F5 and neo 12T cells by more than 99% (data not shown). Thus, the imPACT method has high staining and sorting sensitivity of greater than 98% or 99%.

To test the sensitivity of the imPACT tetramer method, cell doping experiments were performed. T cells expressing neoantigen neo12 were spiked into control PBMC samples at a ratio of 1 to 300,000 (1 neo 12T cells per 300,000 PBMCs). imPACT tetramer analysis was performed using tetramers prepared from neo12 comPACT and 32 unrelated control comPACT. Cells positive for 33 comPACT tetramers were selected from CD 8T cell gating as described above. Cells were sequenced for the relevant TCR and neoID sequences. After sequencing, the signal to noise ratio (S/N) was used to determine the specificity of tetramer binding. The S/N calculation of this example is the DNA copy number of the most predominant neoID divided by the DNA copy number of the second most predominant neoID. In this example, an S/N greater than 10 is considered to be a specific binding of comPACT to T cells. The IP26 stained index flow (indexed flow) results for each cell indicate whether a given cell is genetically edited or naive.

Table 4 summarizes the cells sorted from this experiment. 1686,717 total cells were analyzed by flow cytometry and 11 cells were sorted from the positive gate.

5 of 11 positive cells had an S/N higher than 10 (average S/N83.1), while the other 6 had an S/N lower than 10 (average 1.1). Depending on the ratio of Neo 12-doped cells (1:3000,000) and the number of cells treated (1,686,717), there should be approximately 5-6 Neo12 cells in the sample analyzed. Sequencing showed that 5 neo12 cells were isolated using this method. Thus, the imPACT tetramer method is sensitive enough to isolate antigen-specific cells at a low frequency of 1/300,000. Figure 30 shows gated FACS cells from 1:300,000 doped samples. TCR-represents neo12 positive T cells, while TCR + represents non-specifically bound T cells. The average S/N ratio for specific neo12 cells was 83, while the average S/N ratio for non-specific cells was 1.1.

Specificity of

Next, the new antigen specificity of the imPACT isolation method was evaluated. The Neo12 doping assay was repeated using a second library containing Neo12 comatts added to PBMC samples and 28 unrelated control comatts. PBMC alone were used as negative controls. Double positive cells were isolated, the barcodes sequenced, and the S/N ratio of each barcode associated with a given cell determined. Figure 31 shows PE and ACP FACS gating data for neo12 antigen doped and undoped PBMC samples. A table summarizing the results of barcode S/N sequencing is shown below each test. In the specificity test, the S/N average was 162, indicating 162 copies of the neo12 neoID barcode for each non-neo 12 neoID barcode. This indicates that the neo12 barcode is highly specific for sorted cells from neo12 and PBMC samples. In contrast, the average S/N in the non-specific test was 1.7, indicating that there were only 1.7 copies of the Neo12 neoID barcode for each non-Neo 12 neoID barcode. This indicates the low specificity of the neo12 barcode for sorted cells in PMBC samples only.

The assay was repeated with a comPACT library with 33 elements added to PBMC samples at a ratio of 1 neo 12T cells to 30,000 PBMCs. FIG. 32A shows gated double positive cells, while FIG. 32B shows bar code S/N averages for specific and non-specific T cells. The signal to noise ratio was determined for 33 tetramer positive cells. The average S/N value of the T cells with specific binding was 124.9, whereas the non-specific S/N ratio was 1.2, confirming the high specificity of the imPACT method for the isolation of neoantigen-specific T cells. Table 3 summarizes the information of the sorted cells, and whether the isolated cells were genetically modified (gene editing).

EXAMPLE 11 isolation of neoantigen T cells from patient samples

Materials and methods

Tetramer preparation

Tetramers were prepared as described previously.

CD8 selection and cell staining

Cryopreserved patient PBMCs were thawed and CD8T cells were selected using the CD8+ T cell isolation kit (Miltenyi) according to the manufacturer's recommended protocol. Isolated CD8T cells were used for subsequent staining. Cells were incubated with 40nM fluorescent comPACT tetramer library for NeoE-specific T cell staining. An Fc receptor blocking solution was then added to minimize non-specific antibody staining. The samples were incubated with an antibody cocktail containing FITC CD4, CD14, CD19, CD20, CD40, PerCp-cy5.5 CD8, BV711 CD45RA, BV786 CD95 and BV510 IP26(Biolegend) to identify the phenotype of the T cells. Live/dead near infrared cell stain (Invitrogen) was used to distinguish between viable and non-viable cells. BV605 Annexin-V (Biolegend) was used to further differentiate between live and apoptotic cells.

Single cell sorting and TCR cloning

The fluorescently labeled cells were sorted into single cells using FACSAria III (BD Biosciences). Live CD8+ tetramer positive cells were sorted into 96-well plates containing 10mM Tris and RNAse inhibitor (Promega) lysis buffer. The cells were then frozen for subsequent TCR cloning. A RT-PCR master mix (master mix) was prepared containing the following reagents: nuclease free water (Invitrogen), 5x buffer (Qiagen), 10mM dntps (Qiagen), alpha multi-primer mixture, beta multi-primer mixture, alpha antisense primer, beta antisense primer, DNA barcode sense primer, DNA barcode antisense primer (all primers ordered by IDT), onestrep RT-PCR enzyme (Qiagen), and KOD polymerase (Millipore). The RT-PCR master mix was then added to each well to initiate reverse transcription and polymerase chain amplification of the TCR and DNA barcode sequences. Two additional rounds of PCR were performed to further amplify the TCR and DNA barcode sequences, and to append linker sequences for Next Generation Sequencing (NGS).

Next generation sequencing

Next generation sequencing was performed on a miniseq (illumina) using the recommended reagents. Library preparation was performed according to the protocol recommended by Illumina. Target species and PhiX were mixed in equal amounts to provide diversity.

Results

First, stage IIIA melanoma patient samples (PACT032) were analyzed using a 26 element comPACT library with HLA a02:01 allele type. 3.9X10 in the sample⁶Of the PBMCs, 231 were double positive for APC and PE. Cells were analyzed for neoID barcodes and the signal to noise ratio was determined for all double positive cells. Fig. 33A shows FACS dot plots for double positive T cells. After neoID sequencing, a T cell showed specificity for a mutationAnd the signal-to-noise ratio (S/N1) is larger than 10. The neoantigen TCR was cloned and screened against the predicted neoantigen. The signal-to-noise ratios of the remaining double positive cells were all 1 and were not specific for the neoantigen associated with the bound comPACT. Secondary screening (fig. 33B) confirmed the specificity of the isolated neoantigen TCR by influence analysis. Table 5 below provides a summary of the signal-to-noise ratios of specific and non-specific T cells.

TABLE 5

TCR clonotypes	Counting	S/N(UMI)
			TCR+	230	1
PACT32-TCR75	1	13

Example 12 analysis of S/N1 and S/N2 to identify TCRs

Next, a phase III melanoma patient sample (PACT077) was analyzed using a 138 element comPACT library of the type HLA A02:01, A24:02, B18:01, and C07: 01. 5.1X10 in the sample⁶Of the PBMCs, 250 were double positive for APC and PE. Cells were analyzed for neoID barcodes and the signal to noise ratio was determined for all double positive cells. Fig. 34A shows FACS dot plots for double positive T cells. Figure 34D shows neoantigen-specific T cells identified in peripheral blood. Asterisks indicate that identical TCR clones were found in Tumor Infiltrating Lymphocytes (TILs) by tumor sequencing And (4) molding. After neoID sequencing, 25T cells showed specificity for one mutation with a signal-to-noise ratio (S/N1) greater than 10 (fig. 34C). Figure 34B shows that all candidate cells were from CD95+ cells that have undergone antigen. Neoantigens TCRs were cloned and screened against the predicted neoantigens. Figure 34E shows the percentage of neoTCR gene-edited lymphocytes that are able to recognize homologous antigens. The signal-to-noise ratios of the remaining double positive cells were all 1 and were not specific for the neoantigen associated with the bound comPACT. Table 6 below summarizes the signal to noise ratio of T cells against the selected neoantigens.

TABLE 6

SEQ ID NO:	Novel antigens	Average S/N
			203	EYIPGTTFL	25
204	IYNIIVTTL	43
			205	KTSVALHLI	19
206	HLSLELLGVD	21
			207	DEYIPGTTF	32

Interestingly, analysis of the TCR from PACT077 identified 8 different TCRs. Of these 6 had an S/N1 ratio greater than 10 and were confirmed to be specific neoantigen T cells. For the other 2T cells, the S/N1 ratio was below 10, but the S/N2 ratio was above 10 (FIG. 34C). Cloning of the two TCRs (TCR143 and TCR164) suggested that they could recognize two different neoantigens sharing the same mutation, further explaining the reason for the low S/N1 (fig. 35A). These results indicate that the S/N2 ratio can be used to distinguish between non-specific and specific cells when there are multiple new antigens derived from the same mutation.

Secondary screening (fig. 35B) confirmed the specificity of the neoantigen TCRs isolated by imPACT analysis (i.e., TCR135, TCR136, TCR139, TCR142, TCR144 and TCR 145).

Figure 36 shows that isolated TCRs vary between mutations with different levels of clonality, truncation, and in situ neoantigen expression.

Example 13 isolation of neoantigen T cells Using Dual NEOID barcodes

Materials and methods

Tetramer preparation

Paired fluorescent tetrameric particles were prepared as described above except that each particle pair had a different unique neoID barcode associated with the neo-antigen (see figure 28 for a double barcode paired particle).

CD8 selection and cell staining

Cryopreserved patient PBMCs were thawed and CD8T cells were selected using the CD8+ T cell isolation kit (Miltenyi) according to the manufacturer's recommended protocol. The isolated CD8T cells were used for subsequent staining as described previously.

Single cell sorting and TCR cloning

The fluorescently labeled cells were sorted into single cells using FACSAria III (BD Biosciences) as described previously.

Next generation sequencing

Next generation sequencing was performed on a miniseq (illumina) using the recommended reagents as described previously.

Results

PACT049 (stage 4 CRC, naive) PBMCs were screened using the ipact method and double barcode. Six-membered double barcode comPACT libraries (HLA-B57:01, A01:01 and C06:02) were generated and used to interrogate neoantigens TCR. 352 single cells were sorted. After sequencing analysis, three neoantigen TCR candidates were identified against one HLA-B57:01 neoantigen RCSPEQLKKAW (SEQ ID NO:208) (FIG. 37A). These three candidates were sorted according to tetrameric MFI (mean fluorescence intensity) and were all considered to have undergone antigenic CD95+ (fig. 33B). After cloning PACT049 neoantigen T cells, these TCRs were confirmed by dextramer staining with the relevant predicted neoantigen (fig. 37C and 37D).

EXAMPLE 14 additional isolation of neoantigen T cells from patient samples

Additional patient samples were incubated with the comPACT library and isolated according to the imPACT method described above. Patient samples were analyzed using the imPACT signal-to-noise ratio method and the single or double barcode method. A graph of the number of neoantigens and HLA types identified from each sample and cancer type is provided in fig. 38A, and HLA types are listed in fig. 38B. Among the five cancer types and 13 HLA types from the periphery, 32 neoantigen-specific TCRs were identified. Neoantigens were identified in colorectal cancer (11), melanoma (7), bladder cancer (3), endometrial adenocarcinoma (1), and head and neck cancer (1) samples. Two patient samples (PACT056 and 095) did not produce any neoantigen-specific TCRs. Four patient samples (PACT032,052,053 and 078) had one neoantigen-specific TCR. Multiple neoantigen-specific TCRs were isolated in each of the following seven samples: PACTs 035,036,037,049,077,131 and 133. Multiple neoantigen-specific TCRs enable selection of the optimal TCR for a patient. Two samples (PACT077 and 078) had peripheral TCRs, which were also found in situ by deep sequencing of TILs. These results indicate that the success rate of neoantigen-specific TCR identification was 100% for patients receiving drug treatment and greater than 80% for untreated patients.

The results show that the imPACT technique is able to successfully isolate antigen paired, neoantigen-specific TCRs from patient samples with high accuracy and specificity. Since the imPACT technology targets new antigens and the antigen presentation pathway is versatile, the imPACT platform technology can be applied to different cancer types, enabling the development of personalized NeoTCR-T cell therapies to eradicate solid tumors.

Example 15 reproducibility of T cell isolation procedure

Next, PBMCs of healthy donors were analyzed using a comPACT element library. 15 paired fluorescent comPACTs (HLA A02:01) with novel antigens against Cytomegalovirus (CMV), EB virus (EBV) and influenza were prepared as described previously. The comPACT library was incubated with PBMCs, and double positive T cells were sorted and isolated. The neoID barcode was sequenced as described previously, and the TCR was cloned and sequenced. The experiment was performed in triplicate.

Figure 39A shows the percentage of antigen specificity of isolated T cells with neo-antigens against CMV and EBV in each of the three replicates. Figure 39B shows the number of TCR α chains isolated from each experiment. Table 7 provides a summary of the identified TCR α chains. 14 unique TCR α chains were identified, and 10 of the 14 were shared in all three experiments. This reproducibility experiment shows that the imPACT method can consistently separate antigen-specific T cells at similar levels from the same sample in independent experiments, suggesting that the method of separating double positive T cells by incubating the cells with paired comPACT tetramers with different fluorophores is highly reproducible in a variety of settings.

TABLE 7

Example 16T cell separation comparison Using comPACT tetramers, dextramers, and trimers

The separation efficiency of tetramers, dextramers and trimers of comPACT library elements in the double staining procedure was evaluated. Tetramers (tetramers) of streptavidin core with four copies of each copart element, trimers (trimers + DNA) of streptavidin core with three copies of each copart element and nucleic acid barcode, and dextramers (both dextramers and dextramer + DNA) of dextran multimers (with and without nucleic acid barcodes) with multiple copies of copart elements were incubated with T cells genetically edited to overexpress F5(MART1) or neo12 neo-neoantigen. T cells were isolated based on the gating strategy described in example 10 above and the percentage of double-stained T cells isolated by each staining method was quantified. As shown in fig. 40A (F5T cells) and fig. 40B (neo12 antigen-specific T cells), greater than 98% of the gene-editing cells were stained by the corresponding comPACT elements. The data show that similar staining efficiencies were achieved for T cells with a common TCR (F5 Mart-1) and neoantigen TCR (neo12), tetramer, dextramer, DNA-containing trimer and DNA-containing dextramer.

Example 17 comparison of Signal to noise analysis

PACT Neo 12T cells, PACT M1W T cells, and virus donor PBMCs were incubated with trimeric comPACT particles with nucleic acid barcodes (Trimer + DNA) and Dextramer with multiple copies of comPACT elements with nucleic acid barcodes (Dextramer + DNA). Cells were sorted by FACS into single cells. The TCR alpha/beta and neoID barcodes of each sample were cloned and sequenced. All TCRs were confirmed to be correct for neo12, CMV, or M1W. S/N analysis was performed on each cell as described previously. The S/N ratio for each method (trimer, T; or dextramer, D) is shown in FIG. 41A for each sample. The average of the S/N ratios for each method is provided in FIG. 41B. Notably, analysis of the signal-to-noise ratio of neoID barcode DNA showed that cells isolated with primer + DNA particles had a higher signal-to-noise ratio than cells isolated with Dextramer + DNA. The data show that the Trimer + DNA particles have a better S/N ratio than Dextramer + DNA.

Example 18 isolation and characterization of neoantigen T cells from patient samples following cancer immunotherapy

Subjects with pMMR colorectal cancer (generally considered to be unresponsive to anti-PD 1 antibody therapy) or endometrial adenocarcinoma were treated with AB122 (anti-PD-1 antibody). Pre-treatment blood samples were incubated with comPACT libraries and isolated according to the ipact method described above to identify a baseline repertoire of neoantigen-specific T cells. PBMCs were then collected at different time points and analyzed by the imPACT signal-to-noise method to monitor the therapeutic evolution of the pool of mutant-targeted T cells. Changes in the neoantigen-specific T cell repertoire during AB122 treatment were monitored to correlate immunophenotype with clinical outcome.

Results

FIGS. 42A-C are top views showing PACT157 (FIG. 42A) and PACT132 (FIG. 42B) in the treatment of colorectal cancer; or longitudinal evolution of neoantigen-specific T cells in peripheral blood during patients with endometrial cancer PACT131 (fig. 42C).

42A-C are lower panels showing neoantigen clonality and predicted neoantigen-HLA binding affinity for each sample. The top dots represent clonal mutations, while the bottom dots represent subclonal mutations.

Figures 42A-C also show the gene, HLA type and neoantigen sequence of each TCR identified in each subject by the imPact method.

Longitudinal monitoring of patients during treatment enables analysis of T cells targeting neoantigens and identification of driver mutations associated with therapeutic benefit. In addition, monitoring changes in the repertoire of neoantigen-specific T cells in response to immunotherapy can provide information for further treatment.

Example 19 phenotypic and functional characterization of neoantigen-specific T cells from PACT131

T cells isolated from patient sample PACT131 by the imPACT separation technique method were characterized by flow cytometry for the cell surface markers CD45RA, CD95, CD39 and CD 103. Flow cytometry results for T cells isolated from patients on days 1, 15, and 57 are shown in figure 43. Black dots indicate neoantigen-specific T cells. CD45RA + CD95+ T cells undergo antigen, whereas positive CD39+ CD103+ indicates that T cells have been transported through the tumor compartment.

Next, three TCR clones (TCR200, TCR202, and TCR205) directed to the same PIK3CA neo-antigen target captured from patient samples were characterized. T cells were edited to express selected TCRs. The percentage of live, CD8+ and CD4+ T cells is shown in figure 44A. Activation of neoantigen-specific T cells was determined by incubating edited T cells with increasing amounts of HLA homologous peptides and measuring secretion of IFN γ IL2 and TNF α. Cytokine release from each TCR clone is shown in figure 44B. All T cells are activated by the cognate neoantigen. No cytokine release was detected against the non-homologous neoantigen (data not shown).

Example 20 validation of NEOTCR isolated from melanoma patient samples Using the IMPACT method

Materials and methods

comPACT library preparation

Somatic non-synonymous mutations in patient PACT135 were identified using whole exome sequencing of tumor biopsies and patient normal PBMCs and RNA-Seq transcriptome sequencing of tumor biopsies. The patient had stage IV metastatic melanoma and was undergoing treatment with an anti-PD 1 antibody to nivolumab. 2566 coding mutations were identified. 632 neo-epitopes were predicted from tumor mutation burden and 243 comPACT (neo-epitope-HLA complex) libraries were generated in HLA-A03: 01, A24: 02 and C12: 03 as described in examples 10 and 11. HLA typing was predicted by OptiType program based on patient normal PBMC whole exome sequencing. The library covered 3 of 6 HLAs.

T cell isolation

PBMC and TIL samples were collected from subject PACT135 at different time points before or during anti-PD-1 antibody treatment. PBMC samples were collected at day 14, day 43, and day 84 after initiation of treatment. TIL samples were collected on day-37 before the start of anti-PD 1 treatment and on day 82 after the start of treatment. T cells were incubated with comPACT libraries and neoantigen-specific T cells were isolated using the imPACT method described in examples 10 and 11.

Isolating 14 TCRs specific for 5 neoantigens-HLA; one neoTCR recognized PUM1, one neoTCR recognized TTP2, two neotcrs recognized IL8-HLA-a 24:01, and 10 neotcrs recognized IL8-HLA-a 03: 01. T cells expressing neoTCR were expanded for 14 days in medium containing IL2, IL7, IL15, or a combination thereof. At the end of expansion, the T cells retained the "younger" T cell phenotype, resulting in NeoTCR-P1T cells displaying T memory stem cells and T central memory cell phenotypes.

NeoTCR Gene editing

As described in international patent application No. WO2019089610, published on 9/5/2019 (herein incorporated by reference in its entirety), healthy donor-derived CD4 and CD 8T cells were engineered to express each of the identified neoantigen-specific TCRs using CRISPR-based non-viral methods.

neoTCR expression

Gene edited CD4 and CD 8T cells were analyzed for neoTCR expression using the fluorophore-comPACT trimer dextran complex. Biotinylated comPACT protein was bound to streptavidin dextramer and incubated with neoTCR CD4 and CD 8T cells. The binding of comPACT-dextramers to corresponding T cells expressing neoTCR was determined by methods described in more detail in Bethune, et al (BioTechniques 62: 123-. The dextramer was prepared by using fluorescently labeled streptavidin (Life Technologies, Carlsbad, Calif.).

Matched autologous melanoma cell line production

The matched autologous melanoma cell line was established from a biopsy of the patient (M489). As a negative control, a second cell line from mismatched melanoma was established from biopsies of different patients (M202). The functionality of T cells expressing NeoTCR (expression of activation markers, cytokine secretion, antigen-specific target cell killing and T cell proliferation) was assessed using matched and mismatched autologous melanoma cell lines.

T cell activation

T cells expressing NeoTCR were incubated with or without IFN γ and co-cultured with M489-matched melanoma tumor cell line and neoantigen-matched comPACT-dextramer. As a negative control, neoTCR T cells incubated with or without IFN γ were also co-cultured with the M202-mismatched melanoma tumor cell line and neoantigen-matched comPACT-dextramer. T cells stimulated with the anti-CD 3 antibody OKT3 served as positive controls. Internalization of NeoTCR following comPACT-dextramer binding was assessed by FACS.

Expression of the activation markers 4-1BB and OX-40 was also determined in CD4 and CD8 neoTCR T cells co-cultured with matched and mismatched melanoma cell lines. Expression of the activation marker was determined by FACS. anti-OX-40 antibodies (clone Ber-ACT35, cat #350012) and anti-4-1 BB antibodies (clone 4B4-1, cat #309810) were purchased from Biolegged.

T cell cytotoxicity assay

T cell-induced killing of tumor cells over time was monitored by immunofluorescence using the IncuCyte imaging system (Essen BioSciences). Each of the 14 neoTCR-expressing T cells was co-cultured with patient-matched M489 tumor cells. NeoTCR T cells were also co-cultured with the M202 mismatched cell line as a negative control. M489 and M202 tumor cells were labeled with NucLight Red Lentivirus (Essen BioSciences).

M489 or M202 tumor cells were seeded at 25,000 cells/well in 96-well plates and incubated overnight in an incubator. The next day neoTCR T cells were added at the following concentrations: 25,000T cells/well (1:1T cells: tumor cells ratio) or 100,000T cells/well (5:1T cells: tumor cells ratio). The co-culture formulation was then monitored by collecting time-lapse images at 2 hour intervals for 12 days using an IncuCyte imaging system with a 10X objective.

Cytokine secretion assay

Cytokine production in supernatants of co-cultured T cells and melanoma cell lines was assessed using the cytokine BEAD assay (CBA BEAD-BASED immunnoassay, BD BioSciences). CBA is a flow cytometry multiplex bead-based immunoassay application that allows for the simultaneous quantification of multiple proteins by the efficient capture of analytes using antibody-coated beads. After co-culturing the T cells and target cells for 24 or 48 hours, supernatants were collected and analyzed for secretion of IFN γ, IL-2, and TNF α.

Results

Identification of neoTCR in PACT135 over time

imPACT analysis led to the isolation of 14 TCRs specific for 5 neoantigens-HLA: one neoTCR recognizes PUM1, one neoTCR recognizes TTP2, two neoTCRs recognize IL8-HLA-A24:02, and ten neoTCRs recognize IL8-HLA-A03: 01. The sequence of the novel antigenic peptide isolated from patient PACT135, the α and β TCR CDR3 sequences, and HLA alleles are shown in table 8 below.

Figure 45 provides a summary of the number of neoantigen-specific T cells per CD 8T cells in each sample collected during anti-PD-1 antibody treatment. Each box represents one T cell and each cross represents ten T cells. Each column of boxes or crosses represents a unique neoTCR clonotype.

TCR219, TCR220, TCR223, TCR224, TCR225, TCR228, TCR229, TCR232, TCR240 and TCR241 recognize the IL8-KTYFKPFHPK neoantigen (SEQ ID NO: 256).

TCR221 and TCR227 recognize IL8-YFKPFHPKF neoantigen (SEQ ID NO: 227).

TCR218 recognizes TPP2-CFSEVSAKF neoantigen (SEQ ID NO: 223).

TCR222 recognizes the PUM1-AMMDYFFQR neo-antigen (SEQ ID NO: 226).

neoTCR expression

Figure 46 shows that the T cell gene editing efficiency of 14 neo TCRs was strong in both CD4 and CD 8T cells. For 13 neoTCR T cells, CD4 and CD 8T cells bound to homologous comPACT-dextramer complexes. However, only CD 8T cells expressing neoTCR (TCR222) directed against PUM1 bound to homologous comPACT-dextramer, and no comPACT-dextramer binding was observed in CD4 neoTCR T cells.

T cell activation

The neo TCR was internalized in coculture with the neoTCR T cell syngeneic comPACT-dextramer and the melanoma-matching cell line M489, with and without IFN γ preincubation (FIG. 47). A decrease in the percentage of comPACT-dextramer positive T cells indicates the internalization of neoTCR, which is a surrogate marker of T cell activation. Cells cultured with RPMI medium alone did not internalize the dextramer complex bound to neoTCR, whereas T cells cultured with the anti-CD 3 antibody OKT3 internalized the dextramer complex bound to neoTCR. Neo12 antigen was also used as a negative control for each sample.

neoTCR T cells derived from patient PACT135 also expressed the activation markers 4-1BB (fig. 48) and OX40 (fig. 49) after incubation with the M489 cell line (with and without IFN γ pre-incubation). No expression of 4-1BB or OX40 was observed in TCR222 CD 4T cells, as the neoTCR did not bind the cognate neoantigen.

When tumor cells were pretreated with IFN γ to activate immunoproteasome and enhance HLA expression, 4-1BB expression in IL8-HLA-a03 TCR was increased (fig. 48). OX40 expression in IL8-HLA-a03 TCR was increased when tumor cells were pretreated with IFN γ to activate immunoproteasome and enhance HLA expression (fig. 49).

T cell cytotoxicity assay

All 14T cell preparations expressing the identified neoTCR showed specific cytotoxicity against the matched autologous melanoma cell line M489 as determined by cytotoxicity assays. Figure 50 provides a graph of the percentage of tumor cells confluent after co-culture with all neoTCR T cells identified from PACT135 compared to the percentage of tumor cells confluent after mock treatment or incubation with RPMI media or neo12 TCR T cells. Figures 51A and 51B provide separate graphs of the percentage of tumor cell confluence after co-culture with each neoTCR T cell.

T cells expressing neoTCR both showed strong killing of matched tumor cells at the tested T cell to tumor cell ratios (1:1 and 5:1 (data not shown)). No cytotoxic activity was observed against the mismatched tumor cell line M202 (data not shown). The control sample had a nuclear confluence of 42% compared to less than 20% at 96 hours post incubation in each sample incubated with a 1:1 ratio of neoTCR T cells (p <0.000001 for each neoTCR T cell sample; FIG. 50 and FIGS. 51A-B). Importantly, while the number of tumor cells decreased, the number of T cells increased, indicating that the neoTCR T cells proliferated in response to the homologous antigen endogenously expressed by patient-matched tumor cells (data not shown). After two days of co-culture with the cognate tumor cells, neoTCR T cells were activated, as shown by cluster formation. T cells proliferated, by day 5 they covered the entire well surface, and no tumor cells were detected.

Even the less frequent neotcrs in PBMC or TIL samples, such as neotcrs expressed in only one T cell, were strongly active on patient-matched tumor cell lines, demonstrating the high accuracy and sensitivity of the imPACT technique.

T cell cytokine secretion

T cells expressing NeoTCR were assessed for antigen-specific cytokine production. NeoTCR T cells secrete IFN gamma, IL-2 and TNF alpha cytokines after co-culture in the presence of a patient-matched melanoma cell line M489. When neoTCR T cells were co-cultured with the mismatched melanoma cell line M202, no cytokine secretion was measured. Figure 52 shows that TCR 228T cells secrete IFN γ, IL2, and TNF α after co-culture in the presence of M489. Figure 53 shows that TCR 221T cells secrete IFN γ, IL2 and TNF α and TCR 227T cells secrete IFN γ after co-culture in the presence of M489. Figure 54 shows that TCR 222T cells secrete IFN γ and TNF α after co-culture in the presence of M489. No IL2 was detected at 48 hours. Figure 55 shows that after co-cultivation in the presence of M489 alone (black bars) and one hour of pre-treatment of M489 cells with IFN γ (grey bars), the TCR219, TCR223, TCR224, TCR225, TCR229, TCR240 and TCR 241T cells secreted IFN γ. TCR220, TCR228, and TCR 232T cells secreted IFN γ after co-culture with IFN γ -pretreated M489 cells. No IL2 or TNF α was detected at 48 hours.

Example 21 validation of NEOTCR isolated from colorectal patient samples Using the IMPACT method

Materials and methods

comPACT library preparation

144 new epitopes are predicted for the treatment of primary treatment of patients with colorectal cancerA patient. 61 comPACT (neo-epitope-HLA complex) libraries were generated in HLA-A03: 01, A02: 01, and B07: 03 as described in examples 10 and 11.

T cell isolation

PBMCs collected from subjects (PACT035) were incubated with comPACT library. Neoantigen-specific T cells were isolated using the imPACT method described in examples 10 and 11. Seven neoTCR clonotypes were identified against COX6C protein.

NeoTCR Gene editing

As described in international patent application No. WO2019089610, published on 9/5/2019 (herein incorporated by reference in its entirety), healthy donor-derived CD4 and CD 8T cells were engineered to express seven COX6C neoantigen-specific TCRs using a CRISPR-based, non-viral approach.

COX6C R20Q stable expression cell line

The precise genome engineering expertise of PACT was used to generate stable tumor cell lines that expressed the COX6C R20Q neoantigen under the control of endogenous regulatory elements. The colon cancer cell line SW620 expressing high level of cell surface HLA-A02 was used to express neoantigens. SW620 cells were nuclear transfected with gRNA/Cas9 and HDR template to make COX6C R20Q knock-in cell lines. The edited cells were subjected to a single sort and proliferation. The COX6C locus was sequenced. Sequencing analysis showed that about 80% of single cells were highly edited. Four cell lines expressing COX6C R20Q in endogenous loci were selected: SW620 cells expressing the wild type COX6C gene, SW620 cells heterozygous for the COX6C-R20Q mutation, and two SW620 cell lines homozygous for the COX6C-R20Q mutation (one shown).

Expression of HLA-a02 in the engineered SW620 cell line was confirmed by flow cytometry using BB7.2 anti-HLA a2 antibody. K562 cell lines constitutively expressing HLA-a02 and HLA-C02 were used as positive and negative controls, respectively. SW620 cells were nuclear transfected with GFP construct to confirm transfection efficiency.

T cell activation

The expression of the activation marker Nur77 was also determined in CD4 and CD8TCR089 neoTCR T cells co-cultured with SW620 cells homozygous for the COX6C-R20Q mutation. As a negative control, TCR089 neoTCR T cells were also co-cultured with wild-type SW620 cells, or cultured alone. Cells were stained with Nur77 using anti-Nur 77 mab (ebiosciences) and expression of Nur77 was assessed by flow cytometry.

T cell cytotoxicity assay, Incucyte

The NeoTCR T cell-induced killing of tumor cells over time was also determined by immunofluorescence using the IncuCyte imaging system (Essen BioSciences). T cells expressing each of the seven identified COX6C neoTCR clonotypes were used in this assay. SW620 cells homozygous for the COX6C-R20Q mutation or wild type SW620 cells were labeled Red using NucLight Red lentivirus (Essen). Labeling tumor cells with red color can distinguish them from T cells in coculture and monitor tumor cell killing over time. 40,000 tumor cells/well were seeded in 96-well plates and placed overnight in an incubator. The next day, neoTCR T cells were added at a T cell to tumor cell ratio of 1: 1. Each neoTCR T cell was added to a separate tumor cell sample. RNP (T cells electroporated with Ribonucleoprotein (RNP) complex only), neo12 TCR T cells and medium alone were used as negative controls. Co-cultured samples were monitored by collecting time-delayed images for 5 days at 2 hour intervals using an IncuCyte imaging system with a 10X objective.

T cell cytotoxicity assay, flow cytometry

TCR089 neoTCR T cells were evaluated for antigen-specific T cell-mediated killing. 100,000 TCR089 neoTCR T cells were co-cultured with SW620 cells homozygous for the COX6C-R20Q mutation (+/+) or wild type SW620 cells at different T cell to tumor cell ratios.

After co-culturing the T cells and target cells for 24 hours, the cells were stained for 20 minutes in the dark at 4 ℃ using Live/Dead cell staining kit (Live/Dead Near IR viability stain for flow, cat # NC0584313, ThermoFisher). In cells with damaged cell membranes, the dye reacts with free amines inside and on the cell surface, producing intense fluorescent staining. In living cells, the reactivity of the dye is limited to cell surface amines, resulting in lower fluorescence intensity. The difference in intensity between live and dead cells is typically greater than 50-fold, facilitating differentiation. After incubation, cells were washed, fixed with eBioscience IC fixation buffer (ThermoFisher, Cat. No. 00-8222-49) and analyzed by flow cytometry.

Cytokine secretion assay

TCR089 neoTCR T cells were co-cultured with SW620 cells homozygous for the COX6C-R20Q mutation or SW620 cells heterozygous for the COX6C-R20Q mutation at a T cell to tumor cell ratio of 5: 1. As a positive control, T cells expressing TCR089 were co-cultured with SW620 WT and treated with 1 μ M pulses for 1 hour. As a negative control, T cells expressing TCR089 were co-cultured with SW620 wild-type cells. Supernatants were collected after 24 hours and cytokine production was assessed using the cytokine BEAD assay (CBA, bed-BASED IMMUNOASSAY from BD BioSciences). CBA is a flow cytometry multiplex bead-based immunoassay application that allows for the simultaneous quantification of multiple proteins by the efficient capture of analytes using antibody-coated beads.

Results

Identification of neoTCR in PACT035

Seven neoTCR clonotypes against the COX6C protein were identified in patient samples using the imPACT T cell isolation method (fig. 56, indicated by arrows). When engineered into CD 4T cells, two of the seven bound neoantigen-HLA complexes and were therefore CD8 independent neoTCR. COX6C is a subunit of the mitochondrial enzyme cytochrome C oxidase, which is expressed at moderate levels in essentially all tissues. The neo-antigen target peptide is residues 18-20 with the R20Q mutation. Neotcrs that bind the R20Q COX6C peptide (residues 18-26) are referred to as TCR089, TCR091, TCR092, TCR094, TCR097, TCR098 and TCR 099. The sequence of the novel antigenic peptide isolated from patient PACT035, the α and β TCR CDR3 sequences, and the HLA alleles are shown in table 9 below.

Transfection of SW620 cell line

Figure 57A shows no HLA a2 expression in KV1858 cell line (expressing HLA C2, but not HLA a 2). Figure 57B shows HLA a2 expression (expression of HLA a2) in KV1832 cell lines. Figure 57C shows HLA a2 expression (expression HLA a2) in SW620 cell lines. Fig. 57D shows GFP quantification in nuclear transfected SW620 cells. Isotype control antibodies were used as negative controls.

T cell activation

Nur77 is an immediate early gene whose expression is rapidly upregulated by TCR signaling. Expression of Nur77 was rapidly upregulated by antigen-TCR signaling. Nur77 expression was detected in TCR089 neoTCR T cells co-cultured with SW620 cells homozygous for the COX6C-R20Q mutation (FIG. 58). Nur77 induction was not observed when TCR089 neoTCR T cells were cultured alone or in co-culture with SW620 cells expressing WT COX6C protein.

T cell cytotoxicity assay, IncuCyte

Target cell killing of each COX6C neoTCR T cell was also measured. All T cells expressing neoTCR showed strong killing of SW620 homozygous tumor cells (fig. 59A). No cytotoxic activity was observed against SW620 cells expressing wild-type COX6C protein (fig. 59B). The amount of T cell induced killing is dose dependent and increases with increasing T cell to tumor cell ratio.

IncuCyte images collected during the killing assay using TCR089 neoTCR T cells also showed that the number of T cells increased while the number of tumor cells decreased (data not shown). This indicates that TCR 089T cells proliferated in response to homologous antigens endogenously expressed by SW620 cells homozygous for the COX6C-R20Q mutation.

TCR 089T cell cytotoxicity assay, flow cytometry

TCR089 killed SW620 cells homozygous for the COX6C-R20Q mutation, but not wild type SW620 cells. No cytotoxic activity was observed against SW620 cells expressing wild-type COX6C protein (fig. 60). The amount of T cell-induced killing was dose-dependent and increased with increasing TCR 089T cell to tumor cell ratio.

Cytokine secretion assay

Strong IFN γ secretion was measured in TCR 089T cell samples after co-culture with SW620 cells homozygous for the COX6C-R20Q mutation (fig. 61). Half the amount of IFN γ was measured when TCR 089T cells were co-cultured with SW620 cells heterozygous for the COX6C-R20Q mutation. Similar IFN γ was detected in the absence of tumor cells, for TCR 089T cells alone, or when T cells were co-cultured with wild-type SW620 cells.

EXAMPLE 22 method of treating cancer patients with NEOTCR T cells

A patient with cancer or another proliferative disease may require interventional therapy to slow or stop cell proliferation and kill existing cells that may or do cause harm (e.g., cause pain, discomfort, or disease) to the patient. In particular, the neoTCR T cells disclosed herein can be used to treat cancer.

As shown in figure 62, Neo-E specific TCRs were isolated from patients treated with PD-1 therapy. RNA and DNA were collected from biopsies and tumor cell lines, and RNAseq and WES (whole exome sequencing) were performed on biopsies and cells. DNA was also collected from patient PBMCs for WES to use as a control. Once tumor antigens were identified using RNAseq and WES, algorithms were used to select new epitope candidates for screening using compPACT polypeptides (in which the predicted neoepitope is expressed) and imPACT isolation techniques. The barcoded comact particle library was assembled and combined with the patient sample. The comPACT particles are able to associate and capture new epitope-specific T cells.

Example 23 example of an imPACT separation technique

Based on the computational prediction of patient-specific neoE, hundreds of capture reagents were prepared, consisting of patient HLA class I subtypes loaded with the corresponding predicted neoE (Peng et al, AACR 2019); neoE-specific T cells were then isolated and TCR α and β were sequenced. Functional studies were performed on isolated neotcrs to replace endogenous TCRs by using non-viral precise genome engineering to generate primary human T cells expressing neotcrs (Jacoby et al, AACR 2019, Sennino et al, AACR 2019).

T cell responses were analyzed in two patients with metastatic melanoma who received anti-PD-1 therapy. NeoE-specific T cells were isolated from Peripheral Blood Mononuclear Cells (PBMC) and Tumor Infiltrating Lymphocytes (TIL) at different time points. Patient PT476 had a persistent response; tumor Mutational Burden (TMB) 2556; spanning 3 HLA types were generated: 243 neoE-HLA complexes of HLA-A03:01, A24:01 and C12: 03. This resulted in the isolation of 17 TCRs specific for 5 neoE-HLA. T cells specific for neoE are present in TILs at baseline and are expanded in TILs and PBMCs during treatment. Patient PT461 has rapid disease progression after anti-PD-1; TMB is 61; 78 neoE-HLA complexes covering HLA-A02:01, A03:01, B07:02, C05:01 and C07:02 were generated, resulting in the isolation of 1 neoE-HLA for 2 TCRs.

To further characterize the T cell response, the T cells were genetically edited to express 14 different TCRs isolated from patient PT476, specific for neoE in the mutated IL8, PUMl, and TPP2 genes. All 14T cell preparations showed specific cytotoxicity (50-75% inhibition of tumor growth compared to the growth of melanoma cell lines in mismatched control TCR co-cultures, 96 hours assay using P: T1: 1, P <0.000001 per comparison) against the matched autologous melanoma cell line established from patient PT476 biopsy and no cytotoxic effect against the mismatched control human melanoma cell line. After co-culture with a matched autologous melanoma cell line, neoE TCR T cells up-regulate 4-1BB and OX-40, secrete IFN γ, IL-2, and TNF α, and induce T cell proliferation and degranulation. When T cells were co-cultured with mismatched targets, no response was observed.

These results indicate that anti-PD-1 therapy induces focused neoE-specific T cell responses against a limited number of neoes, and that non-viral precise genome engineering can successfully redirect T cells to tumors expressing neoE, which can be used as a method for personalized ACT treatment.

Similarly, other checkpoint therapies and additional combinations may be used in addition to anti-PD-1 therapy; for example, anti-PD-L1 or anti-CTLA 4 therapy may be used.

Biopsies and PBMCs were collected at various time points after anti-PD-1 treatment as partially described in example 20 (FIG. 45: PT476 responders, and FIG. 63: PT461 non-responders). TILs and cell lines are established from biopsies of patients. imPACT isolation techniques were used to isolate NeoE-specific T cells and monitor their evolution over time. Whole Exome Sequencing (WES) and RNAseq from baseline cell lines were used to predict NeoE derived from non-synonymous mutations and ranked according to predicted HLA binding affinity, truncation of the mutation and expression levels. HLA-NeoE capture reagents for top ranked NeoE were used to isolate NeoE specific T cells. Fig. 45 shows PT 476: 243 neoE-HLA complexes spanning 3 HLA types (HLA-A03:01, A24:01 and C12:03) were generated and 17 TCRs specific for 5 neoE-HLA were isolated. Fig. 63 shows a patient PT 461: 78 neoE-HLA-complexes covering HLA-A02:01, A03:01, B07:02, C05:01 and C07:02 were generated, resulting in the isolation of 1 neoE-HLA for 2 TCRs.

Example 24 ImPACT separation technique example

As described in example 22, NeoE-specific T cells can be isolated from patient samples. In this example, the imPACT isolation technique resulted in the identification of 14 candidate neoTCR-ts, including: 12 IL-8(HLA-A24:02 and HLA-A03:01) neoTCR-T candidates, 1 PUM1(HLA-A3:01) neoTCR-T candidate and 1 TPP2(HLA-A24:02) neoTCR-T candidate.

As shown in fig. 62, the approach of the imPACT separation technique includes three workflows: gene editing, co-culture assays, and cell-based assays.

Gene editing: CD8 and CD 4T cells from healthy donors were precisely genomically engineered to express neoTCR. Briefly, neoE-specific TCR sequences were cloned into a Homologous Recombination (HR) DNA template. These HR templates were used to engineer primary human T cells with site-specific nucleases. Single-step (non-viral) precise genome engineering resulted in seamless replacement of the endogenous TCR with the patient's NeoE-specific TCR (native sequence), the expression of which was endogenously regulated.

Co-culture assay: NeoTCR-P1T cells were co-cultured with melanoma cell line (M489) or mismatched melanoma tumor cell line from baseline biopsy of the same patient at a 1:1 or 5:1 ratio of final product to target (P: T). Target cell killing was assessed within 6 days using the IncuCyte system. The expression of the proliferation marker Ki67 was assessed by flow cytometry at 48 hours. Expression of activation markers was assessed by flow cytometry at 24 hours. Cytokine secretion in cell supernatants was measured at 48 hours using the BD Cytokine Bead Array (CBA) human Th1/Th2 cytokine kit II.

Co-culture assay: peptide-HLA: recognition/stimulation, target cell killing, proliferation, activation markers and cytokine secretion assays were performed.

Example 25: engineered NEOTCR-T cells kill autologous tumor cells

As shown in figure 64, neoTCR-T cells killed autologous melanoma tumor cells. Using time-lapse microscopy of tumor cell death and T cell proliferation, NeoTCR-T cells were co-cultured with autologous melanoma cell lines expressing red fluorescent protein (nuclear RFP) in a stable manner. More specifically, neotcrs were made to target IL-8-HLA-a 03:01 neoantigen on melanoma cell lines and these neotcrs were cultured with IL-8-HLA-a 03:01 neoantigen melanoma cell lines (top three images in fig. 64) and compared to negative controls (bottom three images in fig. 64). The images shown here were collected at time 0 (left panel), 2 days (middle panel) and 5 days (right panel). Thus, NeoTCR is specific for tumor neoantigens and is capable of effectively killing autologous tumor cells.

The ability of neoTCR-T cells to kill autologous tumor cells can also be seen in figure 65A. NeoTCR-T cells were co-cultured with autologous (black bars) or mismatched melanoma tumor cells (white bars), and after 48 hours the percentage of CD8 NeoTCR T cells expressing Ki67 (proliferation marker) was assessed by flow cytometry. P <0.05 compared to mismatched melanoma tumor cells (t-test using Holm-Sidak method for multiple comparative correction). T cells expressing NeoTCR neo12 were used as negative controls.

Similarly, NeoTCR-T cells were shown to express activation markers when co-cultured with autologous tumor cells. This can be seen in fig. 65B. The NeoTCR-T cells were co-cultured with autologous (black bars) or mismatched tumor cell lines (white bars) and the percentage of CD8 NeoTCR T cells expressing the activation marker 4-1BB (top) or the percentage of CD4 NeoTCR T cells expressing the activation marker OX40 (bottom bar) was assessed by flow cytometry after 24 hours. P <0.05 compared to mismatched melanoma tumor cells (t-test using Holm-Sidak method for multiple comparative correction). T cells expressing NeoTCR neo12 were used as negative controls. To measure the upregulation of OX-40 in CD4 neoTCR T cells, melanoma cells were pretreated with IFN γ for 24 hours prior to co-culture with T cells.

Finally, it was shown that NeoTCR-T cells secrete interferon- γ when co-cultured with autologous tumor cells. This can be seen in fig. 65C. NeoTCR-T cells were co-cultured with autologous melanoma tumor cells and IFN γ secretion was assessed by flow Cytometry (CBA) after 48 hours. Mock T cells were used as negative controls. P <0.05 (t-test using Holm-Sidak method for multiple comparison correction).

Together, this suggests that newly generated NeoTCR-T cells expressing TCRs isolated using imPACT isolation techniques up-regulate markers of activation and proliferation when co-cultured with autologous tumor cells. More importantly, all NeoTCR-T cells specifically killed autologous melanoma cells of patient origin.

Example 26 eradication of tumors in mice and in vitro in engineered cell lines with NEOTCR-T cell administration

Tumors expressing the novel antigen were implanted in the flank of 15 NOD Scid Gamma (NSG) mice. When the tumor size reaches 95mm³When, mice were divided into two groups: control group (7 mice) which received PBS; and treatment group (8 mice) administered neoTCR T cells (5 x 10)⁶Total T cells/mouse; gene editing efficiency 50%). As shown in FIG. 66A, following infusion of NeoTCR-T cells (see FIG. C)Medium arrow), tumor size decreased, and tumors were completely eradicated by day 19. Figure 66B shows the number/mL of human CD 8T cells present in mouse blood at day 4 post neoTCR T cell infusion and day 35 post neoTCR-T cell infusion. Although NSG mice lack human cytokines, NeoTCR-T cells remain in circulation after tumor eradication, indicating that the NeoTCR-T cells not only kill target cells, but also proliferate and persist.

EXAMPLE 27 design of NEOTCR-T cells specific for mutations in Stem tumors

The subclone mutation nature of tumor progression in primary tumors and metastases raises a problem in the field of oncology, as tumor drugs are "personalized," i.e., they are designed to target proteins, chemicals, or cells with specific mutations (e.g., many small molecule drugs are designed to target tumors based on point mutations). Since tumors frequently mutate in the late stages and metastatic stages of the primary tumor (mutations accumulate during cancer growth and disease progression), drugs that shrink well or slow tumor progression when the tumor is first detected and treated may lose efficacy over time.

The ability to design and produce neoTCR-T cells specific for a mutation in a primary tumor is disclosed herein. Using algorithms and bioinformatics methods, tumor specific stem mutations expressed by all cancer cells in a patient were determined.

Example 28 NEOTCR-T cells resolve all HLA types in the Global population

There are 13,000 HLAs in the human population. Each person has a set of 6 HLAs. Thus, less than 1% of any NeoE-HLA tumor targets were identical between patients (data generated after analysis of 60,000 patients (Hartmaier et al (2017) Genome Medicine) and 20,000 patients (Schumacher & Schrieber (2015) Science.) analysis performed showed PBMC interrogated HLA allele catalogues > 99% had at least 1 HLA allele coverage, > 90% had 4 to 6 allele coverage, and > 60% of potential test subjects in the United states were expected to have all 6 allele coverage.

Example 29 NEOTCR therapy is useful for tumors with low, moderate, and high mutation loads

Since the imPACT separation technique is extremely sensitive, it is possible to detect neoantigens on tumors at all degrees of mutation load. For example, NeoTCR-T cells can be used to treat tumors with low tumor mutational load such as prostate cancer and breast cancer, tumors with moderate tumor mutational load such as ovarian cancer and colorectal cancer, and tumors with high tumor mutational load such as bladder cancer and melanoma cancer.

Thus, the imPACT isolation techniques described herein can be used to design, engineer and manufacture neotcrs for low, medium and high tumor mutation burden tumors. In certain aspects, imPACT isolation technology methods are useful for detecting neoantigens on low, medium, and high tumor mutation load tumors. In certain aspects, imPACT separation technology methods have been used to detect neoantigens on low, medium and high tumor mutation load tumors. In certain aspects, the imPACT isolation technique methods can be used to prepare compositions comprising neotcrs to treat low, medium, and high tumor mutation burden tumors in patients with such tumors. In certain aspects, the imPACT isolation technique methods can be used to prepare a NeoTCR population to treat low, medium, and high tumor mutation burden tumors in patients with such tumors.

EXAMPLE 30 method of treating patients with NEOTCR-T cell therapy

The initial step in treating a patient with NeoTCR-T cell therapy is to screen the patient. Once screened and biopsied, the patient may be enrolled and leukopheresis performed. During the manufacture of NeoTCR T cells (patient-specific) as described herein, including comPACT library creation, imPACT isolation technique screening, and T cell editing to express NeoTCR, patients may optionally participate in bridging therapy (e.g., a standard of care therapy, including first, second, third, and later line therapies for a particular cancer indication). Such bridging therapy may be prescribed and administered between 0 and 60 days (on average between 21 and 42 days). Following this optional bridging therapy, the patient may be prescribed conditional chemotherapy. This conditional chemotherapy may be performed 5, 4 and 3 days prior to administration of NeoTCR T cell therapy. On the day of administration, the patient will receive an infusion of NeoTCR. Thereafter, the tumor will be evaluated.

Example 31 compositions and methods for treating non-cancer diseases and disorders using NEOTCR T cells

Without limitation, diseases other than cancer may be treated with NeoTCR T cell therapy. In particular, any disease or disorder that results in the production of disease/disorder-specific neoantigens by a diseased cell population can be treated with NeoTCR T cell therapy. Such cells include cells infected with a virus, fungus or bacteria, which due to infection present infection-specific neoantigens that can be detected by NeoTCR T cells. Cells associated with inflammatory or autoimmune diseases may also present disease-specific neoantigens from which NeoTCR T cells may be generated. For example, if a patient suffers from an allergy or inflammatory disease, neotcrs can be prepared that are directed against a novel antigen specific for an inflammatory cytokine ligand presented on inflammatory cells.

Example 32 method of imaging Using NEOTCR T cells

Once comPACT and imPACT separation techniques are applied to a patient tumor sample, the NeoTCR T cells can be used to treat disease, as described herein, and also to image, detect and/or monitor tumor burden, progression, remission and eradication. This can be accomplished by labeling the NeoTCR T cells with a detectable label (e.g., any label that can be imaged, e.g., with a dye or with a zirconium label; see, e.g., U.S. patent No. 8,771,966, which is incorporated herein by reference in its entirety).

For example, NeoTCR T cells can be genetically modified to express a dye or fluorescent protein. Such labeled NeoTCR T cells can be used to determine the efficacy of NeoTCR T cell therapy in eradicating tumors; wherein if labeled NeoTCR T cells can be imaged to proliferate and expand, it can be concluded that the NeoTCR T cell therapy is effective because the cells differentiate into T effector cells upon encountering the target antigen.

For example, NeoTCR T cells can be labeled with an agent such as zirconium 89. Such labeled NeoTCR T cells can be used to determine the presence of any tumor (before, during, or after) NeoTCR T cell therapy based on the interaction or lack of interaction between the NeoTCR T cells and the tumor cells (if present).

Without limitation, cells other than tumor cells can also be imaged, which present neoantigens that allow the production of NeoTCR T cells. Such cells include, but are not limited to, those described in example 30. For example, if a NeoTCR T cell therapy is designed and administered to treat an inflammatory disease by using the methods described herein such that inflammatory cell-specific neo-epitopes (e.g., neo-epitopes on inflammatory cytokine ligands causing inflammation) are discovered and thus NeoTCR T cells are designed and manufactured, the same NeoTCR T cells can be labeled with an imaging agent to later determine whether ligand presenting cells are still present or whether NeoTCR is effective to eradicate or sufficiently reduce such cell populations to improve the disease state of the patient.

Example 33 methods for determining the efficacy of NEOTCR-T cell therapy and methods for adjusting the dose of NEOTCR-T cell therapy

Following administration of neoTCR-T cell therapy to a patient, efficacy can be monitored using imaging methods known in the art. For example, a patient may be infused with neoTCR-T cell therapy as described herein, followed by administration of a tumor tracer that can be imaged 1,2,3,4,5,6,7,8,9,10,11,12,13,14, or 15 days after administration of such tracer. In certain embodiments, the tracer may be administered the same week as the neoTCR-T cell therapy, one week after the neoTCR-T cell therapy, two weeks after the neoTCR-T cell therapy, three weeks after the neoTCR-T cell therapy, or one or more months after the neoTCR-T cells.

In certain embodiments, if the tumor does not progress but does not shrink after the neoTCR-T cell therapy (as observed using imaging), an additional neoTCR-T cell therapy administration may be given. In certain embodiments, additional neoTCR-T cell therapy administration may be given if the tumor does not shrink and optionally increase in size (as observed using imaging) after neoTCR-T cell therapy.

In certain embodiments, the disease state is monitored using tracers and imaging every 3-6 months following neoTCR-T cell therapy. In certain embodiments, the disease state is monitored using tracers and imaging every 6-12 months following neoTCR-T cell therapy.

Example 34 methods of treating patients with NEOTCR T cells

The novel epitope candidates were synthesized and cloned into comPACT polynucleotides as described in examples 1-9. Briefly, polynucleotide sequences encoding candidate antigenic peptides are inserted into the MHC templates depicted in figures 1-5. Mammalian cells are inoculated and transfected with a comPACT polynucleotide comprising a candidate antigenic peptide sequence. The transfected cells express and secrete comPACT polypeptide in cell culture medium. Conditioned medium from the cells was collected and the comPACT polypeptide was purified by size exclusion chromatography. The purified comPACT polypeptide is then assembled with multimeric particles (e.g., tetramers, dextramers, ntamers) comprising multiple copies of the comPACT polypeptide, DNA barcodes, and fluorophores (e.g., APCs or PEs). One of the advantages of this approach is the simultaneous high throughput production and screening of multiple new epitope candidates. Furthermore, the process can be automated. Two different types of particles (first with APC and second with PE as fluorophore) were combined to obtain a collection of particles capable of recognizing patient-specific antigenic peptides.

To identify the sequences of neoTCR T cells and their TCRs, freshly isolated or cryopreserved T cells from patients were stained with multimeric particles and a panel of antibodies for phenotypic characterization. Live and barcoded T cells were sorted into individual cells and their DNA and RNA were extracted and analyzed by next generation sequencing. Sequencing data obtained from comPACT positive T cells were analyzed as described in examples 11-13 to identify and validate the predicted antigenic peptides, neoTCR T cells and validated neoepitope TCR candidates and their sequences.

The identified neoepitope TCR sequences were cloned into a Homologous Directed Recombination (HDR) template for genome editing in T cells. Further details of the sequence and structure of the template can be found in international patent application No. PCT/US2018/058230, the contents of which are incorporated herein by reference. T cells from patients, whether freshly harvested or previously cryopreserved, are engineered to disrupt TCR genes and integrate HDR templates using non-viral methods. CRISPR/Cas9 methods comprising grnas for the endogenous loci of TCR-a and TCR- β gene sequences can be used to disrupt the endogenous TCR loci. The HDR template will recombine with one of the endogenous disrupted TCR gene sequences to introduce the identified neo-epitope TCRs. Thus, engineered T cells lack the expression of endogenous TCRs and express neoepitope TCRs. These neoTCR T cells are then adoptively transferred into the patient and specifically targeted to tumor cells expressing the neoantigen.

This process has significant advantages over other adoptive cell transfer methods, including but not limited to: i) it is flexible in that it allows personalized targeting of tumor-specific mutations presented in a patient-specific HLA context; ii) it provides a clinical tool to attack cancer cells expressing neoantigens that are not expressed on the cell surface; iii) it is effective regardless of the race or cancer type of the patient; and iv) it can be automated in multiple steps and has a small manufacturing footprint.

While the present invention has been particularly shown and described with reference to a preferred embodiment and various alternative embodiments, it will be understood by those skilled in the relevant art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.

All references, published patents and patent applications cited in the text of this specification are hereby incorporated by reference in their entirety for all purposes.