T cell receptors and modified t cells for neurodegenerative and other inflammatory disorders
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2026-02-06
- Publication Date
- 2026-08-13
Smart Images
Figure IMGF000147_0001 
Figure IMGF000045_0001_TABLE 
Figure IMGF000046_0001_TABLE
Abstract
Description
[0001] Attorney Docket No: 046483-7493WOl(04128)
[0002] T CELL RECEPTORS AND MODIFIED T CELLS FOR NEURODEGENERATIVE AND OTHER INFLAMMATORY DISORDERS
[0003] CROSS-REFERENCE TO RELATED APPLICATION
[0004] The present application is entitled to priority under 35 U. S. C. § 119(e) to U. S.
[0005] Provisional Patent Application No. 63 / 755,821 filed on February 7, 2025, which is herein incorporated by reference in its entirety.
[0006] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0007] This invention was made with government support under CA256086 and AI128949 awarded by the National Institutes of Health. The government has certain rights in the invention.
[0008] SEQUENCE LISTING
[0009] The XML file named “046483 -7493xx.xml” created on February 6, 2026, comprising 680,757 bytes, is hereby incorporated by reference in its entirety.
[0010] BACKGROUND
[0011] Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two devastating adult-onset neurodegenerative diseases which can co-occur and are considered to be part of one disease spectrum, ALS / FTD. In ALS, neuronal loss affects primarily upper and lower motor neurons (MNs), leading to a rapidly progressive deterioration of muscle function and ultimately death due to respiratory failure. In FTD, frontal and temporal cortical neurons are preferentially lost, inducing cognitive impairment with language and behavioral changes.
[0012] Highly differentiated and clonal CD8+T cell populations have recently been described as being enriched in ALS, FTD, inclusion body myositis (IBM), Alzheimer's disease (AD), and other neurodegenerative conditions. These cell populations exist both in the peripheral blood mononuclear cells (PBMCs) and in disease relevant tissue such as cerebrospinal fluid (CSF) and skeletal muscle. T cell clonal expansion in addition to the increased expression of proteins associated with activation and cytotoxicity supports an antigen-specific T cell response however,Attorney Docket No: 046483-7493WOl(04128)
[0013] no clear antigenic targets have been described, and attempts at finding them have largely been unsuccessful.
[0014] One shared pathological hallmark between ALS and IBM is the aggregation and nuclear depletion of TAR DNA-binding protein-43 (TDP-43). TDP-43 is a ubiquitously expressed RNA binding protein that resides mostly in the nucleus, where it has been shown to be a repressor of cryptic exons, intronic sequences that are normally not spliced into mRNA, but become derepressed upon TDP-43 mislocalization and included in mature mRNA. Although most of these events generate frameshifts and premature stop codons leading to RNA degradation, some cryptic exons can be translated to form cryptic peptides in the brain of patients with ALS. TDP-43 loss of function leads to the generation of such cryptic peptides, which are foreign to the human immune system as the encoding cryptic exons do not exist in the human thymus, raising the possibility that T cells recognizing these peptides are retained.
[0015] Inasmuch as T cells can recognize novel peptides derived from non-canonical reading frame translation in cancer cells, there is a need in the art to engineer T cells that can mount an effective response against cells expressing cryptic epitopes for improving clinical outcomes in TDP-43 proteinopathies, especially immunotherapy-based treatment strategies that target TDP-43 proteinopathies otherwise resistant to existing treatment strategies.
[0016] SUMMARY
[0017] The present disclosure relates to compositions and methods useful for generating a modified T cell comprising polynucleotides encoding a T cell receptor (TCR) specific for a cryptic epitope associated with a TAR DNA-binding protein-43 (TDP-43) proteinopathy. Also provided are methods and pharmaceutical compositions comprising modified T cells for use in adoptive therapy for the treatment of TDP-43 associated proteinopathies, particularly neurodegenerative conditions.
[0018] In one aspect, the disclosure provides isolated nucleic acid(s) encoding a T cell receptor (TCR), the nucleic acid(s) comprising a first polynucleotide encoding a TCRa (TRA) chain polynucleotide and a second polynucleotide encoding a TCRβ (TRB) chain polypeptide wherein TRA and TRB have an antigenic specificity for a cryptic epitope presented in a complex with a human MHC I polypeptide, wherein:Attorney Docket No: 046483-7493WOl(04128)
[0019] a) the epitope is a cryptic epitope of HDGFL2 comprising the amino acid sequence of SEQ ID NO: 7, the MHC I is HLA-B*08:01, the TRA comprises a third complementarity determining region (CDR3) comprising the amino acid sequence of SEQ ID NO: 3, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 6;
[0020] b) the epitope is a cryptic epitope of HDGFL2 comprising the amino acid sequence of SEQ ID NO: 10, the MHC I is HLA-A*03:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 8, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 9;
[0021] c) the epitope is a cryptic epitope of HDGFL2 comprising the amino acid sequence of SEQ ID NO: 13, the MHC I is HLA-A*03:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 11, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 12;
[0022] d) the epitope is a cryptic epitope of HDGFL2 comprising the amino acid sequence of SEQ ID NO: 13, the MHC I is HLA-A*03:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 14, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 15;
[0023] e) the epitope is a cryptic epitope of HDGFL2 comprising the amino acid sequence of SEQ ID NO: 13, the MHC I is HLA-A*03:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 16, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 17;
[0024] f) the epitope is a cryptic epitope of HDGFL2 comprising the amino acid sequence of SEQ ID NO: 13, the MHC I is HLA-A*03:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 18, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 19;
[0025] g) the epitope is a cryptic epitope of HDGFL2 comprising the amino acid sequence of SEQ ID NO: 13, the MHC I is HLA-A*03:*03:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 20, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 21;
[0026] h) the epitope is a cryptic epitope of HDGFL2 comprising the amino acid sequence of SEQ ID NO: 13, the MHC I is HLA-A*03:*03:01, the TRA comprises a CDR3 comprising theAttorney Docket No: 046483-7493WOl(04128)
[0027] amino acid sequence of SEQ ID NO: 22, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 23;
[0028] i) the epitope is a cryptic epitope of HDGFL2 comprising the amino acid sequence of SEQ ID NO: 13, the MHC I is HLA-A*03:*03:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 24, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 25;
[0029] j) the epitope is a cryptic epitope of HDGFL2 comprising the amino acid sequence of SEQ ID NO: 13, the MHC I is HLA-A*03: *03:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 26, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 27;
[0030] k) the epitope is a cryptic epitope of HDGFL2 comprising the amino acid sequence of SEQ ID NO: 13, the MHC I is HLA-A*03:*03:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 28, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 29;
[0031] l) the epitope is a cryptic epitope of HDGFL2 comprising the amino acid sequence of SEQ ID NO: 13, the MHC I is HLA-A*03:*03:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 30, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 31;
[0032] m) the epitope is a cryptic epitope of IgLON5 comprising the amino acid sequence of SEQ ID NO: 34, the MHC I is HLA-B*35:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 32, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 33;
[0033] n) the epitope is a cryptic epitope of IgLON5 comprising the amino acid sequence of SEQ ID NO: 34, the MHC I is HLA-B*35:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 35, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 36;
[0034] o) the epitope is a cryptic epitope of IgLON5 comprising the amino acid sequence of SEQ ID NO: 43, the MHC I is HLA-A*03: *03:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 39, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 42;Attorney Docket No: 046483-7493WOl(04128)
[0035] p) the epitope is a cryptic epitope of IgLON5 comprising the amino acid sequence of SEQ ID NO: 34, the MHC I is HLA-B*35:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 44, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 45;
[0036] q) the epitope is a cryptic epitope of IgLON5 comprising the amino acid sequence of SEQ ID NO: 34, the MHC I is HLA-B*35:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 46, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 47;
[0037] r) the epitope is a cryptic epitope of IgLON5 comprising the amino acid sequence of SEQ ID NO: 34, the MHC I is HLA-B*35:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 48, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 49;
[0038] s) the epitope is a cryptic epitope of IgLON5 comprising the amino acid sequence of SEQ ID NO: 34, the MHC I is HLA-B*35:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 50, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 51;
[0039] t) the epitope is a cryptic epitope of IgLON5 comprising the amino acid sequence of SEQ ID NO: 34, the MHC I is HLA-B*35:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 52, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 53;
[0040] u) the epitope is a cryptic epitope of IgLON5 comprising the amino acid sequence of SEQ ID NO: 34, the MHC I is HLA-B*35:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 54, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 55;
[0041] v) the epitope is a cryptic epitope of IgLON5 comprising the amino acid sequence of SEQ ID NO: 34, the MHC I is HLA-B*35:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 56, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 57;
[0042] w) the epitope is a cryptic epitope of IgLON5 comprising the amino acid sequence of SEQ ID NO: 60, the MHC I is HLA-A*02:01*, the TRA comprises a CDR3 comprising theAttorney Docket No: 046483-7493WOl(04128)
[0043] amino acid sequence of SEQ ID NO: 58, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 59;
[0044] x) the epitope is a cryptic epitope of IgLON5 comprising the amino acid sequence of SEQ ID NO: 34, the MHC I is HLA-B*35:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 61, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 62;
[0045] y) the epitope is a cryptic epitope of IgLON5 comprising the amino acid sequence of SEQ ID NO: 362, the MHC I is HLA- B*35:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 588, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 589;
[0046] z) the epitope is a cryptic epitope of ZNF423 comprising the amino acid sequence of SEQ ID NO: 491, the MHC I is HLA- A*02:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 562, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 563;
[0047] aa) the epitope is a cryptic epitope of EPB41L4A comprising the amino acid sequence of SEQ ID NO: 292, the MHC I is HLA- A*01:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 564, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 565;
[0048] bb)the epitope is a cryptic epitope of ARHGAP22 comprising the amino acid sequence of SEQ ID NO: 246, the MHC I is HLA- B*40:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 580, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 581;
[0049] cc) the epitope is a cryptic epitope of PTPRZ1 comprising the amino acid sequence of SEQ ID NO: 418, the MHC I is HLA- A*02:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 582, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 583; or
[0050] dd)the epitope is a cryptic epitope of PTPRZ1 comprising the amino acid sequence of SEQ ID NO: 418, the MHC I is HLA- A*02:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 584, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 585.Attorney Docket No: 046483-7493WOl(04128)
[0051] In an embodiment, the isolated nucleic acid(s) comprises at least one non-naturally occurring nucleotide or encode at least one non-naturally occurring amino acid substitution.
[0052] In an embodiment, the TRA and TRB have antigenic specificity for a cryptic epitope of HDGFL2.
[0053] In an embodiment, the cryptic HDGFL2 epitope comprises the amino acid sequence of SEQ ID NO: 10 and the MHC I is HLA-B*03:*03:01. In one embodiment, the cryptic HDGFL2 epitope-binding TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 8, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 9.
[0054] In another embodiment, the cryptic HDGFL2 epitope comprises the amino acid sequence of SEQ ID NO: 13 and the MHC I is HLA-B*03:01. In certain embodiments, the cryptic HDGFL2 epitope-binding TRA comprises a CDR3 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 11, 14, 16, 18, 20, 22, 24, 26, 28, and 30, and the TRB comprises a CDR3 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 12, 15, 17, 19, 21, 23, 25, 27, 29, and 31.
[0055] In another embodiment, the cryptic HDGFL2 epitope comprises the amino acid sequence of SEQ ID NO: 7 and the MHC I is HLA-B*08:*08:01. In certain embodiments, the cryptic HDGFL2 epitope comprises the amino acid sequence of SEQ ID NO: 7, the MHC I is HLA-B*08:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 6. In an embodiment, the TRA further comprises a first complementarity determining region (CDR1) comprising the amino acid sequence of SEQ ID NO: 1 and a second complementarity determining region (CDR2) comprising the amino acid sequence of SEQ ID NO: 2, and the TRB further comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 4 and a CDR2 comprising the amino acid sequence of SEQ ID NO: 5.
[0056] In an embodiment, the cryptic HDGFL2 epitope-binding TRA comprises a TCRa chain variable domain (TRAV or V alpha) comprising at least 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 63. In another embodiment, the TRAV comprises the amino acid sequence of SEQ ID NO: 63. In another embodiment, the TRA comprises a TRAV comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 63 and a TCRa chain constant region (TRAC) comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,Attorney Docket No: 046483-7493WOl(04128)
[0057] 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 65. In another embodiment, the TRA comprises a TRAV comprising the amino acid sequence of SEQ ID NO: 63 and a TRAC comprising the amino acid sequence of SEQ ID NO: 65. In another embodiment, the TRA comprises a TCRa chain comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 67. In another embodiment, the TCRa chain comprises the amino acid sequence of SEQ ID NO: 67.
[0058] In an embodiment, the cryptic HDGFL2 epitope-binding TRB comprises a TCRβ chain variable domain (TRBV or V beta) comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 64. In one embodiment, the TRBV comprises the amino acid sequence of SEQ ID NO: 64. In another embodiment, the TRB comprises a TRBV comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 64 and a TCRβ chain constant region (TRBC) comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 66. In one embodiment, the TCR chain comprises a TRAV comprising the amino acid sequence of SEQ ID NO: 63 and a TRAC comprising the amino acid sequence of SEQ ID NO: 66. In an embodiment, the TRB comprises a TCRβ chain comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 68. In one embodiment, the TCRβ chain comprises the amino acid sequence of SEQ ID NO: 68.
[0059] In an embodiment, the first polynucleotide encoding the TRA and the second polynucleotide encoding the TRB are separated by a third polynucleotide encoding a linker.
[0060] In an embodiment, the third polynucleotide comprises the nucleotide sequence of SEQ ID NO: 106 and / or encodes an amino acid sequence of SEQ ID NO: 105.
[0061] In an embodiment, the first and second polynucleotides are present in a TCRα / β single chain DNA construct. In another embodiment, the first and second polynucleotides are present in a TCRβ / α single chain DNA construct. In one embodiment, the TCRβ / α single chain DNA construct comprises a nucleotide sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to SEQ ID NO: 78 and / or encoding an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%,Attorney Docket No: 046483-7493WOl(04128)
[0062] 97%, 98%, 99%, 99.9%, or 100% sequence identity to SEQ ID NO: 69. In another embodiment, the TCRβ / α single chain DNA construct comprises the nucleotide sequence of SEQ ID NO: 78 and / or encodes the amino acid sequence of SEQ ID NO: 69. In another embodiment, the TCRβ / α single chain DNA construct encoding an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to any one of SEQ ID NOs: 722, 724, 725, 730-733, 741, 745, 748, 749, or 751. In another embodiment, the TCRβ / α single chain DNA construct encodes an amino acid sequence comprising the amino acid sequence of any one of SEQ ID NOs: 722, 724, 725, 730-733, 741, 745, 748, 749, or 751.
[0063] In some embodiments, the TRA and TRB have antigenic specificity for a cryptic epitope of IgLON5.
[0064] In an embodiment, the cryptic IgLON5 epitope comprises the amino acid sequence of SEQ ID NO: 34 and the MHC I is HLB-B*35:01. In certain embodiments, the cryptic IgLON5 epitope comprises the amino acid sequence of SEQ ID NO: 34, the MHC I is HLB-B*35:01, the TRA comprises a CDR3 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 32, 35, 44, 46, 48, 50, 52, 54, 56, 61, and 589, and the TRB comprises a CDR3 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 33, 36, 45, 47, 49, 51, 53, 55, 57, 62, and 589.
[0065] In another embodiment, the cryptic IgLON5 epitope comprises the amino acid sequence of SEQ ID NO: 60 and the MHC I is HLB-A*02:01. In one embodiment, the cryptic IgLON5 epitope comprises the amino acid sequence of SEQ ID NO: 60, the MHC I is HLB-A*02:01, and the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 58 and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 59.
[0066] In another embodiment, the cryptic IgLON5 epitope comprises the amino acid sequence of SEQ ID NO: 43 and the MHC I is HLA-A*03:01. In certain embodiments, the cryptic IgLON5 epitope comprises the amino acid sequence of SEQ ID NO: 43, the MHC I is HLA-A*03:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 39, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 42. In an embodiment, the TRA further comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 37 and a CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and the TRB further comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 40 and a CDR2 comprising the amino acid sequence of SEQ ID NO: 41.Attorney Docket No: 046483-7493WOl(04128)
[0067] In another embodiment, the cryptic IgLON5 epitope-binding TRA comprises a TRAV comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 79. In another embodiment, the TRAV comprises the amino acid sequence of SEQ ID NO: 79. In another embodiment, the TRA comprises a TRAV comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 79 and a TRAC comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 81. In another embodiment, the TRA comprises a TRAV comprising the amino acid sequence of SEQ ID NO: 79 and a TRAC comprising the amino acid sequence of SEQ ID NO: 81. In another embodiment, the TRA comprises a TCRot chain comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 83. In one embodiment, the TCRα chain comprises the amino acid sequence of SEQ ID NO: 83.
[0068] In another embodiment, the cryptic IgLON5 epitope-binding TRB comprises a TCRP chain comprising a TRBV comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 80. In one embodiment, the TRBV comprises the amino acid sequence of SEQ ID NO: 80. In another embodiment, the TRB comprises a TRBV comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 80 and a TRBC comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 82. In one embodiment, the TCRβ chain comprises a TRAV comprising the amino acid sequence of SEQ ID NO: 80 and a TRAC comprising the amino acid sequence of SEQ ID NO: 82. In an embodiment, the TRB comprises a TCRβ chain comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 84. In one embodiment, the TCRβ chain comprises the amino acid sequence of SEQ ID NO: 84.
[0069] In an embodiment, the first polynucleotide encoding the TRA and second polynucleotide encoding the TRB are separated by a third polynucleotide encoding a linker. In an embodiment,Attorney Docket No: 046483-7493WOl(04128)
[0070] the third polynucleotide comprises the nucleotide sequence of SEQ ID NO: 106 and / or encodes the amino acid sequence of SEQ ID NO: 105.
[0071] In an embodiment, the first and second polynucleotides are present in a TCRα / β single chain DNA construct. In another embodiment, the first and second polynucleotides are present in a TCRβ / α single chain DNA construct. In one embodiment, the TCRp / a single chain construct comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 94 and / or encodes an amino acid sequence comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 85, 726, 727, 734-740, 742-744, 746, 747, 750, or 752.
[0072] In some embodiments, the TRA and TRB have antigenic specificity for a cryptic epitope of ZNF423. In an embodiment, the cryptic ZNF423 epitope comprises the amino acid sequence of SEQ ID NO: 491 and the MHC I is HLA-A*02:01. In one embodiment, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 562, and the TRB comprises a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 563. In another embodiment, the TRA comprises a TRAV having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 653. In another embodiment, the TRB comprises a TRBV having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 686. In some embodiments, the TRA comprises a TRAC having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 65 and the TRB comprises a TRBC having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 66. In another embodiment, the TCR having antigenic specificity for the cryptic epitope of ZNF423 comprises a TCRp / a single chain construct comprising at least about 90%, 91%, 92%, 93%>, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 720.
[0073] In some embodiments, the TRA and TRB have antigenic specificity for a cryptic epitope of EPB41L4A. In an embodiment, the cryptic EPB41L4A epitope comprises the amino acid sequence of SEQ ID NO: 292 and the MHC I is HLA-A*01:01. In one embodiment, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 564, and the TRBAttorney Docket No: 046483-7493WOl(04128)
[0074] comprises a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 565. In another embodiment, the TRA comprises a TRAV having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 654 and the TRB comprises a TRBV having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 687. In some embodiments, the TRA comprises a TRAC having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 65 and the TRB comprises a TRBC having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 66. In another embodiment, the TCR having antigenic specificity for the cryptic epitope of EPB41L4A comprises a TCRp / a single chain construct comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 721.
[0075] In some embodiments, the TRA and TRB have antigenic specificity for a cryptic epitope of ARHGAP22. In an embodiment, the cryptic EPB41L4A epitope comprises the amino acid sequence of SEQ ID NO: 246 and the MHC I is HLA-B*40:01. In one embodiment, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 580, and the TRB comprises a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 581. In another embodiment, the TRA comprises a TRAV having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 656 and the TRB comprises a TRBV having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 689. In some embodiments, the TRA comprises a TRAC having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 65 and the TRB comprises a TRBC having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 718. In another embodiment, the TCR having antigenic specificity for the cryptic epitope of EPB41L4A comprises a TCRp / a single chain construct comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 723.Attorney Docket No: 046483-7493WOl(04128)
[0076] In some embodiments, the TRA and TRB have antigenic specificity for a cryptic epitope of PTPRZ1. In an embodiment, the cryptic EPB41L4A epitope comprises the amino acid sequence of SEQ ID NO: 418 and the MHC I is HLA-A*02:01. In one embodiment, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 582 or 584, and the TRB comprises a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 583 or 585. In another embodiment, the TRA comprises a TRAV having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 661 or 662 and the TRB comprises a TRBV having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 694 or 695. In some embodiments, the TRA comprises a TRAC having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 65 and the TRB comprises a TRBC having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 718. In another embodiment, the TCR having antigenic specificity for the cryptic epitope of PTPRZ1 comprises a TCRp / a single chain construct comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 728 or 729.
[0077] In another aspect, the disclosure provides a recombinant expression vector comprising a nucleic acid encoding a TCR as disclosed herein.
[0078] In another aspect, the disclosure provides a T cell receptor encoded by an expression vector comprising one or more nucleic acids encoding a TCR as disclosed herein. In some embodiments, the TCR is a CD8-independent TCR.
[0079] In another aspect, the disclosure provides a modified immune cell comprising a nucleic acid encoding a TCR as disclosed herein. The modified immune cell of claim 45, wherein the immune cell is selected from the group consisting of TCR-Treg (CD4+), TCR-Treg / Tsuppressor (CD8⁺), TCR-TyS, TCR-NK, and TCR-regulatory NK cells. In some embodiments, the immune cell is a T cell. In some embodiments, the T cell is obtained from the group consisting of peripheral blood mononuclear cells, cord blood cells, a purified population of T cells, and a T cell line. In one embodiment, the T cell is a CD4+T cell, a CD4+T reg cell, a CD8+T cell, a CD8+Treg cell, or a CD8+ suppressor cell.Attorney Docket No: 046483-7493WOl(04128)
[0080] In an embodiment, the T cell further comprises a modified endogenous genetic locus. In one embodiment, the modified endogenous genetic locus comprises TCRα chain and / or TCRβ chain polynucleotide sequence(s). In some embodiments, the modified endogenous genetic locus comprises deletion(s) of the TCRa chain and / or TCRp chain polynucleotide sequence(s). In some embodiments, the modified endogenous genetic locus comprises insertion(s) in the TCRa chain and / or TCRP chain polynucleotide sequence! s). In other embodiments, expression from the modified endogenous genetic locus reduces or eliminates expression of the TCRa chain and / or TCR chains. In some embodiments, the modification is accomplished by use of a CRISPR system.
[0081] In another aspect, the disclosure provides a method for generating a modified T cell as described herein. In one embodiment, the method comprises introducing into the T cell a nucleic acid encoding a TCRa chain and / or TCRp chain described herein or a recombinant expression construct encoding a TCRa chain and / or TCRp chain described herein such that the modified T cell expresses a TCR having antigen specificity for a cryptic epitope presented in a complex with a human MHC I polypeptide.
[0082] In some embodiments of the method, the TCR specifically binds a cryptic epitope of HDGFL2. In one embodiment, the HDGFL2 cryptic epitope comprises an amino acid sequence set forth in SEQ ID NO: 7, 10, or 13.
[0083] In other embodiments of the method, the TCR specifically binds a cryptic epitope of IgLON5. In one embodiment, the cryptic epitope comprises an amino acid sequence set forth in SEQ ID NO: 34, 43, or 60.
[0084] In an embodiment of the method, the modified T cell is derived from a biological sample obtained from a patient blood collection or healthy patient donation. In some embodiments, the biological sample is obtained from the group consisting of peripheral blood mononuclear cells, cord blood cells, a purified population of T cells, and a T cell line. In certain embodiments, the T cell is a CD8+cell, CD8+Treg cell, a CD8+ suppressor cell, or a suppressor T cell. In one embodiment, the biological sample comprises PBMCs enriched for CD8+ T cells.
[0085] In some embodiments, the method further comprises the step of modifying expression of endogenous TCRa and / or TCRP chains. In one embodiment, the step of modifying expression of the endogenous TCRa and / or TCR chains is accomplished by use of a CRISPR system.Attorney Docket No: 046483-7493WOl(04128)
[0086] In another aspect, a method for stimulating a T cell-mediated immune response in a subject in need thereof comprises administering to the subject an effective amount of a modified T cell described herein.
[0087] In an embodiment, the subject has a TDP-43 proteinopathy. In exemplary embodiments, the TDP-43 proteinopathy is selected from the group consisting of amyotrophic lateral sclerosis (ALS), inclusion body myositis (IBM), frontotemporal dementia (FTD), frontotemporal lobar degeneration (FTLD), motor neuron disease (MND), Alzheimer’s disease (AD), Parkinson's disease (PD), Huntington’s disease, chronic traumatic encephalopathy (CTE), facial onset sensory and motor neuronopathy (FOSMN), limbic-predominant age-related TDP-43 encephalopathy (LATE), primary progressive aphasia (PPA), Perry disease, Guam parkinsonismdementia, and a Lewy body-related disease.
[0088] In another aspect, a method of treating a subject with a TDP-43 proteinopathy comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a modified T cell described herein and a pharmaceutically acceptable carrier or excipient. In some embodiments, the TDP-43 proteinopathy is selected from the group consisting of amyotrophic lateral sclerosis (ALS), inclusion body myositis (IBM), frontotemporal dementia (FTD), frontotemporal lobar degeneration (FTLD), motor neuron disease (MND), Alzheimer’s disease (AD), Parkinson's disease (PD), Huntington’s disease, chronic traumatic encephalopathy (CTE), facial onset sensory and motor neuronopathy (FOSMN), limbic-predominant age-related TDP-43 encephalopathy (LATE), primary progressive aphasia (PPA), Perry disease, Guam parkinsonism-dementia, and a Lewy body-related disease.
[0089] In some embodiments, the method further provides a comprising administering to the subject one or more additional therapeutic agents. In certain embodiments, the additional therapeutic agent is selected from the group consisting of chemotherapy, chimeric-antigen receptor (CAR)-T cell therapy, monoclonal antibody therapy, biologic therapy, allogeneic stem cell transplant, radiologic therapy, and any combination thereof.
[0090] In another aspect, the disclosure provides a pharmaceutical composition comprising a modified T cell as disclosed herein and a pharmaceutically acceptable carrier.
[0091] In another aspect, the disclosure provides a modified immune cell comprising a T cell receptor that specifically binds a cryptic epitope set forth in Table 3 or Table 4.
[0092] In another aspect, the disclosure provides a T cell receptor set forth in Table 6.Attorney Docket No: 046483-7493WOl(04128)
[0093] In another aspect, the disclosure provides a method for stimulating a cell-mediated immune response in a subject in need thereof, comprising administering to the subject an effective amount of a modified immune cell comprising a T cell receptor that specifically binds a cryptic epitope set forth in Table 3 or Table 4.
[0094] In another aspect, the disclosure provides a method of treating a subject with a TDP-43 proteinopathy, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a modified immune cell comprising a T cell receptor set forth in Table 6.
[0095] In another aspect, the disclosure provides an assay comprising: culturing a biological sample comprising T cells from a subject, wherein the T cells are aliquoted into a plurality of wells; separately stimulating the T cells in each of the plurality of wells with each peptide from a pool of overlapping peptides encompassing a polypeptide of interest under conditions suitable for expansion of the T cells; optionally re-stimulating the T cells in each well with the same peptide to which the T cells in the well were previously exposed; and exposing the T cells in each well to one or more detection reagents suitable for detecting one or more activation-induced T cell markers, wherein detection of the one or more activation-induced T cell markers in a well indicates the presence of a TCR comprising an antigen-specificity for one or more peptides in the pool to which the T cells in the well were exposed.
[0096] In some embodiments, the protein of interest is encoded by a gene selected from the group consisting of AARS, ACTL6B, ARHGAP22, CELSR3, DNM1, EPB41L4A, HDGFL2, IGLON5, MYO18A, NECAB2, PHF2, PTPRZ1, PXDN, SLC24A2, STMN2, SYNJ2, XPO4, and ZNF423.
[0097] In some embodiments, the subject has or is suspected of having a TDP-43 proteinopathy. In some embodiments, the TDP-43 proteinopathy is indicative of the subject having a heterogenous neurodegenerative and inflammatory condition selected from the group consisting of amyotrophic lateral sclerosis (ALS), inclusion body myositis (IBM), frontotemporal dementia (FTD), frontotemporal lobar degeneration (FTLD), motor neuron disease (MND), Alzheimer’s disease (AD), Parkinson's disease (PD), Huntington’s disease, chronic traumatic encephalopathy (CTE), facial onset sensory and motor neuronopathy (FOSMN), limbic-predominant age-related TDP-43 encephalopathy, primary progressive aphasia (PPA), Perry disease, Guam parkinsonism-Attorney Docket No: 046483-7493WOl(04128)
[0098] dementia, and a Lewy body-related disease. In certain embodiments, the TDP-43 proteinopathy is ALS or IBM.
[0099] In some embodiments, the biological sample is from a patient blood collection or healthy patient donation. In some embodiments, the biological sample is selected from the group consisting of whole blood, PBMCs, or cryopreserved PBMCs. In certain embodiments, the biological sample comprises PBMCs enriched for CD8+ cells.
[0100] In some embodiments, the overlapping pool of peptides are between about 10-40 amino acids in length, optionally between about 20-30 amino acids in length. In some embodiments, each peptide in the overlapping pool comprises an overlap with an adjoining peptide of between about 3-10 amino acids in length.
[0101] In some embodiments, the one or more detection reagents are suitable for detecting CD69 and CD 137.
[0102] In some embodiments, the T cells are further cultured in the presence of an antigen-presenting cell (APC) selected from the group consisting of autologous PBMC APCs, dendritic cells, B cells, and engineered APC cell lines.
[0103] In some embodiments, the assay further comprises the step of isolating T cells from the well in which the one or more activation-induced T cell markers were detected.
[0104] In another aspect, the disclosure provides a modified T cell produced by the foregoing assay.
[0105] In a further aspect, the disclosure provides a method of detecting a TDP-43 proteinopathy-associated immune signature in a subject, comprising:
[0106] (a) contacting T cells obtained from a biological sample from the subject with a panel of peptide–HLA multimers presenting a plurality of cryptic epitopes;
[0107] (b) enriching or isolating multimer-positive T cells by cell sorting;
[0108] (c) determining a normalized multimer-positive frequency score based on multimer-positive T cells relative to input T cells and / or relative to a negative-control multimer; and (d) classifying the subject as positive for the TDP-43 proteinopathy-associated immune signature when the normalized multimer-positive frequency score satisfies a predetermined criterion.Attorney Docket No: 046483-7493WOl(04128)
[0109] In some embodiments, the plurality of cryptic epitopes comprises cryptic epitopes derived from at least 10, at least 20, at least 30, or at least 50 proteins associated with a TDP-43 proteinopathy.
[0110] In some embodiments, the cryptic epitopes are selected from proteins encoded by genes selected from the group consisting of AARS, ACTL6B, ARHGAP22, CELSR3, DNM1, EPB41L4A, HDGFL2, IGLON5, MYO18A, NECAB2, PHF2, PTPRZ1, PXDN, SLC24A2, STMN2, SYNJ2, XPO4, and ZNF423.
[0111] In some embodiments, the multimers comprise tetramers, dextramers, pentamers, or streptamers.
[0112] In some embodiments, the method further comprises sequencing nucleic acids from the multimer-positive T cells to obtain TCRa and / or TCRp sequences. In some embodiments, the sequencing comprises single-cell RNA sequencing and the method further comprises determining a phenotype metric of the multimer-positive T cells comprising an effector / cytotoxic state, exhaustion state, activation state, memory / differentiation state, and / or expression of cytotoxic mediators and inflammatory cytokines.
[0113] In some embodiments, the method further comprises determining a clonality metric selected from clone size, clonal expansion, clonal enrichment, repertoire diversity, and / or presence of public / shared clonotypes.
[0114] In some embodiments, the method further comprises performing an activation-induced marker (AIM) assay by contacting T cells from the subject with an overlapping peptide library spanning one or more proteins comprising cryptic epitopes and measuring a frequency of AIMpositive T cells. In some embodiments, the method further comprises AIM-positive T cells are CD69+CD137+T cells. In some embodiments, the AIM assay output comprises ΔAIM relative to a matched negative control condition.
[0115] In some embodiments, the method further comprises isolating AIM-positive T cells and determining TCRa and / or TCRp sequences from the isolated AIM-positive T cells.
[0116] In some embodiments, the overlapping peptide library comprises peptides having a length of about 3 to about 40 amino acids. In some embodiments, adjacent peptides in the library are offset by a step size of about 1 to about 40 amino acids.
[0117] In some embodiments, the biological sample is selected from the group consisting of whole blood, PBMCs, cryopreserved PBMCs, leukapheresis products, buffy coat, cerebrospinalAttorney Docket No: 046483-7493WOl(04128)
[0118] fluid (CSF), lymph node aspirate or biopsy, muscle biopsy, tissue-resident immune cells from a biopsy or surgical specimen, and engineered or transduced T cells or cell lines expressing one or more TCRs.
[0119] In some embodiments, the classification in step (d) comprises performing an early disease diagnosis. In some embodiments, the classification in step (d) comprises patient stratification. In some embodiments, the patient stratification comprises distinguishing (i) Alzheimer’s disease without TDP-43 pathology from (ii) Alzheimer’s disease with concomitant TDP-43 pathology based on one or more of the normalized multimer-positive frequency score, clonality metric, phenotype metric, and / or AIM output
[0120] In some embodiments, the method further comprises repeating steps (a)-(d) at a subsequent time point and using a change in the normalized multimer-positive frequency score, clonality metric, phenotype metric, AIM output, or any combination thereof to monitor disease progression and / or monitor therapeutic effect.
[0121] BRIEF DESCRIPTION OF THE DRAWINGS
[0122] The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings.
[0123] FIGs. 1A-1F: Highly expanded cryptic T cells are enriched in the PBMCs of patients with inclusion body myositis (IBM) and amyotrophic lateral sclerosis (ALS). (A) Schematic of the experimental workflow. Validated cryptic peptides were used to informatically predict binding to patient HLA molecules. Fluorescent pMHC tetramer capture probes were barcoded and used to sort cryptic peptide specific CD8+T cells for downstream single cell analysis and in vitro characterization (top). Muscle tissue analysis overview depicting RNA sequencing, proteomics, and IHC approaches (bottom). (B) Volcano plot showing upregulated splicing junctions upon TDP-43 knockdown in iPSC-derived skeletal muscles (circles) and iPSC-derived glutamatergic cortical neurons (triangles). (C) Representative FACS plot depicting cryptic epitope Tetramer cells (x-axis) and control viral Tetramer cells (y-axis) across three donors. (D) Summary of the complete data represented in (C). Cryptic epitope cell frequency was normalized by the number of total tetramers for each individual donor. Wilcoxon Rank Sum test was used to evaluate significance. (E) T cell clonality per donor. Clones were stratified byAttorney Docket No: 046483-7493WOl(04128)
[0124] CDR3P amino acid according to the following bins: Singleton = 1, Small = 3, Medium = 10, Large = 15, Hyperexpanded = 100. (F) Violin plot of clone size as assigned by CDR3P amino acid across ALS, IBM, and healthy donors. The Wilcoxon Rank Sum test was used to evaluate significance.
[0125] FIGs. 2A-2F: TDP-43 dysfunction leads to a heterogenous and polyclonal CD8+T cell response to cryptic epitopes. (A) Box plot visualization of tetramer UMI counts for a selection of clones with putative specificities to HDGFL2 (top) and IgLON5 (bottom) epitopes. Each dot depicts an individual cell’s signal for each tetramer barcode (x-axis) within the specified clone. All tetramer barcodes were grouped into their parent cryptic protein except for the top hit (represented by the red box). (B) Depiction of identified cryptic epitopes across HDGFL2 and IgLON5. Epitopes found to be targeted are marked by a bar which spans the epitope sequence. The tick marks within each bar represent different clones targeting the same epitope and the distance within each tick represents the relative clone size. The color intensity represents the sum of all clones targeting the particular epitope. (C) Donut graph of the putative cryptic epitope specificities summarized by donor. (D) UMAP representation of single cells by a weighted nearest neighbor calculation of gene and surface protein expression. Clusters were manually annotated based on their differential gene and protein expression. Genes are italicized. (E) Percent discovery of cryptic T cells as measured by the number of cryptic epitope tetramer cells sorted from the total number of starting CD8+T cells. The bar graph is segmented by each group’s phenotype composition. (F) UMAP overlay of cryptic T cell clones.
[0126] FIGs. 3A-3D: T cells with engineered cryptic TCRs can bind and activate in response to cryptic epitopes. (A) TCR:tetramer binding of TCR-14 targeting the HDGFL2 epitope, FGKGHSGM (SEQ ID NO: 7), and TCR-30 targeting the IgLON5 epitope, SSLSAWCQLHR (SEQ ID NO: 43) (B) Flow cytometry histogram plots of %CD69 upregulation following 18 hour incubation in a cryptic epitope tetramer coated plate across the following conditions: no pMHC coated on the plate, pp65 (irrelevant) pMHC coated on the plate, irrelevant TCR cocultured with the matched cryptic epitope pMHC, matched TCR co-cultured with the matched cryptic epitope pMHC. Results for TCR-14 (top row) and TCR-30 (bottom row) are shown. (C / D) Evaluation of tetramer binding and activation of TCR-14 (C) and TCR-30 (D) across various possible proteosomically cleaved versions of their identified cognate cryptic epitopes.Attorney Docket No: 046483-7493WOl(04128)
[0127] The values shown on the heatmaps are background subtracted based on an irrelevant pp65-specific TCR.
[0128] FIGs. 4A-4D: HDGFL2 cryptic peptide specific CD8+T cells can efficiently kill TDP-43 deficient astrocytes. (A) siRNA knockdown and HDGFL2 cryptic exon PCR strategy diagram. (B) Tapestation trace of final HDGFL2 cryptic PCR product across control and TDP-43 knockdown. (C) Depiction of CCF-STTG1-GFP and CCF-STTG1-GFP- 2M-shRNA (HLA-negative) cell death across time as measured by GFP expression. Three T cell conditions across control and siRNA TDP-43 knockdown were measured. CD8-TCR-14: primary activated CD8s transduced to express TCR- 14 and enriched via flow cytometry to be 100% tetramer positive using HDGFL2.17 tetramer, CD8-UT: primary activated and untransduced CD8s from matching donor, No T cell: no T cells were added to the co-culture. The Kolmogorov-Smirnov (two-sided) test was performed to evaluate whether there are differences in the means of the curves between CD8-TCR-14 and CD8-UT for both CCF cell lines. There was a significant difference in the parental cell line (p = 8.305e-07) and no significant difference in the HLA-deficient line (p = 0.5041). (D) representative images of the CCF-STTG1-GFP: CD8-TCR-14 co-culture across TDP-43 (top) and control (bottom) siRNA knockdown. The red mask marks cells undergoing cell death as indicated by caspase 3 / 7 dye.
[0129] FIGs. 5A-5E: Cryptic peptides and MHC I / TCR pathways are found in IBM tissues. (A) Visualization of seriate immunohistochemistry staining for TDP-43, p62, and HDGFL2 cryptic peptide in muscle from one control and two IBM cases. For each case, arrows show colocalization of HDGFL2 cryptic peptide in fibers with p62 and TDP-43 nuclear depletion and / or aggregation. (B) Semi-quantitative scores for immune infiltrates, TDP-43 loss, p62 and HDGFL2 cryptic peptide in control and IBM cases. (C) Proteomics normalized read counts for TDP-43, TCR constant chains (TRBC), CD3E, HLA-A, -B, -C, B2M, and CALR, TAP1 / 2, and ERAP 1 / 2 (MHC I processing pathway). Statistics and boxplots are between cases with high or low HDGFL2 cryptic peptide staining in the IHC, while color is based on disease conditions. (D) Normalized counts of RNA-seq reads covering cryptic exon junctions in controls (n = 15) and IBM (n = 25) cases. (E) Correlation between RNA-seq normalized read counts for TCR constant chains (TRAC, TRBC1, and TRBC2), CD3E, HLA-A, HLA-B, HLA-C, and 02-microglobulin (B2M) and cryptic exon normalized counts in controls (n = 15) and IBM cases (n = 25).Attorney Docket No: 046483-7493WOl(04128)
[0130] FIGs. 6A-6B: Cryptic epitope specific CD8+T cells are clonally expanded and polyclonal. (A) CD8+T cell clonality from cryptic epitope tetramer sorted cells per donor as donut plots. Clones were stratified by CDR3β amino acid according to the following bins:
[0131] Singleton = 1, Small = 3, Medium = 10, Large = 15, Hyperexpanded = 100. Percentages mark clonally expanded cells of size 3 or greater. (B) Chord plots of TRAV and TRBV chain usage of clones binding HDGFL2.11 from donor ALS-UP-2 (left) and IgLON5.48 from donor ALS-UP-5 (right). Clones within each chord plot are assigned a distinct color. Clones missing a paired TRBV or TRAV were removed from the visualization. The arc length is proportional to the relative clone size.
[0132] FIGs. 7A-7B: RNA and surface protein expression show distinct cell states. (A) The top five differentially expressed genes from each seurat cluster. Clusters were downsampled to 300 cells for visualization. (B) Overlay of Abseq surface protein expression across 16 markers.
[0133] FIGs. 8A-8B: Differential expression analysis. (A) Differential expression analysis of cryptic CD8+T cells in ALS / IBM vs. healthy controls. Genes with an absolute value log2 fold change of at least 0.5 and a bonferroni-corrected p-value of 0.05 or less are colored blue (downregulated in ALS / IBM) or red (upregulated in ALS / IBM). (B) Differential expression analysis of TDP-43 siRNA vs. scramble siRNA treated CCF-STTG1 cells. Genes with an absolute value log2 fold change of at least 1 and a bonferroni-corrected p-value of 0.05 or less are colored blue (downregulated in TDP-43 siRNA) or red (upregulated in TDP-43 siRNA). The TDP-43 coding gene, TARDBP, is marked by a red star symbol (log2FC = -1.26).
[0134] FIGs. 9A-9B: TCR similarity analysis reveals conserved clonotypes across patients and datasets. (A) Schematic overview of datasets used to perform GLIPH2 analysis (top). The table shows the GLIPH2 clusters that were retained through the following cluster-wise filtering parameters: Levenshtein distance of CDR3α and CDR3β ≤ 1 alongside a TRBV allele match between at least one TCR from the datasets in literature compared to at least one CE-specific TCRs identified in this paper. (B) Alignment of the TCR identified from ALS-UP-3 and ALS-4 (Campisi, L. et al., Nature 606, 945-952 (2022)) in addition to ALS-UP-5 and MCI7 (Gate, D. et al. Nature 577, 399-404 (2020)). Matched and mismatched amino acids are identified.
[0135] FIGs. 10A-10B: TCR-14 epitope recognition is restrictive and limited to the discovered core epitope. (A) Tetramer binding (top) and percent CD69 expression (bottom) of TCR-14 to its core epitope, FGKGHSGM (SEQ ID NO: 7), derived from HDGFL2 cryptic peptide, in additionAttorney Docket No: 046483-7493WOl(04128)
[0136] to N- and C-terminus extensions of the larger cryptic exon (top). CMV pp65 recognizing TCR used as control. (B) Tetramer binding (top) and percent CD69 expression (bottom) of TCR-14 to N- and C-terminus reductions of the core epitope.
[0137] FIGs. 11A-11B: TCR-30 is able to tolerate epitope variations. (A) Tetramer binding (top) and percent CD69 expression (bottom) of TCR-30 to its discovered epitope, SSLSAWCQLHR (SEQ ID NO: 43) derived from IgLON5 cryptic peptide in addition to N- and C-terminus reductions, including (left to right) AWCQLHR (SEQ ID NO: 143), SAWCQLHR (SEQ ID NO: 99), LSAWCQLHR (SEQ ID NO: 144), SLSAWCQLHR (SEQ ID NO: 145), SSLSAWCQLHR (SEQ ID NO: 43), SSLSAWCQLH (SEQ ID NO: 146), SSLSAWCQL (SEQ ID NO: 147), SSLSAWCQ (SEQ ID NO: 148), and SSLSAWC (SEQ ID NO: 149). (B) Tetramer binding (top) and percent CD69 expression (bottom) of TCR-30 to sliding window and N- and C-terminus extensions of the discovered epitope, including (left to right) AWCQLHR (SEQ ID NO: 143), SAWCQLHR (SEQ ID NO: 99), LSAWCQLHR (SEQ ID NO: 144), SLSAWCQLHR (SEQ ID NO: 145), SSLSAWCQLHR (SEQ ID NO: 43), SSLSAWCQLH (SEQ ID NO: 146), SSLSAWCQL (SEQ ID NO: 147), SSLSAWCQ (SEQ ID NO: 148), SSLSAWC (SEQ ID NO: 149), and CMV pp65 epitope, NLVPMVATV (SEQ ID NO: 97) recognizing TCR used as control.
[0138] FIGs. 12A-12C: Cryptic CD8+T cells kill TDP-43 depleted astrocyte cell line. (A) Representative FACS plots of naive CD45+CCR7+CD4 and CD8 cells sorted for the IncuCyte longitudinal imaging experiment performed in (B). (B) Depiction of CCF-STTG1-GFP cell death across time as measured by GFP expression. Seven T cell conditions across control and siRNA TDP-43 knockdown were measured. CD4 / CD8-UT-naive cells are naive CD45RA+CCR7+cells sorted from PBMCs as shown in (A). CD4 / CD8-UT cells are CD4 or CD8 negatively enriched T cells that were activated using Immunocult. CD4 / CD8-TCR-14 are CD4 or CD8 negatively enriched T cells that were activated using Immunocult and subsequently transduced with TCR-14. TCR-14 transduced cells were not TCR enriched as was done in FIG. 4B. The Kolmogorov-Smirnov test was performed to evaluate whether there are differences in the means of the curves between CD8-UT and CD8-TCR-14 (p=0.04141). (C) Flow cytometry expression of HLA-B*08 between CCF-STTG1-GFP and CCF-STTG1-GFP-p2M-shRNA cell lines used in FIG. 4B and FIG. 12B.Attorney Docket No: 046483-7493WOl(04128)
[0139] FIGs. 13A-13B: All three IBM donors have large clones recognizing CMV pp65 epitope. (A) Heatmap visualization of tetramer UMI counts for a selection of viral-specific clones with putative specificities to CMV pp65 (NLVPMVATV, SEQ ID NO: 97) and SARS-CoV-2 spike epitope (KCYGVSPTK, SEQ ID NO: 98). Each individual map represents the signal for each tetramer barcode (y-axis) across individual cells within a clonotype (x-axis). Corresponding donor and CMVpp65 epitope-binding TCR CDR30 sequences are noted above each heatmap, which include (left to right) CASSPSTGASYGYTF (SEQ ID NO: 150), CASSMSPGPYEQYF (SEQ ID NO: 151), CASSYSNVGALTDTQYF (SEQ ID NO: 152), CASTSTAGYPGELFF (SEQ ID NO: 153), and CASSYATGTSYGYTF (SEQ ID NO: 154). (B) UMAP overlay of selected viral-specific CD8+T cell clones, including (left to right) CASSPSTGASYGYTF (SEQ ID NO: 150), CASTSTAGYPGELFF (SEQ ID NO: 153), CASSYSNVGALTDTQYF (SEQ ID NO: 152), CASSYATGTSYGYTF (SEQ ID NO: 154), and CASSMSPGPYEQYF (SEQ ID NO: 151).
[0140] FIGs. 14A-14E: Additional data from RNA-seq analysis. (A). IGV tracks for MYO18A cryptic exon in a subset of controls and IBM muscle biopsy RNA-seq. (B) PCA for the full RNA-seq cohort. The clear split between two groups in PCI is likely driven by different library preparation methods. (C) Heatmap showing gene expression of genes with cryptic peptides in skeletal muscle and frontal cortex. Expression data obtained from ASCOT(26) (D) IGV tracks for STMN2 cryptic exon in a subset of controls and IBM muscle biopsy RNA-seq. (E) RNA-seq normalized read counts for TCR constant chains (TRAC, TRBC1, and TRBC2), CD3E, HLA-A, HLA-B, HLA-C, and β2-microglobulin (B2M) in control and IBM cases.
[0141] FIGs. 15A-15E: Additional data from the tissue proteomics. (A) MHC-I and -II antigen processing pathway expression in controls and IBM cases, divided by condition and colored based on the upper or lower 50% by TDP-43 levels. (B) Normalized count distribution for the proteomics cohort. (C) GO analysis summary from the proteomics analysis. (D) PCA plot from normalized protein expression from the proteomics experiment. (E) PCA for the NHNN RNA-seq cohort. Note how the PCA from proteomics and that from RNA are fairly overlapping.
[0142] FIGs. 16A-16B: Functional study with TCR-14 on astrocytes (CCF-STTG1) transduced with an HDGFL2 tandem minigene. (A) Tandem mini-gene targets, T cells, and astrocyte targets in assay. (B)Attorney Docket No: 046483-7493WOl(04128)
[0143] FIGs. 17A-17D: Activation-induced marker (AIM) assays using a separate ALS and IBM cohort. (A) Schematic of assay. (B) Representative gating strategy for assay. (C) Representative FACS analysis of CD69+CD137+CD8+T cells % across disease and peptide conditions. (D) Summary of CD69+CD137+readout across ALS, IBM and HC cohorts under HDGFL2, IGLON5, and CEF peptide pool stimulation.
[0144] FIGs. 18A-18B: Validation of in vitro TDP-43 deficient astrocyte killing. (A) Flow cytometry validation of MHC-I manipulation: CCF-STTG1-GFP astrocytes were engineered to separately control (i) HLA restriction element (HLA-A2, HLA-A3, or HLA-B8) and (ii) presence of the HDGFL2 tandem minigene (TMG) antigen cassette versus an irrelevant TMG control. Representative histogram overlays show successful and allele-specific surface expression of HLA-A2, HLA-A3, or HLA-B8 following lentiviral transduction, along with robust β2-microglobulin (β2m) expression consistent with increased total MHC class I complex availability. (B) Functional cytotoxicity distinguishes antigen-specific killing from nonspecific HLA upregulation: The same engineered CCF-STTG1-GFP targets were co-cultured with primary T cells expressing an HDGFL2-epitope-specific TCR (TCR-14) or an irrelevant TCR control, with a No-T condition as baseline. Across multiple “high MHC-I” conditions — including IFNy treatment and forced HLA expression without cognate antigen did not drive substantial GFP loss, indicating that MHC-I upregulation alone is insufficient to trigger killing. In contrast, marked cytotoxicity was observed specifically when the cognate HDGFL2 TMG antigen was present and paired with the permissive restricting allele HLA-B8.
[0145] FIGs. 19A-19B: Evaluation of HDGFL2.17 presentation on a more neuronal cell line neuroblastoma SK-N-BE(2). (A) SK-N-BE(2) neuroblastoma cells were used as a neuronal-like target to test whether the HDGFL2.17 epitope can be processed / presented and recognized by HDGFL2.17-specific T cells. Cells were engineered with the appropriate restricting HLA (absent endogenously) and transduced with either an HDGFL2 tandem minigene (TMG) or an irrelevant TMG control. Longitudinal IncuCyte readout (GFP area normalized to T=0, No-T) shows selective cytotoxicity by TCR-14 T cells only against HDGFL2-TMG targets, with no appreciable killing in WT or irrelevant-TMG targets, or with irrelevant TCR controls — supporting antigen-dependent recognition in this neuronal-like context. (B). Effector program associated with HDGFL2.17-specific recognition: Following co-culture, T cells were harvested for intracellular phenotyping of cytokines (IFNy, TNFα, IL-2) and cytotoxic mediators (GZMK,Attorney Docket No: 046483-7493WOl(04128)
[0146] GZMB). Representative histograms show that antigen-matched conditions (TCR-14 + HDGFL2-TMG) are associated with a pronounced increase in Granzyme B relative to non-killing controls.
[0147] DETAILED DESCRIPTION
[0148] Definitions
[0149] As used herein, the term “autologous” is meant to refer to any material originating from the same individual to which it is later to be re-introduced into the individual.
[0150] “Allogeneic” refers to a graft derived from a different animal of the same species.
[0151] “Xenogeneic” refers to a graft derived from an animal of a different species.
[0152] As used herein, the term “conservative sequence modifications” is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the TCRs of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR regions of the TCR can be replaced with other amino acid residues from the same side chain family and the altered antibody can be tested for the ability to bind antigens using the functional assays described herein.
[0153] “Co-stimulatory ligand,” as the term is used herein, includes a molecule on an antigen presenting cell (e.g., an aAPC, dendritic cell, B cell, and the like) that specifically binds a cognate co-stimulatory molecule on a T cell, thereby providing a signal which, in addition to the primary signal provided by, for instance, binding of a TCR / CD3 complex with an MHC molecule loaded with peptide, mediates a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. A co-stimulatory ligand can include, but isAttorney Docket No: 046483-7493WOl(04128)
[0154] not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, an agonist or antibody that binds Toll ligand receptor and a ligand that specifically binds with B7-H3. A co-stimulatory ligand also encompasses, inter alia, an antibody that specifically binds with a co-stimulatory molecule present on a T cell, such as, but not limited to, CD27, CD28, 4-1BB, 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83.
[0155] A “co-stimulatory molecule” refers to the cognate binding partner on a T cell that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the T cell, such as, but not limited to, proliferation. Co-stimulatory molecules include but are not limited to an MHC class I molecule, BTLA and a Toll ligand receptor.
[0156] A “co-stimulatory signal”, as used herein, refers to a signal, which in combination with a primary signal, such as TCR / CD3 ligation, leads to T cell proliferation and / or upregulation or downregulation of key molecules.
[0157] The term “derived from” refers to being generated, synthesized, or originating from a particular source, such that the derived matter is related to the source. The derived matter does not need to be identical to the particular source. In one embodiment, an antigen is derived from a protein. In another embodiment, a single-chain variable fragment is derived from a monoclonal antibody.
[0158] The term “dextramer”, as used herein, refers to peptide-MHC (pMHC) complexes attached to a dextran backbone (a branched polysaccharide) carrying many more pMHC molecules than tetramers, thereby creating high avidity to T cell receptors. Because they present multiple pMHCs at once, dextramers bind low-affinity TCRs more efficiently and can detect rare or weakly reactive T-cell populations.
[0159] A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate. In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.Attorney Docket No: 046483-7493WOl(04128)
[0160] “Effective amount” or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result or provides a therapeutic or prophylactic benefit. Such results may include, but are not limited to, anti-tumor activity as determined by any means suitable in the art.
[0161] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
[0162] As used herein “endogenous” refers to any material from or produced inside an organism, cell, tissue or system.
[0163] As used herein, the term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue or system.
[0164] The term “expand” as used herein refers to increasing in number, as in an increase in the number of T cells. In one embodiment, the T cells that are expanded ex vivo increase in number relative to the number originally present in the culture. In another embodiment, the T cells that are expanded ex vivo increase in number relative to other cell types in the culture. The term "ex vivo," as used herein, refers to cells that have been removed from a living organism, (e.g., a human) and propagated outside the organism (e.g., in a culture dish, test tube, or bioreactor).
[0165] The term “expression” as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.
[0166] “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.Attorney Docket No: 046483-7493WOl(04128)
[0167] Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.
[0168] The term “human leukocyte antigen” or “HLA” as used herein refers to the cell surface protein complexes responsible for the presentation of antigenic epitopes, typically peptide epitopes, to CD4+and CD8+T cells. HLA complexes comprise two main classes: class I, which are expressed by most cells and present mostly intracellular antigens to CD8+T cells and consist of a monomeric polypeptide alpha chain complexed with a non-epitope-binding protein called β2microglobulin (B2M); and class II, which presents mostly extracellular antigens to CD4+T cells and consists of a hetero dimer of an alpha and beta chains. The genes encoding HLA proteins are highly polymorphic which allow them to present a wide variety of antigens.
[0169] “Identity” as used herein refers to the subunit sequence identity between two polymeric molecules particularly between two amino acid molecules, such as, between two polypeptide molecules. When two amino acid sequences have the same residues at the same positions, e.g., if a position in each of two polypeptide molecules is occupied by an Arginine, then they are identical at that position. The identity or extent to which two amino acid sequences have the same residues at the same positions in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions; e.g., if half (e.g., five positions in a polymer ten amino acids in length) of the positions in two sequences are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 of 10), are matched or identical, the two amino acids sequences are 90% identical.
[0170] The term “immune response” as used herein is defined as a cellular response to an antigen that occurs when lymphocytes identify antigenic molecules as foreign and induce the formation of antibodies and / or activate lymphocytes to remove the antigen.
[0171] The phrases “an immunologically effective amount”, “an anti-immune response effective amount”, “an immune response-inhibiting effective amount”, or “therapeutic amount” refer to the amount of the composition of the present invention to be administered to a subject which amount is determined by a physician, optionally in consultation with a scientist, in consideration of individual differences in age, weight, immune response, type of disease / condition, and the health of the subject (patient) so that the desired result is obtained in the subject.Attorney Docket No: 046483-7493WOl(04128)
[0172] “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
[0173] A “lentivirus” as used herein refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses offer the means to achieve significant levels of gene transfer in vivo.
[0174] A “major histocompatibility complex molecule” (MHC molecule) refers to a glycoprotein that delivers a peptide antigen to a cell surface. MHC class I molecules are heterodimers consisting of a membrane spanning a chain and a non-covalently associated 2 microglobulin. MHC class II molecules are composed of two transmembrane glycoproteins, a and 0, both of which span the membrane. Each chain has two domains. MHC class I molecules deliver peptides originating in the cytosol to the cell surface, where the peptide: MHC complex is recognized by CD8+T cells. MHC class II molecules deliver peptides originating in the vesicular system to the cell surface, where they are recognized by CD4+T cells. An MHC molecule may be from various animal species, including human, mouse, rat, or other mammals.
[0175] The phrase “MHC-peptide tetramer staining" refers to an assay used to detect antigenspecific T cells, which features a tetramer of MHC molecules, each comprising an identical peptide having an amino acid sequence that is cognate (e.g., identical or related to) to at least one antigen, wherein the complex is capable of binding T cells specific for the cognate antigen. Each of the MHC molecules may be tagged with a biotin molecule. Biotinylated MHC / peptides are tetramerized by the addition of streptavidin, which is typically fluorescently labeled. The tetramer may be detected by flow cytometry via the fluorescent label. The fluorescent label, or fluorophore, may be phycoerythrin (PE), allophycocyanin (APC), PE-Cy5, PE-Cy7, APC, APC-Cy7, Qdot® 565, Qdot® 605, Qdot® 655, Qdot® 705, Brilliant® Violet (BV) 421, BV 605, BV 510, BV 711, BV786, PerCP, PerCP / Cy5.5, AlexaFluor® 488, AlexaFluor® 647, FITC, BV570, BV650, DyLight® 488, Dylight® 649, PE / Dazzle® 594.Attorney Docket No: 046483-7493WOl(04128)
[0176] By the term “modified” as used herein, is meant a changed state or structure of a molecule or cell of the invention. Molecules may be modified in many ways, including chemically, structurally, and functionally. Cells of the present invention may be modified by the introduction of nucleic acids, or cellular activation or differentiation.
[0177] By the term “modulating,” as used herein, is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and / or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and / or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human.
[0178] In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.
[0179] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s). Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.
[0180] The term “operably linked” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.
[0181] The term “overexpressed” tumor antigen or “overexpression” of a tumor antigen is intended to indicate an abnormal level of expression of a tumor antigen in a cell from a diseaseAttorney Docket No: 046483-7493WOl(04128)
[0182] area like a solid tumor within a specific tissue or organ of the patient relative to the level of expression in a normal cell from that tissue or organ. Patients having solid tumors, or a hematological malignancy characterized by overexpression of the tumor antigen can be determined by standard assays known in the art.
[0183] “Parenteral” administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrastemal injection, or infusion techniques.
[0184] The term “polynucleotide” as used herein is defined as a chain of nucleotides.
[0185] Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR™, and the like, and by synthetic means.
[0186] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
[0187] The term “peptide antigen” refers to an amino acid sequence, ranging from about 7 amino acids to about 25 amino acids in length that is specifically recognized by a TCR, or binding domains thereof, as an antigen, and which may be derived from or based on a fragment of aAttorney Docket No: 046483-7493WOl(04128)
[0188] longer target biological molecule (e.g., polypeptide, protein) or derivative thereof. An antigen may be expressed on a cell surface, within a cell, or as an integral membrane protein. An antigen may be a host-derived (e.g., tumor antigen, autoimmune antigen) or have an exogenous origin (e.g., bacterial, viral).
[0189] The term “pentamer” as used herein refers to 5 MHC class I molecules, each loaded with the same peptide antigen and bound together around a central scaffold. In some embodiments, the pentamer is fluorescently labeled.
[0190] The term “promoter” as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.
[0191] As used herein, the term “promoter / regulatory sequence” means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter / regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter / regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.
[0192] A “constitutive” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.
[0193] An “inducible” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell.
[0194] A “signal transduction pathway” refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell. The phrase “cell surface receptor” includes molecules and complexes of molecules capable of receiving a signal and transmitting signal across the plasma membrane of a cell. An example of a “cell surface receptor” is human FSHR.
[0195] “Similarity” as used herein, refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both ofAttorney Docket No: 046483-7493WOl(04128)
[0196] the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are similar at that position. The similarity between two sequences is a direct function of the number of matching or similar positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are similar, the two sequences are 50% similar; if 90% of the positions (e.g., 9 of 10), are matched or similar, the two sequences are 90% similar.
[0197] By the term “specifically binds,” as used herein with respect to an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen.
[0198] However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.
[0199] By the term “stimulation,” is meant a primary response induced by binding of a stimulatory molecule (e.g., a TCR / CD3 complex) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR / CD3 complex. Stimulation can mediate altered expression of certain molecules, such as downregulation of TGF-beta, and / or reorganization of cytoskeletal structures, and the like.
[0200] A “stimulatory molecule,” as the term is used herein, means a molecule on a T cell that specifically binds with a cognate stimulatory ligand present on an antigen presenting cell.
[0201] A “stimulatory ligand,” as used herein, means a ligand that when present on an antigen presenting cell (e.g., an aAPC, a dendritic cell, a B-cell, and the like) can specifically bind with a cognate binding partner (referred to herein as a “stimulatory molecule”) on a T cell, therebyAttorney Docket No: 046483-7493WOl(04128)
[0202] mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, and the like. Stimulatory ligands are well-known in the art and encompass, inter alia, an MHC Class I molecule loaded with a peptide, an anti-CD3 antibody, a super agoni)st anti-CD28 antibody, and a super agonist anti-CD2 antibody.
[0203] A ’’streptamer,” as used herein refers to a reversible MHC multimer used to detect, isolate, and study antigen-specific T cells. Streptamers bind T-cell receptors and are made of peptide-MHC (pMHC) complexes (pMHC) that are linked together via streptavidin using a reversible binding system. Streptamers can be removed after staining by adding e.g., biotin, which disrupts the streptaviding interaction. After removal, the T cell is left functionally unaltered.
[0204] The term “subject” is intended to include living organisms in which an immune response can be elicited (e.g., mammals). A “subject” or “patient,” as used therein, may be a human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. Preferably, the subject is human.
[0205] As used herein, a “substantially purified” cell is a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state. In some instances, a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cells that have been separated from the cells with which they are naturally associated in their natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.
[0206] As used herein, the term “tetramer,” refers to a 4 MHC molecules, each loaded with the same peptide antigen. In some embodiments, the tetramer is linked together via streptavidinbiotin chemistry and / or conjugated to a fluorophore.
[0207] A “T cell” as used herein denotes a lymphocyte that is maintained in the thymus and has either an a:[B or y:8 heterodimeric receptor.” Naive T cells have not encountered specific antigens and T cells are naive when leaving the thymus. Naive T cells are identified as CD45RO-, CD45RA+, and CD62L+Memory T cells mediate immunological memory to respond rapidly on re-exposure to the antigen that originally induced their expansion and can be “CD8+” (T cytotoxic cells) or “CD4+” (T helper cells). Memory CD4 T cells are identified as CD4+, CD45RCC cells and memory CD8 cells are identified as CD8+CD45RO+In some aspects,Attorney Docket No: 046483-7493WOl(04128)
[0208] “precursor T cells” refers to cells found in individuals without an immune response to antigen targets. The antigen targets may be HIV-specific T cells in healthy HIV negative blood donors or pre-proinsulin- specific T cells in healthy blood donors who are not diabetic.
[0209] As used herein, the term “T cell receptor” or “TCR” refers to a complex of membrane proteins that participate in the activation of T cells in response to the recognition of an antigen. The TCR, in association with CD3, is generally sponsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. The TCR is composed of a heterodimer of an alpha (a) and beta (P) chain, although in some cells the TCR is composed of gamma and delta (y / 6) chains. TCRs may exist in alpha / beta and gamma / delta forms, which are structurally similar but have distinct anatomical locations and functions. Each chain of the TCR is a member of the immunoglobulin superfamily and possesses one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminal end.
[0210] TCRs are described using the International Immunogenetics (IMGT) TCR nomenclature, and links to the IMGT public database of TCR sequences. Native alpha-beta heterodimeric TCRs have an alpha chain and a beta chain. Broadly, each chain comprises variablejoining and constant regions, and the beta chain also usually contains a short diversity region between the variable and joining regions, but this diversity region is often considered as part of the joining region. Each variable region comprises three CDRs (Complementarity Determining Regions) embedded in a framework sequence, one being the hypervariable region named CDR3. There are several types of alpha chain variable (Va) regions and several types of beta chain variable (VP) regions distinguished by their framework, CDR1 and CDR2 sequences, and by a partly defined CDR3 sequence. The Va types are referred to in IMGT nomenclature by a unique TRAV number. Thus “TRAV21” defines a TCR Va region having unique framework and CDR1 and CDR2 sequences, and a CDR3 sequence which is partly defined by an amino acid sequence which is preserved from TCR to TCR but which also includes an amino acid sequence which varies from TCR to TCR. In the same way, “TRBV5-1” defines a TCR VP region having unique framework and CDR1 and CDR2 sequences, but with only a partly defined CDR3 sequence.
[0211] The joining regions of the TCR are similarly defined by the unique IMGT TRAJ and TRBJ nomenclature, and the constant regions by the IMGT TRAC and TRBC nomenclature.Attorney Docket No: 046483-7493WOl(04128)
[0212] The beta chain diversity region is referred to in IMGT nomenclature by the abbreviation TRBD, and, as mentioned, the concatenated TRBD / TRBJ regions are often considered together as the joining region.
[0213] The a and 0 chains of αβ TCR's are generally regarded as each having two “domains”, namely variable and constant domains. The variable domain consists of a concatenation of variable region and joining region. In the present specification and claims, the term “TCR alpha variable domain” therefore refers to the concatenation of TRAV and TRAJ regions, and the term TCR alpha constant domain refers to the extracellular TRAC region, or to a C-terminal truncated TRAC sequence. Likewise, the term “TCR beta variable domain” refers to the concatenation of TRBV and TRBD / TRBJ regions, and the term TCR beta constant domain refers to the extracellular TRBC region, or to a C-terminal truncated TRBC sequence.
[0214] In some embodiments, the TCR may be modified on any cell comprising a TCR, including, for example, a helper T cell, a cytotoxic T cell, a regulatory T cell, a memory T cell, regulatory T cell, natural killer T cell, and gamma delta T cell. A TCR of this disclosure can be “immunospecific” or capable of binding to a desired degree, including “specifically or selectively binding” a target while not significantly binding other components present in a test sample.
[0215] A “target site” or “target sequence” refers to a genomic nucleic acid sequence that defines a region of a nucleic acid to which a binding molecule may specifically bind under conditions sufficient for binding to occur.
[0216] The term “therapeutic” as used herein means a treatment and / or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.
[0217] A “tissue-specific” promoter is a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.
[0218] The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.Attorney Docket No: 046483-7493WOl(04128)
[0219] To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.
[0220] The phrase “under transcriptional control” or “operatively linked” as used herein means that the promoter is in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.
[0221] A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.
[0222] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
[0223] Description
[0224] The invention of the present disclosure is based on the discovery of T cell receptors (TCRs) which are specific for cryptic epitopes from HDGFL2 or IgLON5 that are presented in complexes with human MHC I in patients with TDP-43 -associated proteinopathies. As such, in certain aspects, the present disclosure includes recombinant T cell receptor (TCR) alpha (a) and beta (P) chains which are specific for cryptic epitopes from HDGFL2 or IgLON5. Also included are isolated nucleic acids encoding HDGFL2 or IgLON5-specific TCR alpha and beta chains. InAttorney Docket No: 046483-7493WOl(04128)
[0225] certain aspects, the invention also includes TCR compositions and methods for generating modified T cells expressing a TCR specific for a cryptic epitope of HDGFL2 or IgLON5. In other aspects, the invention includes modified T cells and compositions comprising modified T cells which comprise recombinant, HDGFL2 or IgLON5-specific TCR alpha and beta chains, which, in some embodiments, also comprise deletions in endogenous TCR-encoding genetic loci as well as methods for generating the modified T cells. The present disclosure further provides methods for treating TDP-43 -associated proteinopathies which comprise administering effective amounts of modified T cells expressing the HDGFL2- or IgLON5-specific TCR alpha and beta chains.
[0226] HDGFL2, IgLON5 and TDP-43 -Associated Proteinopathies
[0227] In some embodiments, the TCRs of the present invention target cryptic exons derived from HDGFL2 or IgLON5. Hepatoma-derived growth factor-like protein 2 (HDGFL2) is a ubiquitously expressed histone-binding protein. Cryptic exons derived from HDGFL2 are reproducibly detected via transcript analysis and proteomics in various cell lines including human iPSC-derived neurons, HeLa cells, in addition to CSF samples from patients with amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer’s disease (AD) (Seddighi, S. et al., Sci Transl Med, eadg7162 (2024); Irwin, K. E. et al., Nat Med, doi: 10.1038 / s41591-023-02788-5 (2024); Calliari, A. et &\., Mol Neurodegener 19, 29 (2024).
[0228] Antibodies binding to a cryptic peptide within HDGFL2 (Irwin et al., Calliari et al.) have been used to show that HDGFL2 cryptic peptide can be detected in the presymptomatic and early-stage of ALS / FTD (Irwin et al.) and that it is significantly increased in brain regions with TDP-43 pathology in FTLD-TDP and AD-TDP, compared to non-TDP-43 controls (Calliari et al.).
[0229] IgLON5 is a cell adhesion molecule belonging to the Ig superfamily that is primarily expressed in the brain and testis with poorly understood function. The IgLON5 protein has been recently implicated as a target in an autoimmune and neurodegenerative condition that is characterized by autoantibodies against the IgLON5 protein that is concurrent with neuronal tau deposits (Zhang, Y. H. et al., Neural Regen Res 18, 1017-1022 (2023)). Additionally, a case of a patient with co-existence of neuronal tau pathology along with microglia and neuronal TDP-43 pathology with autoantibodies against IgLON5 has been reported (Cagnin, A. et al., J Alzheimer s Dis 59, 13-20 (2017)).Attorney Docket No: 046483-7493WOl(04128)
[0230] One shared pathological hallmark between neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and inclusion body myositis (IBM) is the aggregation and nuclear depletion of TAR DNA-binding protein-43 (TDP-43). TDP-43 is a ubiquitously expressed RNA binding protein that resides mostly in the nucleus, where it has been shown to be a repressor of cryptic exons, intronic sequences that are normally not spliced into mRNA, but become derepressed upon TDP-43 mislocalization and included in mature mRNA. Although most of these events generate frameshifts and premature stop codons leading to RNA degradation, some cryptic exons are translated to form cryptic peptides in the brain of patients with neurodegenerative diseases, such as ALS. TDP-43 loss of function leads to the generation of such cryptic peptides, which are foreign to the human immune system as the encoding cryptic exons do not exist in the human thymus, consistent with recent data indicating that T cells recognizing these peptides are retained.
[0231] T Cell Receptors
[0232] In certain aspects, the invention of the current disclosure includes recombinant T cell receptor alpha and beta chains which can associate with each other in order to form functional recombinant T cell receptors (TCRs) specific for cryptic epitopes of HDGFL2 or IgLON5 protein. In another aspect, the invention includes a method for generating a modified T cell comprising expanding a population of T cells and introducing a nucleic acid encoding modified TCR alpha and beta chains with binding affinity for cryptic epitopes of HDGFL2 or IgLON5 protein expressed by a target cell into the expanded T cells. In this embodiment, the T cells are capable of expressing the modified TCR.
[0233] A T cell receptor is a complex of membrane proteins that participate in the activation of T cells in response to the recognition of presented antigen. Stimulation of the TCR is triggered by major histocompatibility complex molecules (MHC) or human leukocyte antigen complex molecules (HLA) on either normal cells (in the case of HLA / MHC class I) or professional antigen presenting cells (in the case of HLA / MHC class II) that present antigenic peptides to the T cells and bind to the TCR alpha / beta heterodimer to induce a series of intracellular signaling cascades.
[0234] The TCR complex is generally composed of six different membrane bound proteins that form the TCR heterodimer complex. Antigen recognition is provided by the TCR alpha (a) andAttorney Docket No: 046483-7493WOl(04128)
[0235] TCR beta (β) chains, while signal transduction is provided by a CD3δ chain, two CD3ε chains, and the CD3ζ chain. TCRs exist in alpha / beta or gamma / delta forms, which are structurally similar but have distinct anatomical locations and functions. In one embodiment, the TCR comprises a TCR alpha and beta chain, such as the nucleic acid encoding the TCR comprises a nucleic acid encoding a TCR alpha and a TCR beta chain. In another embodiment, an alpha or beta chain or both comprises at least one N-deglycosylation.
[0236] Each chain is composed of two extracellular domains, a variable and constant domain. The constant domain is proximal to the cell membrane, followed by a transmembrane domain and a short cytoplasmic tail. The variable domain contributes to the determination of the particular antigen and HLA molecule to which the TCR has binding specificity. In turn, the specificity of a T cell for a unique antigen-HLA complex resides in the particular TCR expressed by the T cell.
[0237] The variable domains include the highly polymorphic loops analogous to the complementarity determining regions (CDRs) of antibodies. The diversity of TCR sequences is generated via somatic rearrangement of linked variable (V), diversity (D), joining (J), and constant genes.
[0238] Functional alpha and gamma chain polypeptides are formed by rearranged V-J-C regions, whereas beta and delta chains consist of V-D-J-C regions. The extracellular constant domain includes a membrane proximal region and an immunoglobulin region.
[0239] TCR Encoded Nucleic Acids and Expression Vectors
[0240] In one aspect, the present invention the disclosure provides isolated nucleic acid(s) encoding a T cell receptor (TCR), the nucleic acid(s) comprising a first polynucleotide encoding a TCRa (TRA) chain polynucleotide and a second polynucleotide encoding a TCRβ (TRB) chain polypeptide, wherein TRA and TRB have an antigenic specificity for a cryptic epitope presented in a complex with a human MHC I polypeptide, wherein:
[0241] a) the epitope is a cryptic epitope of HDGFL2 comprising the amino acid sequence of SEQ ID NO: 7, the MHC I is HLA-B*08:01, the TRA comprises a third complementarity determining region (CDR3) comprising the amino acid sequence of SEQ ID NO: 3, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 6;Attorney Docket No: 046483-7493WOl(04128)
[0242] b) the epitope is a cryptic epitope of HDGFL2 comprising the amino acid sequence of SEQ ID NO: 10, the MHC I is HLA-A*03:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 8, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 9;
[0243] c) the epitope is a cryptic epitope of HDGFL2 comprising the amino acid sequence of SEQ ID NO: 13, the MHC I is HLA-A*03:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 11, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 12;
[0244] d) the epitope is a cryptic epitope of HDGFL2 comprising the amino acid sequence of SEQ ID NO: 13, the MHC I is HLA-A*03:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 14, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 15;
[0245] e) the epitope is a cryptic epitope of HDGFL2 comprising the amino acid sequence of SEQ ID NO: 13, the MHC I is HLA-A*03:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 16, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 17;
[0246] f) the epitope is a cryptic epitope of HDGFL2 comprising the amino acid sequence of SEQ ID NO: 13, the MHC I is HLA-A*03:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 18, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 19;
[0247] g) the epitope is a cryptic epitope of HDGFL2 comprising the amino acid sequence of SEQ ID NO: 13, the MHC I is HLA-A*03:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 20, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 21;
[0248] h) the epitope is a cryptic epitope of HDGFL2 comprising the amino acid sequence of SEQ ID NO: 13, the MHC I is HLA-A*03:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 22, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 23;
[0249] i) the epitope is a cryptic epitope of HDGFL2 comprising the amino acid sequence of SEQ ID NO: 13, the MHC I is HLA-A*03:01, the TRA comprises a CDR3 comprising theAttorney Docket No: 046483-7493WOl(04128)
[0250] amino acid sequence of SEQ ID NO: 24, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 25;
[0251] j) the epitope is a cryptic epitope of HDGFL2 comprising the amino acid sequence of SEQ ID NO: 13, the MHC I is HLA-A*03:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 26, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 27;
[0252] k) the epitope is a cryptic epitope of HDGFL2 comprising the amino acid sequence of SEQ ID NO: 13, the MHC I is HLA-A*03:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 28, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 29;
[0253] l) the epitope is a cryptic epitope of HDGFL2 comprising the amino acid sequence of SEQ ID NO: 13, the MHC I is HLA-A*03:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 30, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 31;
[0254] m) the epitope is a cryptic epitope of IgLON5 comprising the amino acid sequence of SEQ ID NO: 34, the MHC I is HLA-B*35:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 32, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 33;
[0255] n) the epitope is a cryptic epitope of IgLON5 comprising the amino acid sequence of SEQ ID NO: 34, the MHC I is HLA-B*35:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 35, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 36;
[0256] o) the epitope is a cryptic epitope of IgLON5 comprising the amino acid sequence of SEQ ID NO: 43, the MHC I is HLA-A*03:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 39, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 42;
[0257] p) the epitope is a cryptic epitope of IgLON5 comprising the amino acid sequence of SEQ ID NO: 34, the MHC I is HLA-B*35:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 44, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 45;Attorney Docket No: 046483-7493WOl(04128)
[0258] q) the epitope is a cryptic epitope of IgLON5 comprising the amino acid sequence of SEQ ID NO: 34, the MHC I is HLA-B*35:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 46, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 47;
[0259] r) the epitope is a cryptic epitope of IgLON5 comprising the amino acid sequence of SEQ ID NO: 34, the MHC I is HLA-B*35:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 48, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 49;
[0260] s) the epitope is a cryptic epitope of IgLON5 comprising the amino acid sequence of SEQ ID NO: 34, the MHC I is HLA-B*35:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 50, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 51;
[0261] t) the epitope is a cryptic epitope of IgLON5 comprising the amino acid sequence of SEQ ID NO: 34, the MHC I is HLA-B*35:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 52, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 53;
[0262] u) the epitope is a cryptic epitope of IgLON5 comprising the amino acid sequence of SEQ ID NO: 34, the MHC I is HLA-B*35:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 54, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 55;
[0263] v) the epitope is a cryptic epitope of IgLON5 comprising the amino acid sequence of SEQ ID NO: 34, the MHC I is HLA-B*35:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 56, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 57;
[0264] w) the epitope is a cryptic epitope of IgLON5 comprising the amino acid sequence of SEQ ID NO: 60, the MHC I is HLA-A*02:01*, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 58, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 59;
[0265] x) the epitope is a cryptic epitope of IgLON5 comprising the amino acid sequence of SEQ ID NO: 34, the MHC I is HLA-B*35:01, the TRA comprises a CDR3 comprising the aminoAttorney Docket No: 046483-7493WOl(04128)
[0266] acid sequence of SEQ ID NO: 61, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 62;
[0267] y) the epitope is a cryptic epitope of IgLON5 comprising the amino acid sequence of SEQ ID NO: 362, the MHC I is HLA- B*35:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 588, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 589;
[0268] z) the epitope is a cryptic epitope of ZNF423 comprising the amino acid sequence of SEQ ID NO: 491, the MHC I is HLA- A*02:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 562, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 563;
[0269] aa) the epitope is a cryptic epitope of EPB41L4A comprising the amino acid sequence of SEQ ID NO: 292, the MHC I is HLA- A*01:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 564, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 565;
[0270] bb)the epitope is a cryptic epitope of ARHGAP22 comprising the amino acid sequence of SEQ ID NO: 246, the MHC I is HLA- B*40:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 580, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 581;
[0271] cc) the epitope is a cryptic epitope of PTPRZ1 comprising the amino acid sequence of SEQ ID NO: 418, the MHC I is HLA- A*02:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 582, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 583; or
[0272] dd)the epitope is a cryptic epitope of PTPRZ1 comprising the amino acid sequence of SEQ ID NO: 418, the MHC I is HLA- A*02:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 584, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 585.
[0273] Table 1 provides a summary of selected TCRs according to certain aspects of the present disclosure:
[0274] TCR SEQ SEQ SEQ CDR3 alpha ID CDR3 beta ID Antigen'HLA Antigen AA ID Alias NO NO NO TCR-8 AFAFSGNTPLV 562 ASSLLRGGPGLAGRNIQY 563 ZNF423.9.16. HLA. A.02.01.110 VLGSAWDS 491 TCR-9 ALSAMDSNYQLI 564 ASSQTSVSGGELF 565 EPB41L4A.23.33. HLA. A.01.01.26 QAEVCESVCAY 292
[0275]
[0276] TCR- 14 AYRPRDDKII 3 ASSSGQGDSYEQY 6 HDGFL2.14.22. HLA. B.08.01.58 FGKGHSGM 7Attorney Docket No: 046483-7493WOl(04128)
[0277] TCR-18 AFGGNKLV 8 SARDPQGRFSPLH 9 HDGFL2.33.41. HLA. A.0301.124 RLHESERVR 10 TCR-19 AWGTYKYI 580 ASSSHQGNTIY 581 ARHGAP22.85.96 HLA. B.40.01.113 AEEAEEHHEEL 246 TCR-20 AAGYDKII 11 ASSLGESGYGYT 12 HDGFL2.39.49. HLA. A.03.01.126 RVRKQERERDT 13 TCR-21 ATESSSYKLI 14 ASSRSDSPNEKLF 15 HDGFL2.39.49. HLA. A.0301.126 RVRKQERERDT 13 TCR-24 AMREGRDSGSARQLT 582 ASSQDRGLLSEGYT 583 PTPRZ1.32.40. HLA. A.02.01.104 GLTDPPTSA 418 TCR-25 AVRPDSGAGSYQLT 584 ASSQDVSYGYT 585 PTPRZ1.32.40. HLA. A.02.01.104 GLTDPPTSA 418 TCR-26 AVAGGTSYGKLT 16 ASRLPGGVNEQF 17 HDGFL2.39.49. HLA. A.0301.126 RVRKQERERDT 13 TCR-27 VVSAGYDYKLS 18 ASSQDAEGDEQY 19 HDGFL2.39.49. HLA. A.0301.126 RVRKQERERDT 13 TCR-28 AYGRRRARLM 20 ASSLAGGFPEQY 21 HDGFL2.39.49. HLA. A.0301.126 RVRKQERERDT 13 TCR-29 GTESNQAGTALI 22 ASSVGQKPYEQY 23 HDGFL2.39.49. HLA. A.0301.126 RVRKQERERDT 13 TCR-39 CLVVRGAGNMLTF 24 SAYRPGQGDSTEAF 25 HDGFL2.39.49. HLA. A.0301.126 RVRKQERERDT 13 TCR-43 AYGRRRARLM 20 ASSLAGGFPEQY 21 HDGFL2.39.49. HLA. A.03.01 126 RVRKQERERDT 13 TCR-46 AATNAGNMLT 26 ASYLTEGGEQF 27 HDGFL2.39.49. HLA. A.0301.126 RVRKQERERDT 13 TCR-47 AVEGSSYKLI 28 AISGAGGRVEQY 29 HDGFL2.39.49. HLA. A.0301.126 RVRKQERERDT 13 TCR-49 ALQTGTASKLT 30 ASSPDGLPSDEQY 31 HDGFL2.39.49. HLA. A.0301.126 RVRKQERERDT 13 TCR-22 LVGDSGWGGGGNKLT 32 ASSLEGDQPQH 33 IGLON5.76.85. HLA. B.35.01.98 WAWAHTCKH 34 TCR-23 AVRELSGTY KYI 35 ASSPGQGNYGYT 36 IGLON5.76.85. HLA. B.35.01.98 WAWAHTCKH 34 TCR-30 AMRGVLSGTYKYI 39 ASSLTRTTYYEQY 42 IGLON5.27.37. HLA. A.03.01.129 SSLSAWCQLHR 43 TCR-31 AVGANSNYQLI 44 ASSLAWQDTQY 45 IGLON5.76.85. HLA. B.35.01.98 WAWAHTCKH 34 TCR-32 AGPREEGGGADGLT 46 SARDVQPGVSWDEQF 47 IGLON5.76.85. HLA. B.35.01.98 WAWAHTCKH 34 TCR-33 FY 48 ASSGGDRDYYEQY 49 IGLON5.76.85. HLA. B.35.01.98 WAWAHTCKH 34 TCR-34 AGPMDSNYQLI 50 ASSVEGLAGVDGTGELF 51 IGLON5.76.85. HLA. B.35.01.98 WAWAHTCKH 34 TCR-36 AGQEDTNAGKST 52 SARGAEGLNNEQF 53 IGLON5.76.85. HLA. B.35.01.98 WAWAHTCKH 34 TCR-37 ALGELPDYKLS 54 ASSGTGTEAF 55 IGLON5.76.85. HLA. B.35.01.98 WAWAHTCKH 34 TCR-38 ALGELGNQFY 56 ASSEEETGGSEAF 57 IGLON5.76.85. HLA. B.35.01.98 WAWAHTCKH 34 TCR-40 AVRDDNAGNMLT 58 ASSSTLGSYEQY 59 IGLON5.28.38. HLA. A.02.01.89 SLSAWCQLHRL 60 TCR-41 FY 48 ASSGGDRDYYEQY 49 IGLON5.76.85. HLA. B.35.01.98 WAWAHTCKH 34 TCR-42 AGPREEGGGADGLT 46 SARDVQPGVSWDEQF 47 IGLON5.76.85. HLA. B.35.01.98 WAWAHTCKH 34 TCR-44 AVNSPGGGADGLT 61 ASSPRPPMVSYEQY 62 IGLON5.76.85. HLA. B.35.01.98 WAWAHTCKH 34 TCR-45 AGPMDSNYQLI 50 ASSVEGLAGVDGTGELF 51 IGLON5.76.85. HLA. B.35.01.98 WAWAHTCKH 34 TCR-48 AGQEDTNAGKST 52 SARGAEGLNNEQF 53 IGLON5.76.85. HLA. B.35.01.98 WAWAHTCKH 34
[0278]
[0279] TCR-50 AGLSYNTDKLI 588 ASSLGAGPQNEQF 589 IGLON5.40.49. HLA. B.35:01.96 MPSPHSLAL 362
[0280] In an embodiment, the isolated nucleic acid(s) comprise at least one non-naturally occurring nucleotide or encode at least one non-naturally occurring amino acid substitution.
[0281] In an embodiment, the TRA and TRB have antigenic specificity for a cryptic epitope of HDGFL2.
[0282] In one embodiment, the cryptic HDGFL2 epitope comprises the amino acid sequence of SEQ ID NO: 7 and the MHC 1 is HLA-B*08:01.
[0283] In another embodiment, the cryptic HDGFL2 epitope comprises the amino acid sequence of SEQ ID NO: 13 and the MHC I is HLA-B*03:01. In one embodiment, the cryptic HDGFL2 epitope-directed TRA comprises a CDR3 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 11, 14, 16, 18, 20, 22, 24, 26, and 28, and the cryptic HDGFL2 epitopedirected TRB comprises a CDR3 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 12, 15, 17, 19, 21, 23, 25, 27, 29, and 31.
[0284] In another embodiment, the cryptic HDGFL2 epitope comprises the amino acid sequence of SEQ ID NO: 10 and the MHC I is HLA-B*03:01. In one embodiment, the TRA comprises aAttorney Docket No: 046483-7493WOl(04128)
[0285] CDR3 comprising the amino acid sequence of SEQ ID NO: 8 and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 9.
[0286] In another embodiment, the cryptic HDGFL2 epitope comprises the amino acid sequence of SEQ ID NO: 7, the MHC I is HLA-B*08:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 6.
[0287] In certain embodiments, the cryptic HDGFL2 epitope-directed TRA further comprises a first complementarity determining region (CDR1) comprising the amino acid sequence of SEQ ID NO: 1 and a second complementarity determining region (CDR2) comprising the amino acid sequence of SEQ ID NO: 2, and the cryptic HDGFL2 epitope-directed TRB further comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 4 and a CDR2 comprising the amino acid sequence of SEQ ID NO: 5.
[0288] In some embodiments, a TCR of the present invention targets a cryptic peptide from a gene identified in Table 3. In other embodiments, a TCR of the present invention targets an HLA-restricted cryptic peptide from a gene as set forth in Table 4. Exemplary genes expressing cryptic epitopes according to the present invention include AARS, ACTL6B, ARHGAP22, CELSR3, DNM1, EPB41L4A, HDGFL2, IGLON5, MYO18A, NECAB2, PHF2, PTPRZ1, PXDN, SLC24A2, STMN2, SYNJ2, XPO4, and ZNF423 as set forth in Tables 3 and 4.
[0289] In some embodiments, the cryptic peptide is expressed in muscle. In other embodiments, the cryptic peptide is expressed in a nervous system tissue. In some embodiments, the cryptic peptide may be detected in blood or cerebrospinal fluid.
[0290] Tolerable variations of the nucleic acid sequences encoding the TCRs of the present invention will be known to those of skill in the art. For example, in some embodiments the nucleic acid(s) encoding the cryptic HDGFL2 epitope-directed TRA comprises a TCRa chain variable domain (TRAV) comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 63. In another embodiment, the TRAV comprises the amino acid sequence of SEQ ID NO: 63. In another embodiment, the TRA comprises a TRAV comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 63 and a TCRa chain constant region (TRAC) comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequenceAttorney Docket No: 046483-7493WOl(04128)
[0291] identity to the amino acid sequence of SEQ ID NO: 65. In another embodiment, the TRA comprises a TRAV comprising the amino acid sequence of SEQ ID NO: 63 and a TRAC comprising the amino acid sequence of SEQ ID NO: 65.
[0292] In another embodiment, the cryptic HDGFL2-directed TRA comprises a TCRa chain comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 67. In another embodiment, the TCRa chain comprises the amino acid sequence of SEQ ID NO: 67. In another embodiment, the cryptic HDGFL2 epitope-directed TRB comprises a TCRβ chain variable domain (TRBV) comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity sequence identity to the amino acid sequence of SEQ ID NO: 64. In another embodiment, the TRBV comprises the amino acid sequence of SEQ ID NO: 64.
[0293] In another embodiment, the cryptic HDGFL2 epitope-directed TRB comprises a TCRβ chain comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 68. In another embodiment, the TCRβ chain comprises the amino acid sequence of SEQ ID NO: 68.
[0294] In some embodiments, the TRA and TRB have antigenic specificity for a cryptic epitope of IgLON5.
[0295] In an embodiment, the cryptic IgLON5 epitope comprises the amino acid sequence of SEQ ID NO: 43 and the MHC I is HLA-A*03:01.
[0296] In another embodiment, the cryptic IgLON5 epitope comprises the amino acid sequence of SEQ ID NO: 34 or 362 and the MHC I is HLB-B*35:01.
[0297] In another embodiment, the cryptic IgLON5 epitope comprises the amino acid sequence of SEQ ID NO: 60 and the MHC I is HLB-A*02:01.
[0298] In certain embodiments, the cryptic IgLON5 epitope-directed TRA comprises a CDR3 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 32, 35, 44, 46, 48, 50, 52, 54, 56, and 61, and the cryptic IgLON5 epitope-directed TRB comprises a CDR3 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 33, 36, 45, 47, 49, 51, 53, 55, 57, and 62.
[0299] In an embodiment, the cryptic IgLON5 epitope comprises the amino acid sequence of SEQ ID NO: 60, the MHC I is HLB-A*02:01, and the TRA comprises a CDR3 comprising theAttorney Docket No: 046483-7493WOl(04128)
[0300] amino acid sequence of SEQ ID NO: 58 and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 59.
[0301] In another embodiment, the cryptic IgLON5 epitope comprises the amino acid sequence of SEQ ID NO: 43, the MHC I is HLA-A*03:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 39, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 42. In a further embodiment, the cryptic IgLON5 epitope-directed TRA further comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 37 and a CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and the cryptic IgLON5 epitopedirected TRB further comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 40 and a CDR2 comprising the amino acid sequence of SEQ ID NO: 41.
[0302] In an embodiment, the cryptic IgLON5 epitope-directed TRA comprises a TRAV comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 79. In another embodiment, the TRAV comprises the amino acid sequence of SEQ ID NO: 79. In another embodiment, the TRA comprises the amino acid sequence of SEQ ID NO: 79. In another embodiment, the TRA comprises a TRAV comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 79 and a TRAC comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 81. In another embodiment, the TRA comprises a TRAV comprising the amino acid sequence of SEQ ID NO: 79 and a TRAC comprising the amino acid sequence of SEQ ID NO: 81.
[0303] In another embodiment, the cryptic IgLON5 epitope-directed TRA comprises a TCRa chain comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 83. In one embodiment, the TCRα chain comprises the amino acid sequence of SEQ ID NO: 83.
[0304] In another embodiment, the cryptic IgLON5 epitope-directed TRB comprises a TRBV comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 80. In one embodiment, the TRBV comprises the amino acid sequence of SEQ ID NO: 80.
[0305] In another embodiment, the cryptic IgLON5 epitope-directed TRB comprises a TCRβ chain comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%,Attorney Docket No: 046483-7493WOl(04128)
[0306] or 100% sequence identity to the amino acid sequence of SEQ ID NO: 84. In one embodiment, the TCRβ chain comprises the amino acid sequence of SEQ ID NO: 84.
[0307] In some embodiments, the TRA and TRB have antigenic specificity for a cryptic epitope of ZNF423. In an embodiment, the cryptic ZNF423 epitope comprises the amino acid sequence of SEQ ID NO: 491 and the MHC I is HLA-A*02:01. In one embodiment, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 562, and the TRB comprises a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 563. In another embodiment, the TRA comprises a TRAV having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 653. In another embodiment, the TRB comprises a TRBV having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 686. In some embodiments, the TRA comprises a TRAC having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 65 and the TRB comprises a TRBC having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 66.
[0308] In some embodiments, the TRA and TRB have antigenic specificity for a cryptic epitope of EPB41L4A. In an embodiment, the cryptic EPB41L4A epitope comprises the amino acid sequence of SEQ ID NO: 292 and the MHC I is HLA-A*01:01. In one embodiment, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 564, and the TRB comprises a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 565. In another embodiment, the TRA comprises a TRAV having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 654 and the TRB comprises a TRBV having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 687. In some embodiments, the TRA comprises a TRAC having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 65 and the TRB comprises a TRBC having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 66.Attorney Docket No: 046483-7493WOl(04128)
[0309] In some embodiments, the TRA and TRB have antigenic specificity for a cryptic epitope of ARHGAP22. In an embodiment, the cryptic ARHGAP22 epitope comprises the amino acid sequence of SEQ ID NO: 246 and the MHC I is HLA-B*40:01. In one embodiment, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 580, and the TRB comprises a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 581. In another embodiment, the TRA comprises a TRAV having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 656 and the TRB comprises a TRBV having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 689. In some embodiments, the TRA comprises a TRAC having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 65 and the TRB comprises a TRBC having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 718.
[0310] In some embodiments, the TRA and TRB have antigenic specificity for a cryptic epitope of PTPRZ1. In an embodiment, the cryptic PTPRZ1 epitope comprises the amino acid sequence of SEQ ID NO: 418 and the MHC I is HLA-A*02:01. In one embodiment, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 582 or 584, and the TRB comprises a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 583 or 585. In another embodiment, the TRA comprises a TRAV having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 661 or 662 and the TRB comprises a TRBV having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 694 or 695. In some embodiments, the TRA comprises a TRAC having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 65 and the TRB comprises a TRBC having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 718.
[0311] In some embodiments, the first and second polynucleotide sequences encoding a cryptic epitope-directed TCR are present in a single DNA construct encoding both of the TCRa and TCR-P chains. In an exemplary embodiment, a nucleic acid encoding a TCR according to theAttorney Docket No: 046483-7493WOl(04128)
[0312] present invention encodes TCRa (TRA) and TCR0 (TRB) chains encoded by the first and second polynucleotides which are separated by a third polynucleotide sequence encoding a linker such that separate TCRa and TCR chains are expressed from a single nucleic acid. Thus, in one embodiment, a single nucleic acid encodes a TCR according to the present invention, where the TCR is encoded from 5’ to 3’ a first polynucleotide sequence encoding a TCRa chain, the third polynucleotide sequence encoding the linker, and a second polynucleotide sequence encoding a TCRp chain, thereby producing separate TCRa and TCRp chains that form the TCRa / p heterodimer. In another embodiment, the nucleic acid encodes a TCR according to the present invention, where the TCR is encoded from 5’ to 3’ a first polynucleotide sequence encoding a TCRp chain, the third polynucleotide sequence encoding the linker, and the second polynucleotide sequence encoding a TCRa chain, thereby producing separate TCRa and TCRp chains that form the TCRa / p heterodimer.
[0313] In some embodiments, the third polynucleotide sequence encoding the linker encodes a self-cleaving peptide. As used herein, a “self-cleaving peptide” or “self-cleaving 2A peptide” refers to an oligopeptide that allow multiple proteins to be encoded as polyproteins, which dissociate into component proteins upon translation. Use of the term “self-cleaving” is not intended to imply a proteolytic cleavage reaction. Various self-cleaving or 2A peptides are known to those of skill in the art, including, without limitation, those found in members of the Picornaviridae virus family, e.g., foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAVO, Thosea asigna virus (TaV), and porcine tescho virus-1 (PTV-1); and cardioviruses such as Theilovirus and encephalomyocarditis viruses. In one embodiment, the self-cleaving peptide is a 2A peptide. 2A peptides are known in the art as 12-22 amino acid-long peptides which induce ribosomal skipping during translation and share a core sequence motif of DxExNPGP (SEQ ID NO: 100). Non-limiting examples of 2A peptides for use in the invention include P2A (ATNFSLLKQAGDVEENPGP, SEQ ID NO: 101), T2A (EGRGSLLTCGDVEENPGP, SEQ ID NO: 102), E2A (QCTNYALLKLAGDVESNPGP, SEQ ID NO: 103), and F2A (VKQTLNFDLLKLAGDVESNPGP, SEQ ID NO: 104).
[0314] In some embodiments, the polynucleotide encoding the linker polypeptide further includes a polynucleotide encoding a furin cleavage site that is upstream and adjacent to the selfcleaving peptide sequence. In some embodiments, a nucleic acid encoding a cryptic HDGFL2-directed TCR comprises TCRa and TCRp chain coding regions separated by a furin-P2A self-Attorney Docket No: 046483-7493WOl(04128)
[0315] cleaving peptide sequence such that expression from the nucleic acid results in expression of separate TCRa and TCR0 chains that form the TCRa / 0 heterodimer. In one embodiment, a furin-P2A linker comprises the amino acid sequence of SEQ ID NO: 105 and / or is encoded by the nucleotide sequence of SEQ ID NO: 106. In some embodiments, the furin-P2A linker contains a spacer. For example, in some embodiments the spacer has an amino acid sequence set forth in SEQ ID NO: 156.
[0316] In an exemplary embodiment, a single nucleic acid encoding a cryptic HDGFL2-directed TCRp / a heterodimer comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 78 and / or encodes an amino acid sequence comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 69. In another embodiment, the cryptic HDGFL2-directed TCRp / a heterodimer comprises the nucleotide sequence of SEQ ID NO: 78 and / or encodes the amino acid sequence of SEQ ID NO: 69.
[0317] In another embodiment, a single nucleic acid encoding a cryptic IgLON5-directed TCRp / a heterodimer comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 94 and / or encodes an amino acid sequence comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 85.
[0318] In another embodiment, a TCR having antigenic specificity for the cryptic epitope of ZNF423 set forth in SEQ ID NO: 491 comprises a TCRp / single chain construct comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 720.
[0319] In another embodiment, the TCR having antigenic specificity for the cryptic epitope of EPB41L4A set forth in SEQ ID NO: 292 comprises a TCRp / a single chain construct comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 721.
[0320] In another embodiment, the TCR having antigenic specificity for the cryptic epitope of ARHGAP22 set forth in SEQ ID NO: 246 comprises a TCRp / a single chain constructAttorney Docket No: 046483-7493WOl(04128)
[0321] comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 723.
[0322] In another embodiment, the TCR having antigenic specificity for the cryptic epitope of PTPRZ1 set forth in SEQ ID NO: 418 comprises a TCRp / a single chain construct comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 728 or 729.
[0323] In some embodiments, the linker polynucleotide encodes an internal ribosome entry site (IRES) interposed between the two TCR subunits. As used herein, “an internal ribosome entry site” or “IRES” refers to an element that promotes direct internal ribosome entry to the initiation codon, such as ATG, of a protein coding region, thereby leading to cap-independent translation of the gene. Various internal ribosome entry sites are known to those of skill in the art. In one embodiment, the IRES comprises the nucleotide sequence of SEQ ID NO: 114.
[0324] In some embodiments, the antigen-specific TCR, including the TRAV and TRBC sequences, is a recombinant TCR, i.e., an antigen-specific TCR that is not native to (not naturally-occurring on) the T cell. A recombinant TCR can be a TCR which has been generated through recombinant expression of one or more exogenous TCR a-, 0-, y-, and / or 8-chain encoding genes. A recombinant TCR can comprise polypeptide chains derived entirely from a single mammalian species, or the antigen-specific TCR can be a chimeric or hybrid TCR comprised of amino acid sequences derived from TCRs from two different mammalian species. For example, the antigen-specific TCR can comprise a variable region derived from a murine TCR, and a constant region of a human TCR.
[0325] In some embodiments, the antigen-specific TCR, including the TRAV and TRBC comprise or consist of sequences from human subjects.
[0326] Thus, in some embodiment, the TCRs of the present invention can comprise one or more portions of a human TCR, such that the TCR, when administered to a human, is not rejected by the immune system of the human (as in e.g., graft vs. host disease). The portion can be, for example, a variable region of a human TCR and / or or a constant region of a human TCR.
[0327] Desirably, the portion is a constant region of a human TCR. The constant region of a human TCR can, for example, comprise the amino acid sequence set forth in SEQ ID NO: 65 (alpha chain) and / or SEQ ID NO: 66 or 719 (beta chain). Methods of making such hybrid TCRs are known in the art. See, for example, Cohen et al., Cancer Res. 66: 8878-8886 (2006). Methods ofAttorney Docket No: 046483-7493WOl(04128)
[0328] making recombinant TCRs are known in the art. See, for example, U. S. Pat. Nos. 7,820,174 B2, 8,785,601 B2, and 8,216,565 B2; and U. S. Patent Application Publication Nos. 2013 / 0274203 Al, US, 2016 / 0137715 Al, and 2019 / 0092834A1.
[0329] In some embodiments, the recombinant TCRs, including the TRAV and TRBC sequences of the present invention are affinity-enhanced. Methods for generating humanized TCRs and affinity-enhanced TCRs are known in the art. Affinity-enhanced TCRs are TCRs with enhanced affinity for a peptide-MHC complex (including e.g. the isolation of TCR genes that encode TCRs from patient samples (e.g. patient peripheral blood or TILs) and the improvement of TCR affinity for a peptide-MHC complex via modification of TCR sequences (e.g. by in vitro mutagenesis and selection of enhanced affinity (or affinity matured) TCRs). Methods of introducing such TCR genes into cells (such as T cells, NK cells or NKT cells) are known in the art. Methods of identifying optimal affinity TCRs involving the immunization of antigennegative humanized transgenic mice which have a diverse human TCR repertoire (e g.
[0330] TCR / MHC humanized mice such as ABabDII mice) with antigen, and isolation of antigenspecific TCRs from such immunized transgenic mice are also known in the art (see e.g. Obenaus M et al., Nat Biotechnol. 33(4):402-7, 2015). In some aspects high-affinity TCRs are generated, for example by the methods described in WO 2004 / 044004 or other methods.
[0331] Affinity-enhanced TCRs may be generated by identifying a T cell clone from which the TCR a and P chains with the desired target specificity are cloned and subjected to PCR directed mutagenesis at the complimentary determining regions of the a and P chains. The mutations in each CDR region may be screened to select for mutants with enhanced affinity over the native TCR. Once complete, lead candidates are cloned into vectors to allow functional testing in T cells expressing the affinity-enhanced TCR.
[0332] In some embodiments, the nucleic acid(s) encoding recombinant TCR alpha and / or beta chain sequences include codon-optimized sequence(s) comprising a plurality of non-naturally occurring nucleotide substitutions. Codon optimization may be accomplished using any available technology and algorithms designed to optimize codons in an amino acid sequence. In certain embodiments, the TCR alpha and beta chain sequences are codon optimized for expression in human cells.
[0333] In another aspect, the present invention provides cloning vectors, expression vectors, or expression constructs encoding a TCR as described herein. Suitable expression vectors include,Attorney Docket No: 046483-7493WOl(04128)
[0334] e.g., plasmids, viral vectors, and the like. Large numbers of suitable vectors and promoters are known to those of skill in the art; many are commercially available for generating a subject recombinant construct. The following vectors are provided by way of example, and should not be construed in any way as limiting: Bacterial: pBS, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia).
[0335] Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker operative in the expression host may be present. Suitable expression vectors include, but are not limited to, viral vectors e.g., viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest. Opthalmol. Vis. Sci. (1994) 35: 2543-2549; Borras et al., Gene Thvc. (1999) 6: 515-524; Li and Davidson, Proc. Natl. Acad. Sci. USA (1995) 92: 7700-7704; Sakamoto et al., H. Gene Ther. (1999) 5: 1088-1097; WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655); adeno-associated virus (see, e.g., Ali et al., Hum. Gene Ther. (1998) 9: 81-86, Flannery et al., Proc. Natl. Acad. Sci. USA (1997) 94: 6916-6921; Bennett et al., Invest. Opthalmol. Vis. Sci. (1997) 38: 2857-2863; Jomary et al., Gene Ther. (1997) 4:683 690, Rolling et al., Hum. Gene Ther. (1999) 10: 641-648; Ali et al., Hum. Mol. Genet. (1996) 5: 591-594; Srivastava in WO 93 / 09239, Samulski et al., Vir. (1989) 63: 3822-3828; Mendelson et al., Virol. (1988) 166: 154-165; and Flotte et al., Proc. Natl. Acad. Sci. USA (1993) 90: 10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., Proc. Natl. Acad. Sci. USA (1997) 94: 10319-23; Takahashi et al., J. Virol. (1999) 73: 7812-7816); a retroviral vector (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like.
[0336] Additional expression vectors suitable for use are, e.g., without limitation, a lentivirus vector, a gamma retrovirus vector, a foamy virus vector, an adeno-associated virus vector, an adenovirus vector, a pox virus vector, a herpes virus vector, an engineered hybrid virus vector, a transposon mediated vector, and the like. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual,Attorney Docket No: 046483-7493WOl(04128)
[0337] volumes 1 -4, Cold Spring Harbor Press, NY), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno- associated viruses, herpes viruses, and lentiviruses.
[0338] In some embodiments, the expression vector or expression construct comprises a N-terminal leader sequence encoding a signal peptide for translocating the TCRa and TCRp chains to the cell membrane. Suitable signal peptide sequences are known to those of skill in the art.
[0339] In certain embodiments, the vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g, WO 01 / 96584; WO 01 / 29058; and U. S. Pat. No. 6,326,193).
[0340] In some embodiments, an expression vector (e.g., a lentiviral vector) may be used for expressing a TCR of the present invention and / or any other additional transgenes. In some embodiments, the expression vector (e.g, lentiviral vector) comprises additional elements that aid in the functional expression of the targeting protein or targeting protein encoded therein. In some embodiments, an expression vector comprises a mammalian promoter. In one embodiment, the vector comprises an elongation-factor- 1 -alpha promoter (EF-la promoter. Use of an EF- la promoter may increase the efficiency in expression of downstream transgenes, such as the nucleic acid encoding the targeting protein. Physiologic promoters e.g., an EF-l promoter) may be less likely to induce integration mediated genotoxicity, and may abrogate the ability of the retroviral vector to transform stem cells Other physiological promoters suitable for use in a vector (e.g., a lentiviral vector) are known to those of skill in the art and may be incorporated into a vector of the present invention.
[0341] In some embodiments, a lentivirus expression vector includes a non-requisite cz -acting sequence that may improve titers and gene expression. One non-limiting example of a nonrequisite cis acting sequence is the central polypurine tract and central termination sequence (cPPT / CTS) which is important for efficient reverse transcription and nuclear import. Other non-requisite cis acting sequences are known to those of skill in the art and may be incorporated into a vector (e.g., lentiviral vector) of the present invention.
[0342] In some embodiments, the vector further comprises a posttranscriptional regulatory element. Posttranscriptional regulatory elements may improve RNA translation, improve transgene expression, and stabilize RNA transcripts. One example of a posttranscriptional regulatory element is the woodchuck hepatitis virus posttranscriptional regulatory elementAttorney Docket No: 046483-7493WOl(04128)
[0343] (WPRE). Accordingly, in some embodiments a vector for the present invention further comprises a WPRE sequence. Various posttranscriptional regulator elements are known to those of skill in the art and may be incorporated into a vector (e.g., a lentiviral vector) of the present invention.
[0344] A vector of the present invention may further comprise additional elements such as a rev response element (RRE) for RNA transport, packaging sequences, and 5’ and 3’ long terminal repeats (LTRs). The term “long terminal repeat” or “LTR” refers to domains of base pairs located at the ends of retroviral DNAs which comprise U3, R and U5 regions. LTRs generally provide functions required for the expression of retroviral genes (e.g., promotion, initiation, and polyadenylation of gene transcripts) and viral replication. In one embodiment, a vector (e.g., lentiviral vector) of the present invention includes a 3’ U3 deleted LTR. Accordingly, a vector (e.g., lentiviral vector) of the present invention may comprise any combination of the elements described herein to enhance the efficiency of functional expression of transgenes. For example, a vector (e.g., lentiviral vector) of the present invention may comprise a WPRE sequence, cPPT sequence, RRE sequence, 5 ’LTR, 3’ U3 deleted LTR’ in addition to a nucleic acid encoding the TCR polypeptides.
[0345] Vectors of the present invention may be self-inactivating vectors. As used herein, the term “self-inactivating vector” refers to vectors in which the 3’ LTR enhancer promoter region (U3 region) has been modified (e.g., by deletion or substitution). A self-inactivating vector may prevent viral transcription beyond the first round of viral replication. Consequently, a selfinactivating vector may be capable of infecting and then integrating into a host genome (e.g., a mammalian genome) only once, and cannot be passed further. Accordingly, self-inactivating vectors may greatly reduce the risk of creating a replication-competent virus.
[0346] In order to assess the expression of a polypeptide or portions thereof, the expression vector or construct to be introduced into a cell may also contain either a selectable marker gene or a reporter gene, or both, to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In some embodiments, the selectable marker may be carried on a separate piece of DNA and used in a cotransfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, without limitation, antibiotic-resistance genes.Attorney Docket No: 046483-7493WOl(04128)
[0347] Reporter genes may be used e.g., for tracking T cells expressing the TCRs as described herein. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., flow cytometry or enzymatic activity. Expression of the reporter gene is assessed at a suitable time after the DNA has been introduced into the recipient cells.
[0348] In some embodiments, a nucleic acid of the present invention may be RNA, e.g., in vitro synthesized RNA. Methods for in vitro synthesis of RNA are known to those of skill in the art; any known method can be used to synthesize RNA comprising a sequence encoding a chimeric receptor of the present disclosure. Methods for introducing RNA into a host cell are known in the art. See, e.g., Zhao et al. Cancer Res. (2010) 15: 9053. Introducing RNA comprising a nucleotide sequence encoding a chimeric receptor of the present disclosure into a host cell can be carried out in vitro, ex vivo or in vivo. For example, a host cell (e.g., a Treg cell, an NK cell, a cytotoxic T lymphocyte, etc.) can be electroporated in vitro or ex vivo with RNA comprising a nucleotide sequence encoding a chimeric receptor of the present disclosure.
[0349] Exemplary amino acid and nucleotide sequences cited in this disclosure are described in Table 2 below.
[0350] Table 2: Amino Acid and Nucleotide Sequences
[0351] SEQ Description Sequence
[0352] ID NO:
[0353] 1 TCR14 / TCR28 / TSESDYY
[0354] TCR43 CDR1 alpha
[0355] 2 TCR14 / TCR8 / QEAYKQQN
[0356] TCR18 / TCR28 /
[0357] TCR43 CDR2 alpha
[0358] 3 TCR14 CDR3 alpha AYRPRDDKII
[0359] 4 TCR14 / TCR40 SEHNR
[0360] CDR1 beta
[0361] 5 TCR14 / TCR40 FQNEAQ
[0362] CDR2 beta
[0363] 6 TCR14 CDR3 beta ASSSGQGDSYEQY
[0364] 7 TCR14 HDGFL2 FGKGHSGM
[0365] epitope
[0366] 8 TCR18 CDR3 alpha AFGGNKLV
[0367] 9 TCR18 CDR3 beta SARDPQGRFSPLH
[0368] 10 TCR18 HDGFL2 RLHESERVR
[0369] epitope
[0370] 11 TCR20 CDR3 alpha AAGYDKII
[0371]
[0372] 12 TCR20 CDR3 beta ASSLGESGYGYTAttorney Docket No: 046483-7493WOl(04128)
[0373] TCR20 / 21 / 26- RVRKQERERDT
[0374] 29 / 39 / 43, 46 / 47 / 49
[0375] HDGFL2 epitope
[0376] TCR21 CDR3 alpha ATESSSYKLI
[0377] TCR21 CDR3 beta ASSRSDSPNEKLF
[0378] TCR26 CDR3 alpha AVAGGTSYGKLT
[0379] TCR26 CDR3 beta ASRLPGGVNEQF
[0380] TCR27 CDR3 alpha VVSAGYDYKLS
[0381] TCR27 CDR3 beta ASSQDAEGDEQY
[0382] TCR28 / 43 CDR3 AYGRRRARLM
[0383] alpha
[0384] TCR28 / 43 CDR3 ASSLAGGFPEQY
[0385] beta
[0386] TCR29 CDR3 alpha GTESNQAGTALI
[0387] TCR29 CDR3 beta ASSVGQKPYEQY
[0388] TCR39 CDR3 alpha CLVVRGAGNMLTF
[0389] TCR39 CDR3 beta SAYRPGQGDSTEAF
[0390] TCR46 CDR3 alpha AATNAGNMLT
[0391] TCR46 CDR3 beta ASYLTEGGEQF
[0392] TCR47 CDR3 alpha AVEGSSYKLI
[0393] TCR47 CDR3 beta AISGAGGRVEQY
[0394] TCR49 CDR3 alpha ALQTGTASKLT
[0395] TCR49 CDR3 beta ASSPDGLPSDEQY
[0396] TCR22 CDR3 alpha LVGDSGWGGGGNKLT
[0397] TCR22 CDR3 beta ASSLEGDQPQH
[0398] TCR22 / 23 / 31- WAWAHTCKH
[0399] 34 / 36-38 /
[0400] 41 / 42 / 44 / 45 / 48
[0401] IgLON5 epitope
[0402] TCR23 CDR3 alpha AVRELSGTYKYI
[0403] TCR23 CDR3 beta ASSPGQGNYGYT
[0404] TCR30 CDR1 alpha TSDQSYG
[0405] TCR30 CDR2 alpha QGSYDEQN
[0406] TCR30 CDR3 alpha AMRGVLSGTYKYI
[0407] TCR30 / TCR8 / MNHEY
[0408] TCR22 / TCR23 /
[0409] TCR28 / TCR37 /
[0410] TCR43 / TCR44 /
[0411] TCR46 / TCR49
[0412] CDR1 beta
[0413] TCR30 / TCR37 SVGEGT
[0414] CDR2 beta
[0415] TCR30 CDR3 beta ASSPRRGSNQPQH
[0416] TCR30 lgLON5 SSLSAWCQLHR
[0417] epitope
[0418] TCR31 CDR3 alpha AVGANSNYQLI
[0419] TCR31 CDR3 beta ASSLAWQDTQY
[0420] TCR32 / 42 CDR3 AGPREEGGGADGLT
[0421]
[0422] alphaAttorney Docket No: 046483-7493WOl(04128)
[0423] TCR32 / 42 CDR3 SARDVQPGVSWDEQF
[0424] beta
[0425] TCR33 / 41 CDR3 FY
[0426] alpha
[0427] TCR33 / 41 CDR3 ASSGGDRDYYEQY
[0428] beta
[0429] TCR34 / 45 CDR3 AGPMDSNYQLI
[0430] alpha
[0431] TCR34 / 45 CDR3 ASSVEGLAGVDGTGELF
[0432] beta
[0433] TCR36 / 48 CDR3 AGQEDTNAGKST
[0434] alpha
[0435] TCR36 / 48 CDR3 SARGAEGLNNEQF
[0436] beta
[0437] TCR37 CDR3 alpha ALGELPDYKLS
[0438] TCR37 CDR3 beta ASSGTGTEAF
[0439] TCR38 CDR3 alpha ALGELGNQFY
[0440] TCR38 CDR3 beta ASSEEETGGSEAF
[0441] TCR40 CDR3 alpha AVRDDNAGNMLT
[0442] TCR40 CDR3 beta ASSSTLGSYEQY
[0443] TCR40 IgLON5 SLSAWCQLHRL
[0444] epitope
[0445] TCR44 CDR3 alpha AVNSPGGGADGLT
[0446] TCR44 CDR3 beta ASSPRPPMVSYEQY
[0447] TCR14 V alpha MACPGFLWALVISTCLEFSMAQTVTQSQPEMSVQEAETVTLSCT YDTSESDYYLFWYKQPPSRQM1LV1RQEAYKQQNATENRFSVNF QKAAKSFSLKISDSQLGDAAMYFCAYRPRDDKIIFGKGTRLHILP TCR14 V beta MGTSLLCWMALCLLGADHADTGVSQNPRHKITKRGQNVTFRCD PISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAER PKGSFSTLEIQRTEQGDSAMYLCASSSGQGDSYEQYFGPGTRLTV T TCR14 TRAC NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYIT DKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFP SPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMT LRLWSS TRBC-1 EDLKNVFPPKVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSW WVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQ NPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADC GFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRK DSRG TCR14 alpha chain MACPGFLWALVISTCLEFSMAQTVTQSQPEMSVQEAETVTLSCT YDTSESDYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNF QKAAKSFSLKISDSQLGDAAMYFCAYRPRDDKIIFGKGTRLHILP NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYIT DKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFP SPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMT LRLWSS TCR14 beta chain MGTSLLCWMALCLLGADHADTGVSQNPRHKITKRGQNVTFRCD
[0448] PISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAER
[0449]
[0450] PKGSFSTLEIQRTEQGDSAMYLCASSSGQGDSYEQYFGPGTRLTVAttorney Docket No: 046483-7493WOl(04128)
[0451] TEDLKNVFPPKVAVFEPSEAEISHTQKATLVCLATGFYPDHVELS WWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFW QNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD CGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKR KDSRG TCR14 beta-alpha MGTSLLCWMALCLLGADHADTGVSQNPRHKITKRGQNVTFRCD chain PISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAER PKGSFSTLEIQRTEQGDSAMYLCASSSGQGDSYEQYFGPGTRLTV TEDLKNVFPPKVAVFEPSEAEISHTQKATLVCLATGFYPDHVELS WWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFW QNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD CGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKR KDSRGRAKRSGSGATNFSLLKQAGDVEENPGPMACPGFLWALVI STCLEFSMAQTVTQSQPEMSVQEAETVTLSCTYDTSESDYYLFW YKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISD SQLGDAAMYFCAYRPRDDKIIFGKGTRLHILPNIQNPDPAVYQLR DSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDF KSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEK SFETDTNLNFQNLSVIGFRTLLLKVAGFNLLMTLRLWSS TCR14 CDR1 alpha ACCAGTGAGAGTGATTATTAT
[0452] TCR14 CDR2 alpha CAAGAAGCTTATAAGCAACAGAAT
[0453] TCR14 CDR3 alpha GCTTATAGGCCCCGAGATGACAAGATCATC
[0454] TCR14 CDR1 beta TCTGAACACAACCGC
[0455] TCR14 CDR2 beta TTCCAGAATGAAGCTCAA
[0456] TCR14 CDR3 beta GCCAGCAGCTCTGGACAGGGGGATAGCTACGAGCAGTAC TCR14 V alpha ATGGCATGCCCTGGCTTCCTGTGGGCACTTGTGATCTCCACCT GTCTTGAATTTAGCATGGCTCAGACAGTCACTCAGTCTCAACC AGAGATGTCTGTGCAGGAGGCAGAGACCGTGACCCTGAGCTG CACATATGACACCAGTGAGAGTGATTATTATTTATTCTGGTAC AAGCAGCCTCCCAGCAGGCAGATGATTCTCGTTATTCGCCAA GAAGCTTATAAGCAACAGAATGCAACAGAGAATCGTTTCTCT GTGAACTTCCAGAAAGCAGCCAAATCCTTCAGTCTCAAGATCT CAGACTCACAGCTGGGGGATGCCGCGATGTATTTCTGTGCTTA TAGGCCCCGAGATGACAAGATCATCTTTGGAAAAGGGACACG ACTTCATATTCTCCCCA TCR14 V beta ATGGGCACCAGCCTCCTCTGCTGGATGGCCCTGTGTCTCCTGG GGGCAGATCACGCAGATACTGGAGTCTCCCAGAACCCCAGAC ACAAGATCACAAAGAGGGGACAGAATGTAACTTTCAGGTGTG ATCCAATTTCTGAACACAACCGCCTTTATTGGTACCGACAGAC CCTGGGGCAGGGCCCAGAGTTTCTGACTTACTTCCAGAATGA AGCTCAACTAGAAAAATCAAGGCTGCTCAGTGATCGGTTCTCT GCAGAGAGGCCTAAGGGATCTTTCTCCACCTTGGAGATCCAG CGCACAGAGCAGGGGGACTCGGCCATGTATCTCTGTGCCAGC AGCTCTGGACAGGGGGATAGCTACGAGCAGTACTTCGGGCCG GGCACCAGGCTCACGGTCACAG TCR14 beta-alpha ATGGGCACCAGCCTTCTATGCTGGATGGCCCTGTGCCTGCTGG single chain GCGCGGACCATGCAGACACCGGCGTGTCCCAGAATCCACGTC ACAAGATTACCAAGCGCGGCCAGAACGTGACCTTCAGGTGTG ATCCCATCTCGGAGCACAACCGCTTGTATTGGTACCGCCAGAC
[0457]
[0458] CCTGGGTCAGGGCCCTGAGTTCCTGACCTACTTCCAGAACGAGAttorney Docket No: 046483-7493WOl(04128)
[0459] GCTCAGCTGGAGAAATCCCGCCTGCTCAGTGACCGCTTTTCAG CCGAGCGGCCCAAGGGCTCCTTCTCGACCCTGGAGATCCAGC GTACTGAACAGGGTGATTCTGCCATGTACCTGTGCGCCTCCTC TTCTGGACAGGGGGACAGCTACGAGCAATACTTTGGGCCGGG AACACGACTCACTGTCACCGAGGACCTGAAGAACGTGTTTCC TCCTAAGGTGGCCGTGTTCGAGCCATCAGAGGCTGAAATCAG CCACACGCAGAAAGCTACACTGGTATGCCTGGCCACCGGCTT TTATCCGGATCATGTGGAACTGTCTTGGTGGGTGAACGGCAA GGAAGTGCACAGCGGCGTGTCCACCGACCCTCAGCCTCTGAA AGAGCAGCCCGCTCTGAACGATAGCCGCTACTGTCTGAGCAG CAGGCTGAGAGTGTCCGCAACCTTCTGGCAGAACCCCCGCAA TCACTTCAGATGTCAGGTGCAGTTCTACGGCCTGTCCGAGAAC GACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACACAGATC GTCTCTGCCGAGGCCTGGGGCAGAGCCGACTGCGGCTTCACC AGCGAGAGCTACCAGCAAGGAGTCCTTTCCGCGACTATTCTGT ACGAGATCCTGCTGGGCAAGGCCACCTTGTACGCGGTGCTGG TCTCGGCTCTCGTGCTGATGGCCATGGTGAAGCGGAAGGACA GCCGCGGAAGGGCAAAGCGGAGCGGAAGCGGAGCAACCAAT TTCAGCCTGCTGAAGCAGGCCGGCGATGTGGAGGAGAATCCT GGACCAATGGCTTGTCCAGGCTTCCTTTGGGCGCTGGTGATTA GTACCTGCCTGGAGTTCTCTATGGCTCAGACGGTCACCCAGAG CCAACCTGAGATGTCTGTGCAGGAGGCCGAGACCGTGACCCT CTCATGCACTTACGACACCTCCGAGAGCGATTACTACCTGTTT TGGTACAAGCAGCCGCCTTCCCGCCAGATGATCTTGGTGATCC GTCAGGAAGCGTACAAGCAGCAGAACGCGACAGAGAACCGC TTCTCGGTTAATTTCCAGAAGGCTGCCAAGTCCTTTTCGCTCA AGATTTCCGACAGCCAGCTGGGGGACGCAGCCATGTATTTCT GTGCCTACCGGCCCAGGGATGACAAAATCATCTTCGGCAAAG GTACTCGCCTGCACATCCTGCCCAACATTCAGAACCCGGATCC TGCCGTGTACCAGCTGCGCGATAGCAAATCTAGCGACAAGAG TGTGTGCCTGTTCACCGATTTTGACAGCCAGACCAACGTGTCC CAGAGCAAGGACTCTGATGTCTACATCACCGACAAGACCGTG CTGGACATGCGCTCCATGGACTTCAAGTCCAACTCTGCTGTCG CTTGGAGCAACAAGAGCGACTTCGCCTGTGCCAACGCCTTCA ACAACAGCATCATCCCCGAGGACACCTTCTTCCCTTCTCCAGA GAGCAGCTGCGACGTGAAACTGGTGGAAAAGTCCTTCGAGAC AGACACTAATCTGAACTTCCAGAATCTGAGCGTGATCGGCTTC AGAATCCTGCTGCTGAAGGTGGCGGGATTTAATCTCCTGATGA CCCTGAGACTGTGGTCGTCGtaa
[0460] TCR30 TRAC PRT MSLSSLLKVVTASLWLGPGIAQKITQTQPGMFVQEKEAVTLDCT YDTSDQSYGLFWYKQPSSGEM1FLIYQGSYDEQNATEGRYSLNF QKARKSANLVISASQLGDSAMYFCAMRGVLSGTYKYIFGTGTRL KVLA TCR30 TRBC PRT MSLGLLCCGAFSLLWAGPVNAGVTQTPKFRVLKTGQSMTLLCA QDMNHEYMYWYRQDPGMGLRLIHYSVGEGTTAKGEVPDGYNV SRLKKQNFLLGLESAAPSQTSVYFCASSLTRTTYYEQYFGPGTRL TVT TRAC PRT NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYIT DKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFP SPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMT
[0461]
[0462] LRLWSSAttorney Docket No: 046483-7493WOl(04128)
[0463] TCR30 TRBC EDLKNVFPPKVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSW WVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQ NPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADC GFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRK DSRG TCR30 alpha chain MSLSSLLKVVTASLWLGPGIAQKITQTQPGMFVQEKEAVTLDCT YDTSDQSYGLFWYKQPSSGEMIFLIYQGSYDEQNATEGRYSLNF QKARKSANLVISASQLGDSAMYFCAMRGVLSGTYKYIFGTGTRL KVLANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDS DVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPE DTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGF NLLMTLRLWSS TCR30 beta chain MSLGLLCCGAFSLLWAGPVNAGVTQTPKFRVLKTGQSMTLLCA QDMNHEYMYWYRQDPGMGERL1HYSVGEGTTAKGEVPDGYNV SRLKKQNFLLGLESAAPSQTSVYFCASSLTRTTYYEQYFGPGTRL TVTEDLKNVFPPKVAVFEPSEAEISHTQKATLVCLATGFYPDHVE LSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATF WQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGR ADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMV KRKDSRG TCR30 beta-alpha MSLGLLCCGAFSLLWAGPVNAGVTQTPKFRVLKTGQSMTLLCA single chain PRT QDMNHEYMYWYRQDPGMGLRLIHYSVGEGTTAKGEVPDGYNV SRLKKQNFLLGLESAAPSQTSVYFCASSLTRTTYYEQYFGPGTRL TVTEDLKNVFPPKVAVFEPSEAEISHTQKATLVCLATGFYPDHVE LSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATF WQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGR ADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMV KRKDSRGRAKRSGSGATNFSLLKQAGDVEENPGPMSLSSLLKVV TASLWLGPGIAQKITQTQPGMFVQEKEAVTLDCTYDTSDQSYGL FWYKQPSSGEMIFLIYQGSYDEQNATEGRYSLNFQKARKSANLV ISASQLGDSAMYFCAMRGVLSGTYKYIFGTGTRLKVLANIQNPD PAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVL DMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSC DVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWS S TCR30 CDR1 alpha ACCAGTGATCAAAGTTATGGT
[0464] TCR30 CDR2 alpha CAGGGGTCTTATGACGAGCAAAAT
[0465] TCR30 CDR3 alpha GCAATGAGAGGGGTCCTCTCAGGAACCTACAAATACATC TCR30 CDR1 beta ATGAACCATGAATAC
[0466] TCR30 CDR2 beta TCAGTTGGTGAGGGTACA
[0467] TCR30 CDR3 beta GCCAGCAGCCTTACCCGGACTACTTACTACGAGCAGTAC TCR30 V alpha ATGGCATGCCCTGGCTTCCTGTGGGCACTTGTGATCTCCACCT DNA GTCTTGAATTTAGCATGGCTCAGACAGTCACTCAGTCTCAACC AGAGATGTCTGTGCAGGAGGCAGAGACCGTGACCCTGAGCTG CACATATGACACCAGTGAGAGTGATTATTATTTATTCTGGTAC AAGCAGCCTCCCAGCAGGCAGATGATTCTCGTTATTCGCCAA GAAGCTTATAAGCAACAGAATGCAACAGAGAATCGTTTCTCT GTGAACTTCCAGAAAGCAGCCAAATCCTTCAGTCTCAAGATCT
[0468]
[0469] CAGACTCACAGCTGGGGGATGCCGCGATGTATTTCTGTGCTTAAttorney Docket No: 046483-7493WOl(04128)
[0470] TAGGCCCCGAGATGACAAGATCATCTTTGGAAAAGGGACACG ACTTCATATTCTCCCCA TCR30 Vbeta DNA ATGGGCACCAGCCTCCTCTGCTGGATGGCCCTGTGTCTCCTGG GGGCAGATCACGCAGATACTGGAGTCTCCCAGAACCCCAGAC ACAAGATCACAAAGAGGGGACAGAATGTAACTTTCAGGTGTG ATCCAATTTCTGAACACAACCGCCTTTATTGGTACCGACAGAC CCTGGGGCAGGGCCCAGAGTTTCTGACTTACTTCCAGAATGA AGCTCAACTAGAAAAATCAAGGCTGCTCAGTGATCGGTTCTCT GCAGAGAGGCCTAAGGGATCTTTCTCCACCTTGGAGATCCAG CGCACAGAGCAGGGGGACTCGGCCATGTATCTCTGTGCCAGC AGCTCTGGACAGGGGGATAGCTACGAGCAGTACTTCGGGCCG GGCACCAGGCTCACGGTCACAG TCR30 beta-alpha ATGGGCACCAGCCTTCTATGCTGGATGGCCCTGTGCCTGCTGG single chain DNA GCGCGGACCATGCAGACACCGGCGTGTCCCAGAATCCACGTC ACAAGATTACCAAGCGCGGCCAGAACGTGACCTTCAGGTGTG ATCCCATCTCGGAGCACAACCGCTTGTATTGGTACCGCCAGAC CCTGGGTCAGGGCCCTGAGTTCCTGACCTACTTCCAGAACGAG GCTCAGCTGGAGAAATCCCGCCTGCTCAGTGACCGCTTTTCAG CCGAGCGGCCCAAGGGCTCCTTCTCGACCCTGGAGATCCAGC GTACTGAACAGGGTGATTCTGCCATGTACCTGTGCGCCTCCTC TTCTGGACAGGGGGACAGCTACGAGCAATACTTTGGGCCGGG AACACGACTCACTGTCACCGAGGACCTGAAGAACGTGTTTCC TCCTAAGGTGGCCGTGTTCGAGCCATCAGAGGCTGAAATCAG CCACACGCAGAAAGCTACACTGGTATGCCTGGCCACCGGCTT TTATCCGGATCATGTGGAACTGTCTTGGTGGGTGAACGGCAA GGAAGTGCACAGCGGCGTGTCCACCGACCCTCAGCCTCTGAA AGAGCAGCCCGCTCTGAACGATAGCCGCTACTGTCTGAGCAG CAGGCTGAGAGTGTCCGCAACCTTCTGGCAGAACCCCCGCAA TCACTTCAGATGTCAGGTGCAGTTCTACGGCCTGTCCGAGAAC GACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACACAGATC GTCTCTGCCGAGGCCTGGGGCAGAGCCGACTGCGGCTTCACC AGCGAGAGCTACCAGCAAGGAGTCCTTTCCGCGACTATTCTGT ACGAGATCCTGCTGGGCAAGGCCACCTTGTACGCGGTGCTGG TCTCGGCTCTCGTGCTGATGGCCATGGTGAAGCGGAAGGACA GCCGCGGAAGGGCAAAGCGGAGCGGAAGCGGAGCAACCAAT TTCAGCCTGCTGAAGCAGGCCGGCGATGTGGAGGAGAATCCT GGACCAATGGCTTGTCCAGGCTTCCTTTGGGCGCTGGTGATTA GTACCTGCCTGGAGTTCTCTATGGCTCAGACGGTCACCCAGAG CCAACCTGAGATGTCTGTGCAGGAGGCCGAGACCGTGACCCT CTCATGCACTTACGACACCTCCGAGAGCGATTACTACCTGTTT TGGTACAAGCAGCCGCCTTCCCGCCAGATGATCTTGGTGATCC GTCAGGAAGCGTACAAGCAGCAGAACGCGACAGAGAACCGC TTCTCGGTTAATTTCCAGAAGGCTGCCAAGTCCTTTTCGCTCA AGATTTCCGACAGCCAGCTGGGGGACGCAGCCATGTATTTCT GTGCCTACCGGCCCAGGGATGACAAAATCATCTTCGGCAAAG GTACTCGCCTGCACATCCTGCCCAACATTCAGAACCCGGATCC TGCCGTGTACCAGCTGCGCGATAGCAAATCTAGCGACAAGAG TGTGTGCCTGTTCACCGATTTTGACAGCCAGACCAACGTGTCC CAGAGCAAGGACTCTGATGTCTACATCACCGACAAGACCGTG CTGGACATGCGCTCCATGGACTTCAAGTCCAACTCTGCTGTCG
[0471]
[0472] CTTGGAGCAACAAGAGCGACTTCGCCTGTGCCAACGCCTTCAAttorney Docket No: 046483-7493WOl(04128)
[0473] ACAACAGCATCATCCCCGAGGACACCTTCTTCCCTTCTCCAGA GAGCAGCTGCGACGTGAAACTGGTGGAAAAGTCCTTCGAGAC AGACACTAATCTGAACTTCCAGAATCTGAGCGTGATCGGCTTC AGAATCCTGCTGCTGAAGGTGGCGGGATTTAATCTCCTGATGA CCCTGAGACTGTGGTCGTCGtaa
[0474] CMVpp65 betaATGGGCCCTCAGCTGCTGGGCTACGTGGTTCTGTGCCTGCTTG alpha single chain GCGCGGGGCCCCTGGAGGCTCAGGTCACCCAGAATCCACGCT DNA ACCTGATCACCGTCACCGGCAAGAAGCTGACCGTGACCTGCT CTCAGAACATGAACCACGAGTACATGAGTTGGTACAGGCAGG ACCCGGGGCTAGGTTTGCGGCAAATTTACTACTCAATGAACGT GGAGGTGACAGACAAAGGTGATGTGCCGGAGGGATACAAGG TGTCCCGCAAGGAGAAGCGCAACTTTCCTCTCATCCTGGAGA GCCCCTCTCCCAACCAGACGAGCCTTTATTTCTGTGCCTCGAC CTCCACTGCCGGCTATCCCGGCGAGTTGTTCTTCGGAGAGGGC TCCCGTCTCACTGTACTGGAAGACCTGAAGAACGTGTTTCCTC CTAAGGTGGCCGTGTTCGAGCCATCAGAGGCTGAAATCAGCC ACACGCAGAAAGCTACACTGGTATGCCTGGCCACCGGCTTTT ATCCGGATCATGTGGAACTGTCTTGGTGGGTGAACGGCAAGG AAGTGCACAGCGGCGTGTCCACCGACCCTCAGCCTCTGAAAG AGCAGCCCGCTCTGAACGATAGCCGCTACTGTCTGAGCAGCA GGCTGAGAGTGTCCGCAACCTTCTGGCAGAACCCCCGCAATC ACTTCAGATGTCAGGTGCAGTTCTACGGCCTGTCCGAGAACG ACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACACAGATCG TCTCTGCCGAGGCCTGGGGCAGAGCCGACTGCGGCTTCACCA GCGAGAGCTACCAGCAAGGAGTCCTTTCCGCGACTATTCTGTA CGAGATCCTGCTGGGCAAGGCCACCTTGTACGCGGTGCTGGT CTCGGCTCTCGTGCTGATGGCCATGGTGAAGCGGAAGGACAG CCGCGGAAGGGCAAAGCGGAGCGGAAGCGGAGCAACCAATT TCAGCCTGCTGAAGCAGGCCGGCGATGTGGAGGAGAATCCTG GACCAATGGCCTCCGCTCCCATCTCCATGCTCGCCATGCTGTT TACCCTTTCGGGTCTCCGTGCGCAAAGTGTAGCCCAGCCGGAA GACCAGGTGAATGTGGCTGAGGGCAACCCCCTGACCGTCAAG TGCACTTACTCCGTCTCGGGCAACCCTTATCTGTTTTGGTACGT GCAGTACCCTAACCGCGGCCTACAGTTCCTGCTGAAGTACATC ACAGGGGATAACCTGGTGAAAGGCAGCTACGGCTTCGAGGCC GAGTTCAACAAGTCACAGACCTCCTTCCACCTGAAGAAGCCA TCTGCTTTGGTTTCCGACAGCGCACTTTATTTCTGCGCGGTGTA CTTCGGAAACGTGCTGCACTGTGGTAGCGGGACCCAGGTGAT TGTCCTGCCCCATATTCAGAACCCGGATCCTGCCGTGTACCAG CTGCGCGATAGCAAATCTAGCGACAAGAGTGTGTGCCTGTTC ACCGATTTTGACAGCCAGACCAACGTGTCCCAGAGCAAGGAC TCTGATGTCTACATCACCGACAAGACCGTGCTGGACATGCGCT CCATGGACTTCAAGTCCAACTCTGCTGTCGCTTGGAGCAACAA GAGCGACTTCGCCTGTGCCAACGCCTTCAACAACAGCATCATC CCCGAGGACACCTTCTTCCCTTCTCCAGAGAGCAGCTGCGACG TGAAACTGGTGGAAAAGTCCTTCGAGACAGACACTAATCTGA ACTTCCAGAATCTGAGCGTGATCGGCTTCAGAATCCTGCTGCT GAAGGTGGCGGGATTTAATCTCCTGATGACCCTGAGACTGTG
[0475]
[0476] GTCGTCGtaaAttorney Docket No: 046483-7493WOl(04128)
[0477] 96 CMVpp65 betaMGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQ alpha single chain NMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVS PRT RKEKRNFPLILESPSPNQTSLYFCASTSTAGYPGELFFGEGSRLTV LEDLKNVFPPKVAVFEPSEAEISHTQKATLVCLATGFYPDHVELS WWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFW QNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD CGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKR KDSRGRAKRSGSGATNFSLLKQAGDVEENPGPMASAPISMLAML FTLSGLRAQSVAQPEDQVNVAEGNPLTVKCTYSVSGNPYLFWY VQYPNRGLQFLLKYITGDNLVKGSYGFEAEFNKSQTSFHLKKPS ALVSDSALYFCAVYFGNVLHCGSGTQVIVLPHIQNPDPAVYQLR DSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDF KSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEK SFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
[0478] 97 CMV pp65 epitope NLVPMVATV
[0479] 98 SARS-CoV-2 spike KCYGVSPTK
[0480] epitope
[0481] 99 Secondary TCR-30 SAWCQLHR
[0482] IgLON5 epitope
[0483] too Self-cleaving core DxExNPGP
[0484] 2A sequence motif
[0485] 101 P2A linker ATNFSLLKQAGDVEENPGP
[0486] 102 T2A linker EGRGSLLTCGDVEENPGP
[0487] 103 E2A linker QCTNYALLKLAGDVESNPGP
[0488] 104 F2A linker VKQTLNFDLLKLAGDVESNPGP
[0489] 105 Furin P2A linker RAKRSGSGATNFSLLKQAGDVEENPGP
[0490] PRT
[0491] 106 Furin P2A linker AGGGCAAAGCGGAGCGGAAGCGGAGCAACCAATTTCAGCCT DNA GCTGAAGCAGGCCGGCGATGTGGAGGAGAATCCTGGACCA
[0492] 107 Oct4 miRNA GTCCGAGTGTGGTTCTGTA
[0493] sequence
[0494] 108 non-targeting GAGGCAACCGCUCUAGCGCG
[0495] sgRNA-1
[0496] 109 non-targeting GGUUGAACCGCCCCCGCCGA
[0497] sgRNA-2
[0498] 110 TDP-43 sgRNA-1 GGGAAGUCAGCCGUGAGACC
[0499] 111 TDP-43 sgRNA-2 GAGGACUAGGCCCGGUCUCA
[0500] 112 HDGFL2 cryptic CTCGGAGGCTTCTTCACAGACA
[0501] exon primer
[0502] 113 HDGFL2 cryptic AAGACGCCTGCGCTAAAGAT
[0503] exon PCR forward
[0504] primer
[0505] 114 HDGFL2 cryptic CTCGGAGGCTTCTTCACAGACA
[0506] exon PCR reverse
[0507] primer
[0508] 115 HDGFL2 cryptic AAGACGCCTGCGCTAAAGAT
[0509] exon PCR forward
[0510]
[0511] primerAttorney Docket No: 046483-7493WOl(04128)
[0512] HDGFL2 cryptic GCTTCCCTCCCTTCTGATGC
[0513] exon PCR reverse
[0514] primer
[0515] pLKO.l shRNA AGTTAAGCGTGCATAAGTTAACTCGAGTTAACTTATGCACGCT targeted sequence TAACT
[0516] pLKO.l shRNA TTCAATCTCTTGCACTCAAAGCTCGAGCTTTGAGTGCAAGAGA targeted sequence TTGAA
[0517] EMCV IRES GGGCGGCTATAGGGGCGGCTCGAGCGGGATCAATTCCGCCCC CCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGG TGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCGCCCGGA AACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTC CCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAA GGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTC TGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGA CAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACC TGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGAT AGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAA CAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGG ATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAG TCGAGGTTAAAAAAACGTCTAGGCCCCCCGAACCACGGGGAC GTGGTTTTCCTTTGAAAAACACGATGATAATATGGCCACAACC HDGFL2 EKKAAVRAPRRGPLGGRKKKEPTIWFGKGHSGMLASEGREAVL TRLHESERVRKQERERDTEERREKAPSASDSDSKADSDGAKPE
[0518] IgLON5 ARLRLLAAAALAGLAVISRGQPRAGPSHSRSPRSSSRPSSLSAWC QLHRLRMPSPHSLALVQPAGHGLQRDDCVGVTEPACCAEVCEW AWAHTCKHALANLSTQCACVDVCVCKGLLSQSLEFNSPADNYT VCEG TCR-14 epitope TIWFGKGHSGMLAS
[0519] TCR-30 epitope PSSLSAWCQLHRL
[0520] CDR3a CAVMEYGNKLVF
[0521] CDR3b CATHDGNTGELFF
[0522] CDR3a CAYMEYGNKLVF
[0523] CDR3b CATHSGNTGELFF
[0524] CDR3a CAVATGGGADGLTF
[0525] CDR3a CLVGDRGSNYQLIW
[0526] CDR3b CASSTWTASTDTQYF
[0527] CDR3b CAS SVWTASTDTQYF
[0528] TCR14-related TIWFGKGHSGM
[0529]
[0530] epitopeAttorney Docket No: 046483-7493WOl(04128)
[0531] TCR14-related IWFGKGHSGM
[0532] epitope
[0533] TCR14-related WFGKGHSGM
[0534] epitope
[0535] TCR14-related FGKGHSGM
[0536] epitope
[0537] TCR14-related FGKGHSGMLA
[0538] epitope
[0539] TCR14-related FGKGHSGMLAS
[0540] epitope
[0541] TCR14-related FGKGHSG
[0542] epitope
[0543] TCR14-related GKGHSGM
[0544] epitope
[0545] TCR14-related GKGHSGML
[0546] epitope
[0547] TCR14-related GKGHSGMLA
[0548] epitope
[0549] TCR14-related KGHSGMLA
[0550] epitope
[0551] TCR30-related AWCQLHR
[0552] epitope
[0553] TCR30-rclatcd LSAWCQLHR
[0554] epitope
[0555] TCR30-related SLSAWCQLHR
[0556] epitope
[0557] TCR30-related SSLSAWCQLH
[0558] epitope
[0559] TCR30-related SSLSAWCQL
[0560] epitope
[0561] TCR30-related SSLSAWCQ
[0562] epitope
[0563] TCR30-related SSLSAWC
[0564] epitope
[0565] CMV pp65 -related CASSPSTGASYGYTF
[0566] epitope
[0567] CMV pp65 -related CASSMSPGPYEQYF
[0568] epitope
[0569] CMV pp65 -related CASSYSNVGALTDTQYF
[0570] epitope
[0571] CMV pp65 -related CASTSTAGYPGELFF
[0572] epitope
[0573] CMV pp65 -related CAS SYATGTSYGYTF
[0574] epitope
[0575] HDGFL2 cryptic RLHESERVRKQERERD
[0576] epitope
[0577] Spacer SGSG
[0578]
[0579] Attorney Docket No: 046483-7493WOl(04128)
[0580] TCR30-related SAWCQLHR
[0581] epitope
[0582] TCR14-related SLSAWCQLH
[0583] epitope
[0584] TCR30-related SLSAWCQL
[0585] epitope
[0586] TCR30-related LSAWCQLH
[0587] epitope
[0588] TCR30-related PSSLSAWCQLHR
[0589] epitope
[0590] TCR30-related PS SLSAWCQLH
[0591] epitope
[0592] TCR30-related SSLSAWCQLHRL
[0593] epitope
[0594] TCR19 CDR3 alpha AWGTYKYI
[0595] TCR19 CDR3 beta ASSSHQGNTIY
[0596] TCR50 CDR3 alpha AGLSYNTDKLI
[0597] TCR50 CDR3 beta ASSLGAGPQNEQF
[0598] TCR51 (CMV pp65) AVYFGNVLH
[0599] CDR3 alpha
[0600] TCR51 (CMV pp65) ASTSTAGYPGELF
[0601] CDR3 beta
[0602] TCR8 / TCR18 TSENNYY
[0603] CDR1 alpha
[0604] TCR9 CDR1 alpha ATGYPS
[0605] TCR26 / TCR44 DRGSQS
[0606] CDR1 alpha
[0607] TCR25 / TCR31 / DSAIYN
[0608] TCR51 CDR1 alpha
[0609] TCR20 / TCR46 DSASNY
[0610] CDR1 alpha
[0611] TCR47 CDR1 alpha DSVNN
[0612] TCR29 CDR1 alpha KALYS
[0613] TCR22 / TCR39 NIATNDY
[0614] CDR1 alpha
[0615] TCR34 / TCR36. SIFNT
[0616] TCR38 / TCR45 /
[0617] TCR48 CDR1 alpha
[0618] TCR19 CDR1 alpha SSNFYA
[0619]
[0620] Attorney Docket No: 046483-7493WOl(04128)
[0621] TCR24 CDR1 alpha TSDPSYG
[0622] TCR8 / TCR18 TSENNYY
[0623] CDR1 alpha
[0624] TCR32 / TCR40 / TSGFNG
[0625] TCR42
[0626] TCR21 CDR1 alpha TSINN
[0627] TCR37 / TCR49 TSWWSYY
[0628] CDR1 alpha
[0629] TCR33 / TCR41 TTSDR
[0630] CDR1 alpha
[0631] TCR23 CDR1 alpha VSGNPY
[0632] TCR27 CDR1 alpha VSPFSN
[0633] TCR9 CDR2 alpha ATKADDK
[0634] TCR22 / TCR39 GYKTK
[0635] CDR2 alpha
[0636] TCR47 CDR2 alpha IPSGT
[0637] TCR25 / TCR31 / IQSSQRE
[0638] TCR51 CDR2 alpha
[0639] TCR21 CDR2 alpha IRSNERE
[0640] TCR20 / TCR46 IRSNVGE
[0641] CDR2 alpha
[0642] TCR26 / TCR44 IYSNGD
[0643] CDR2 alpha
[0644] TCR29 CDR2 alpha LLKGGEQ
[0645] TCR33 / TCR41 LLSNGAV
[0646] CDR2 alpha
[0647] TCR34 / TCR36 / LYKAGEL
[0648] TCR38 / TCR45 /
[0649] TCR48 CDR2 alpha
[0650] TCR27 CDR2 alpha MTFSENT
[0651] TCR19 CDR2 alpha MTLNGDE
[0652] TCR32 / TCR40 / NVLDGL
[0653] TCR42 CDR2 alpha
[0654] TCR37 / TCR49 QGS
[0655] CDR2 alpha
[0656] TCR24 CDR2 alpha QGSYDQQN
[0657]
[0658] Attorney Docket No: 046483-7493WOl(04128)
[0659] TCR23 CDR2 alpha YITGDNLV
[0660] TCR18 / TCR32 / DFQATT
[0661] TCR39 / TCR42
[0662] CDR1 beta
[0663] TCR47 CDR1 beta ENHRY
[0664] TCR24 / TCR25 / LGHNA
[0665] TCR27 CDR1 beta
[0666] TCR20 / TCR21 / MDHEN
[0667] TCR26 CDR1 beta
[0668] TCR29 / TCR34 / SGDLS
[0669] TCR45 CDR1 beta
[0670] TCR33 / TCR41 SGHDT
[0671] CDR1 beta
[0672] TCR31 CDR1 beta SGHNS
[0673] TCR51 CDR1 beta SGHNT
[0674] TCR19 CDR1 beta SGHTA
[0675] TCR38 CDR1 beta SNHLY
[0676] TCR36 / TCR48 SQVTM
[0677] CDR1 beta
[0678] TCR9 CDR1 beta SSHAT
[0679] TCR36 / TCR48 ANQGSEA
[0680] CDR2 beta
[0681] TCR31 CDR2 beta FNNNVP
[0682] TCR9 CDR2 beta FNYEAQ
[0683] TCR19 CDR2 beta FQGTGA
[0684] TCR38 CDR2 beta FYNNEI
[0685] TCR8 / TCR22 / SMNEV
[0686] TCR46 CDR2 beta
[0687] TCR18 / TCR32 SNEGSKA
[0688] TCR39 / TCR42
[0689] CDR2 beta
[0690] TCR23 / TCR28 / SVGAGI
[0691] TCR43 / TCR44 /
[0692] TCR49 CDR2 beta
[0693] TCR20 / TCR21 / SYDVKM
[0694] TCR26 CDR2 beta
[0695] TCR47 CDR2 beta SYGVKD
[0696]
[0697] 'llAttorney Docket No: 046483-7493WOl(04128)
[0698] TCR24 CDR2 beta YNFKEQ
[0699] TCR25 / TCR27 YSLEER
[0700] CDR2 beta
[0701] TCR33 / TCR41 YYEEEE
[0702] CDR2 beta
[0703] TCR29 / TCR34 / YYNGEE
[0704] TCR45 / TCR50
[0705] CDR2 beta
[0706] TCR50 CDR2 beta YYREEE
[0707] TCR8 V alpha MTRVSLLWAVVVSTCLESGMAQTVTQSQPEMSVQEAETVTLSC TYDTSENNYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVN FQKAAKSFSLKISDSQLGDTAMYFCAFAFSGNTPLVFGKGTRLSV IA TCR9 V alpha MNYSPGLVSLILLLLGRTRGNSVTQMEGPVTLSEEAFLTINCTYT ATGYPSLFWYVQYPGEGLQLLLKATKADDKGSNKGFEATYRKE TTSFHLEKGSVQVSDSAVYFCALSAMDSNYQLIWGAGTKLIIKP TCR18 V alpha MTRVSLLWAVVVSTCLESGMAQTVTQSQPEMSVQEAETVTLSC TYDTSENNYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVN FQKAAKSFSLKISDSQLGDTAMYFCAFGGNKLVFGAGTILRVKS TCR19 V alpha MEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTC SFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLN TKEGYSYLYIKGSQPEDSATYLCAWGTYKYIFGTGTRLKVLA TCR20 V alpha MTSIRAVFIFLWLQLDLVNGENVEQHPSTLSVQEGDSAVIKCTYS DSASNYFPWYKQELGKGPQLIIDIRSNVGEKKDQRIAVTLNKTAK HFSLHITETQPEDSAVYFCAAGYDKIIFGKGTRLHILP TCR21 V alpha METLLGVSLVILWLQLARVNSQQGEEDPQALSIQEGENATMNCS YKTSINNLQWYRQNSGRGLVHLILIRSNEREKHSGRLRVTLDTSK KSSSLLITASRAADTASYFCATESSSYKLIFGSGTRLLVRP TCR22 V alpha MRQVARVIVFLTLSTLSLAKTTQPISMDSYEGQEVNITCSHNNIA TNDYITWYQQFPSQGPRFIIQGYKTKVTNEVASLFIPADRKSSTLS LPRVSLSDTAVYYCLVGDSGWGGGGNKLTFGTGTQLKVEL TCR23 V alpha MASAPISMLAMLFTLSGLRAQSVAQPEDQVNVAEGNPLTVKCT YSVSGNPYLFWYVQYPNRGLQFLLKYITGDNLVKGSYGFEAEFN KSQTSFHLKKPSALVSDSALYFCAVRELSGTYKYIFGTGTRLKVL A TCR24 V alpha MSLSSLLKVVTASLWLGPGIAQKITQTQPGMFVQEKEAVTLDCT YDTSDPSYGLFWYKQPSSGEMIFLIYQGSYDQQNATEGRYSLNF QKARKSANLVISASQLGDSAMYFCAMREGRDSGSARQLTFGSGT QLTVLP TCR25 V alpha METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFT DSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSG RSTLYIAASQPGDSATYLCAVRPDSGAGSYQLTFGKGTKLSVIP TCR26 V alpha MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNC
[0708] TYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNK
[0709]
[0710] ASQYVSLLIRDSQPSDSATYLCAVAGGTSYGKLTFGQGTILTVHPAttorney Docket No: 046483-7493WOl(04128)
[0711] TCR27 V alpha MKKHLTTFLVILWLYFYRGNGKNQVEQSPQSLIILEGKNCTLQC NYTVSPFSNLRWYKQDTGRGPVSLTIMTFSENTKSNGRYTATLD ADTKQSSLHITASQLSDSASYICVVSAGYDYKLSFGAGTTVTVRA TCR28 V alpha MACPGFLWALVISTCLEFSMAQTVTQSQPEMSVQEAETVTLSCT YDTSESDYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNF QKAAKSFSLKISDSQLGDAAMYFCAYGRRRARLMFGDGTQLVV KP TCR29 V alpha METLLKVLSGTLLWQLTWVRSQQPVQSPQAVILREGEDAVINCS SSKALYSVHWYRQKHGEAPVFLMILLKGGEQKGHEKISASFNEK KQQSSLYLTASQLSYSGTYFCGTESNQAGTALIFGKGTTLSVSS TCR31 V alpha METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFT DSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSG RSTLYIAASQPGDSATYLCAVGANSNYQLIWGAGTKLIIKP TCR32 V alpha MWGVFLLYVSMKMGGTTGQNIDQPTEMTATEGAIVQINCTYQT SGFNGLFWYQQHAGEAPTFLSYNVLDGLEEKGRFSSFLSRSKGY SYLLLKELQMKDSASYLCAGPREEGGGADGLTFGKGTHLIIQP TCR33 V alpha MKKLLAMILWLQLDRLSGELKVEQNPLFLSMQEGKNYTIYCNY STTSDRLYWYRQDPGKSLESLFVLLSNGAVKQEGRLMASLDTK ARLSTLHITAAVHDLSATPPQFYFGTGTSLTVIP TCR34 V alpha MLLEHLLIILWMQLTWVSGQQLNQSPQSMFIQEGEDVSMNCTSS SIFNTWLWYKQEPGEGPVLLIALYKAGELTSNGRLTAQFGITRKD SFLNISASIPSDVGIYFCAGPMDSNYQLIWGAGTKLIIKP TCR36 V alpha MLLEHLLIILWMQLTWVSGQQLNQSPQSMFIQEGEDVSMNCTSS SIFNTWLWYKQEPGEGPVLLIALYKAGELTSNGRLTAQFGITRKD SFLNISASIPSDVGIYFCAGQEDTNAGKSTFGDGTTLTVKP TCR37 V alpha MLFSSLLCVFVAFSYSGSSVAQKVTQAQSSVSMPVRKAVTLNCL YETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFK KAAKSVALTISALQLEDSAKYFCALGELPDYKLSFGAGTTVTVR A TCR38 V alpha MLFSSLLCVFVAFSYSGSSVAQKVTQAQSSVSMPVRKAVTLNCL YETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFK KAAKSVALTISALQLEDSAKYFCALGELGNQFYFGTGTSLTVIP TCR39 V alpha MRQVARVIVFLTLSTLSLAKTTQPISMDSYEGQEVNITCSHNNIA TNDYITWYQQFPSQGPRFIIQGYKTKVTNEVASLFIPADRKSSTLS LPRVSLSDTAVYYCLVVRGAGNMLTFGGGTRLMVKP TCR40 V alpha MWGVFLLYVSMKMGGTTGQNIDQPTEMTATEGAIVQINCTYQT SGFNGLFWYQQHAGEAPTFLSYNVLDGLEEKGRFSSFLSRSKGY SYLLLKELQMKDSASYLCAVRDDNAGNMLTFGGGTRLMVKP TCR41 V alpha MKKLLAMILWLQLDRLSGELKVEQNPLFLSMQEGKNYTIYCNY STTSDRLYWYRQDPGKSLESLFVLLSNGAVKQEGRLMASLDTK ARLSTLHITAAVHDLSATPPQFYFGTGTSLTVIP TCR42 V alpha MWGVFLLYVSMKMGGTTGQNIDQPTEMTATEGAIVQINCTYQT SGFNGLFWYQQHAGEAPTFLSYNVLDGLEEKGRFSSFLSRSKGY SYLLLKELQMKDSASYLCAGPREEGGGADGLTFGKGTHLIIQP TCR43 V alpha MACPGFLWALVISTCLEFSMAQTVTQSQPEMSVQEAETVTLSCT YDTSESDYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNF QKAAKSFSLKISDSQLGDAAMYFCAYGRRRARLMFGDGTQLVV KP TCR44 V alpha MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNC
[0712]
[0713] TYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKAttorney Docket No: 046483-7493WOl(04128)
[0714] ASQYVSLLIRDSQPSDSATYLCAVNSPGGGADGLTFGKGTHLIIQ P TCR45 V alpha MLLEHLLIILWMQLTWVSGQQLNQSPQSMFIQEGEDVSMNCTSS SIFNTWLWYKQDPGGGPVLLIALYKAGELTSNGRLTAQFGITRK DSFLNISASIPSDVGIYFCAGPMDSNYQLIWGAGTKLIIKP TCR46 V alpha MTSIRAVFIFLWLQLDLVNGENVEQHPSTLSVQEGDSAVIKCTYS DSASNYFPWYKQELGKGPQLIIDIRSNVGEKKDQRIAVTLNKTAK HFSLHITETQPEDSAVYFCAATNAGNMLTFGGGTRLMVKP TCR47 V alpha MKRILGALLGLLSAQVCCVRGIQVEQSPPDLILQEGANSTLRCNF SDSVNNLQWFHQNPWGQLINLFYIPSGTKQNGRLSATTVATERY SLLYISSSQTTDSGVYFCAVEGSSYKLIFGSGTRLLVRP TCR48 V alpha MLLEHLLIILWMQLTWVSGQQLNQSPQSMFIQEGEDVSMNCTSS SIFNTWLWYKQDPGEGPVLLIALYKAGELTSNGRLTAQFGITRK DSFLNISASIPSDVGIYFCAGQEDTNAGKSTFGDGTTLTVKP TCR49 V alpha MLFSSLLCVFVAFSYSGSSVAQKVTQAQSSVSMPVRKAVTLNCL YETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFK KAAKSVALTISALQLEDSAKYFCALQTGTASKLTFGTGTRLQVT TCR50 V alpha METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFT DSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSG RSTLYIAASQPGDSATYLCAGLSYNTDKLIFGTGTRLQVFP TCR8 V beta MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQ NMNHEYMSWYRQDPGLGLRQ1YYSMNVEVTDKGDVPEGYKVS RKEKRNFPLILESPSPNQTSLYFCASSLLRGGPGLAGRNIQYFGAG TRLSVL TCR9 V beta MGTSLLCWVVLGFLGTDHTGAGVSQSPRYKVTKRGQDVTLRCD PISSHATLYWYQQALGQGPEFLTYFNYEAQPDKSGLPSDRFSAER PEGSISTLTIQRTEQRDSAMYRCASSQTSVSGGELFFGEGSRLTVL TCR18 Vbeta MLLLLLLLGPGISLLLPGSLAGSGLGAVVSQHPSWVICKSGTSVKI ECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQGVEK DKFLINHASLTLSTLTVTSAHPEDSSFYICSARDPQGRFSPLHFGN GTRLTVT TCR19 Vbeta MGTRLLCWAALCLLGADHTGAGVSQTPSNKVTEKGKYVELRC DPISGHTALYWYRQSLGQGPEFLIYFQGTGAADDSGLPNDRFFA VRPEGSVSTLKIQRTERGDSAVYLCASSSHQGNTIYFGEGSWLTV V TCR20 V beta MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQ DMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSR EKKERFSLILESASTNQTSMYLCASSLGESGYGYTFGSGTRLTVV TCR21 Vbeta MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQ DMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSR EKKERFSLILESASTNQTSMYLCASSRSDSPNEKLFFGSGTQLSVL TCR22 V beta MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQ NMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVS RKEKRNFPLILESPSPNQTSLYFCASSLEGDQPQHFGDGTRLSIL TCR23 V beta MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCA QDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNV SRSTTEDFPLRLLSAAPSQTSVYFCASSPGQGNYGYTFGSGTRLT VV TCR24 V beta MGCRLLCCAVLCLLGAVPMETGVTQTPRHLVMGMTNKKSLKC
[0715]
[0716] EQHLGHNAMYWYKQSAKKPLELMFVYNFKEQTENNSVPSRFSPAttorney Docket No: 046483-7493WOl(04128)
[0717] ECPNSSHLFLHLHTLQPEDSALYLCASSQDRGLLSEGYTFGSGTR LEW TCR25 V beta MGCRLLCCAVLCLLGAGELVPMETGVTQTPRHLVMGMTNKKS LKCEQHLGHNAMYWYKQSAKKPLELMFVYSLEERVENNSVPSR FSPECPNSSHLFLHLHTLQPEDSALYLCASSQDVSYGYTFGSGTR LTVV TCR26 V beta MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASRLPGGVNEQFFGPGTRLTVL TCR27 V beta MGCRLLCCAVLCLLGAGELVPMETGVTQTPRHLVMGMTNKKS LKCEQHLGHNAMYWYKQSAKKPLELMFVYSLEERVENNSVPSR FSPECPNSSHLFLHLHTLQPEDSALYLCASSQDAEGDEQYFGPGT RLTVT TCR28 V beta MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCA QDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNV SRSTTEDFPLRLLSAAPSQTSVYFCASSLAGGFPEQYFGPGTRLTV T TCR29 V beta MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSP RSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSAQQF PDLHSELNLSSLELGDSALYFCASSVGQKPYEQYFGPGTRETVT TCR31 Vbeta MDSWTFCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKP ISGHNSLFWYRQTMMRGLELLIYFNNNVPIDDSGMPEDRFSAKM PNASFSTLKIQPSEPRDSAVYFCASSLAWQDTQYFGPGTRLTVL TCR32 V beta MLLLLLLLGPGISLLLPGSLAGSGLGAVVSQHPSWVICKSGTSVKI ECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQGVEK DKFLINHASLTLSTLTVTSAHPEDSSFYICSARDVQPGVSWDEQFF GPGTRLTVL TCR33 V beta MGPGLLCWALLCLLGAGLVDAGVTQSPTHLIKTRGQQVTLRCSP KSGHDTVSWYQQALGQGPQFIFQYYEEEERQRGNFPDRFSGHQF PNYSSELNVNALLLGDSALYLCASSGGDRDYYEQYFGPGTRLTV T TCR34 V beta MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSP RSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSAQQF PDLHSELNLSSLELGDSALYFCASSVEGLAGVDGTGELFFGEGSR LTVL TCR36 V beta MLSLLLLLLGLGSVFSAVISQKPSRDICQRGTSLTIQCQVDSQVT MMFWYRQQPGQSLTLIATANQGSEATYESGFVIDKFPISRPNLTF STLTVSNMSPEDSSIYLCSARGAEGLNNEQFFGPGTRLTVL TCR37 Vbeta MSLGLLCCGAFSLLWAGPVNAGVTQTPKFRVLKTGQSMTLLCA QDMNHEYMYWYRQDPGMGLRLIHYSVGEGTTAKGEVPDGYNV SRLKKQNFLLGLESAAPSQTSVYFCASSGTGTEAFFGQGTRLTVV TCR38 Vbeta MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVP ISNHLYFYWYRQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPD GSNFTLKIRSTKLEDSAMYFCASSEEETGGSEAFFGQGTRLTVV TCR39 V beta MLLLLLLLGPGSGLGAVVSQHPSWVICKSGTSVKIECRSLDFQAT TMFWYRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASL TLSTLTVTSAHPEDSSFYICSAYRPGQGDSTEAFFGQGTRLTVV TCR40 V beta MGTSLLCWMALCLLGADHADTGVSQNPRHKITKRGQNVTFRCD
[0718] PISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAER
[0719]
[0720] PKGSFSTLEIQRTEQGDSAMYLCASSSTLGSYEQYFGPGTRLTVTAttorney Docket No: 046483-7493WOl(04128)
[0721] TCR41 Vbeta MGPGLLCWALLCLLGAGLVDAGVTQSPTHLIKTRGQQVTLRCSP KSGHDTVSWYQQALGQGPQFIFQYYEEEERQRGNFPDRFSGHQF PNYS SELNVN ALLLGDSALYLCAS SGGDRDYYEQYFGPGTRLTV T TCR42 V beta MLLLLLLLGPGSGLGAVVSQHPSRVICKSGTSVKIECRSLDFQAT TMFWYRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASL TLSTLTVTSAHPEDSSFYICSARDVQPGVSWDEQFFGPGTRLTVL TCR43 V beta MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCA QDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNV SRSTTEDFPLRLLSAAPSQTSVYFCASSLAGGFPEQYFGPGTRLTV T TCR44 V beta MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCA QDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNV SRSTTEDFPLRLLSAAPSQTSVYFCASSPRPPMVSYEQYFGPGTRL TVT TCR45 V beta MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSP RSGDLSVYWYQQSLDQGLQFLIHYYNGEERAKGNILERFSAQQF PDLHSELNLSSLELGDSALYFCASSVEGLAGVDGTGELFFGEGSR LTVL TCR46 V beta MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQ NMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVS RKEKRNFPLILESPSPNQTSLYFCASYLTEGGEQFFGPGTRLTVL TCR47 V beta MGTRLFFYVALCLLWTGHMDAGITQSPRHKVTETGTPVTLRCH QTENHRYMYWYRQDPGHGLRLIHYSYGVKDTDKGEVSYGYSV SRSKTEDFLLTLESATSSQTSVYFCAISGAGGRVEQYFGPGTRLT VT TCR48 V beta MLSLLLLLLGLGSVFSAVISQKPSRDICQRGTSLTIQCQVDSQVT MMFWYRQQPGQSLTLIATANQGSEATYESGFVIDKFPISRPNLTF STLTVSNMSPEDSSIYLCSARGAEGLNNEQFFGPGTRLTVL TCR49 V beta MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCA QDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSPDGLPSDEQYFGPGTRLTVT TCR50 V beta MGPGLLCWVLLCLLGAGSVETGVTQSPTHLIKTRGQQVTLRCSS QSGHNTVSWYQQALGQGPQFIFQYYREEENGRGNFPPRFSGLQF PNYSSELNVNALELDDSALYLCASSLGAGPQNEQFFGPGTRLTVL TRBC-2 EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSW WVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQ NPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADC GFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRK DF TCR8 beta-alpha MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQ single chain PRT NMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVS RKEKRNFPLILESPSPNQTSLYFCASSLLRGGPGLAGRNIQYFGAG TRLSVLRAKRSGSGATNFSLLKQAGDVEENPGPMTRVSLLWAV VVSTCLESGMAQTVTQSQPEMSVQEAETVTLSCTYDTSENNYYL FWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLK ISDSQLGDTAMYFCAFAFSGNTPLVFGKGTRLSVIA TCR9 beta-alpha MGTSLLCWVVLGFLGTDHTGAGVSQSPRYKVTKRGQDVTLRCD
[0722]
[0723] single chain PRT PISSHATLYWYQQALGQGPEFLTYFNYEAQPDKSGLPSDRFSAERAttorney Docket No: 046483-7493WOl(04128)
[0724] PEGSISTLTIQRTEQRDSAMYRCASSQTSVSGGELFFGEGSRLTVL RAKRSGSGATNFSLLKQAGDVEENPGPMNYSPGLVSLILLLLGRT RGNSVTQMEGPVTLSEEAFLTINCTYTATGYPSLFWYVQYPGEG LQLLLKATKADDKGSNKGFEATYRKETTSFHLEKGSVQVSDSAV YFCALSAMDSNYQLIWGAGTKLIIKP TCR18 beta-alpha MLLLLLLLGPGISLLLPGSLAGSGLGAVVSQHPSWVICKSGTSVKI single chain PRT ECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQGVEK DKFLINHASLTLSTLTVTSAHPEDSSFYICSARDPQGRFSPLHFGN GTRLTVTRAKRSGSGATNFSLLKQAGDVEENPGPMTRVSLLWA VVVSTCLESGMAQTVTQSQPEMSVQEAETVTLSCTYDTSENNYY LFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSL KISDSQLGDTAMYFCAFGGNKLVFGAGTILRVKS TCR19 beta-alpha MGTRLLCWAALCLLGADHTGAGVSQTPSNKVTEKGKYVELRC single chain PRT DPISGHTALYWYRQSLGQGPEFLIYFQGTGAADDSGLPNDRFFA VRPEGSVSTLKIQRTERGDSAVYLCASSSHQGNTIYFGEGSWLTV VRAKRSGSGATNFSLLKQAGDVEENPGPMEKNPLAAPLLILWFH LDCVSSILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWE TAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPE DSATYLCAWGTYKYIFGTGTRLKVLA TCR20 beta-alpha MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQ single chain PRT DMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSR EKKERFSLILESASTNQTSMYLCASSLGESGYGYTFGSGTRLTVV RAKRSGSGATNFSLLKQAGDVEENPGPMTSIRAVFIFLWLQLDL VNGENVEQHPSTLSVQEGDSAVIKCTYSDSASNYFPWYKQELGK GPQLIIDIRSNVGEKKDQRIAVTLNKTAKHFSLHITETQPEDSAVY FCAAGYDKIIFGKGTRLHILP TCR21 beta-alpha MG1RLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQ single chain PRT DMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSR EKKERFSLILESASTNQTSMYLCASSRSDSPNEKLFFGSGTQLSVL RAKRSGSGATNFSLLKQAGDVEENPGPMETLLGVSLVILWLQLA RVNSQQGEEDPQALSIQEGENATMNCSYKTSINNLQWYRQNSGR GLVHLILIRSNEREKHSGRLRVTLDTSKKSSSLLITASRAADTASY FCATESSSYKLIFGSGTRLLVRP TCR22 beta-alpha MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQ single chain PRT NMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVS RKEKRNFPLILESPSPNQTSLYFCASSLEGDQPQHFGDGTRLSILR AKRSGSGATNFSLLKQAGDVEENPGPMRQVARVIVFLTLSTLSL AKTTQPISMDSYEGQEVNITCSHNNIATNDYITWYQQFPSQGPRFI IQGYKTKVTNEVASLFIPADRKSSTLSLPRVSLSDTAVYYCLVGD SGWGGGGNKLTFGTGTQLKVEL TCR23 beta-alpha MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCA single chain PRT QDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNV SRSTTEDFPLRLLSAAPSQTSVYFCASSPGQGNYGYTFGSGTRLT VVRAKRSGSGATNFSLLKQAGDVEENPGPMASAPISMLAMLFTL SGLRAQSVAQPEDQVNVAEGNPLTVKCTYSVSGNPYLFWYVQY PNRGLQFLLKYITGDNLVKGSYGFEAEFNKSQTSFHLKKPSALVS DSALYFCAVRELSGTYKYIFGTGTRLKVLA TCR24 beta-alpha MGCRLLCCAVLCLLGAVPMETGVTQTPRHLVMGMTNKKSLKC single chain PRT EQHLGHNAMYWYKQSAKKPLELMFVYNFKEQTENNSVPSRFSP
[0725]
[0726] ECPNSSHLFLHLHTLQPEDSALYLCASSQDRGLLSEGYTFGSGTRAttorney Docket No: 046483-7493WOl(04128)
[0727] LTVVRAKRSGSGATNFSLLKQAGDVEENPGPMSLSSLLKVVTAS LWLGPGIAQKITQTQPGMFVQEKEAVTLDCTYDTSDPSYGLFWY KQPSSGEMIFLIYQGSYDQQNATEGRYSLNFQKARKSANLVISAS QLGDSAMYFCAMREGRDSGSARQLTFGSGTQLTVLP TCR25 beta-alpha MGCRLLCCAVLCLLGAGELVPMETGVTQTPRHLVMGMTNKKS single chain PRT LKCEQHLGHNAMYWYKQSAKKPLELMFVYSLEERVENNSVPSR FSPECPNSSHLFLHLHTLQPEDSALYLCASSQDVSYGYTFGSGTR LTVVRAKRSGSGATNFSLLKQAGDVEENPGPMETLLGLLILWLQ LQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQ DPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGD SATYLCAVRPDSGAGSYQLTFGKGTKLSVIP TCR26 beta-alpha MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQ single chain PRT DMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSR EKKERFSLILESASTNQTSMYLCASRLPGGVNEQFFGPGTRLTVL RAKRSGSGATNFSLLKQAGDVEENPGPMKSLRVLLVILWLQLSW VWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYS GKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSA TYLCAVAGGTSYGKLTFGQGTILTVHP TCR27 beta-alpha MGCRLLCCAVLCLLGAGELVPMETGVTQTPRHLVMGMTNKKS single chain PRT LKCEQHLGHNAMYWYKQSAKKPLELMFVYSLEERVENNSVPSR FSPECPNSSHLFLHLHTLQPEDSALYLCASSQDAEGDEQYFGPGT RLTVTRAKRSGSGATNFSLLKQAGDVEENPGPMKKHLTTFLVIL WLYFYRGNGKNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLRW YKQDTGRGPVSLTIMTFSENTKSNGRYTATLDADTKQSSLHITAS QLSDSASYICVVSAGYDYKLSFGAGTTVTVRA TCR28 beta-alpha MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCA single chain PRT QDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSLAGGFPEQYFGPGTRLTVTRAKRSGSGATNFSLLKQAGDVEENPGPMACPGFLWALVISTCL EFSMAQTVTQSQPEMSVQEAETVTLSCTYDTSESDYYLFWYKQP PSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLG DAAMYFCAYGRRRARLMFGDGTQLVVKP TCR29 beta-alpha MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSP single chain PRT RSGDLSVYWYQQSLDQGLQFLTQYYNGEERAKGNTLERFSAQQF PDLHSELNLSSLELGDSALYFCASSVGQKPYEQYFGPGTRLTVTR AKRSGSGATNFSLLKQAGDVEENPGPMETLLKVLSGTLLWQLT WVRSQQPVQSPQAVILREGEDAVINCSSSKALYSVHWYRQKHG EAPVFLMILLKGGEQKGHEKISASFNEKKQQSSLYLTASQLSYSG TYFCGTESNQAGTALIFGKGTTLSVS S TCR31 beta-alpha MDSWTFCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKP single chain PRT ISGHNSLFWYRQTMMRGLELLIYFNNNVPIDDSGMPEDRFSAKM PNASFSTLKIQPSEPRDSAVYFCASSLAWQDTQYFGPGTRLTVLR AKRSGSGATNFSLLKQAGDVEENPGPMETLLGLLILWLQLQWVS SKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGL TSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLC AVGANSNYQLIWGAGTKLIIKP TCR32 beta-alpha MLLLLLLLGPGISLLLPGSLAGSGLGAVVSQHPSWVICKSGTSVKI single chain PRT ECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQGVEK
[0728] DKFLINHASLTLSTLTVTSAHPEDSSFYICSARDVQPGVSWDEQFF
[0729]
[0730] GPGTRLTVLRAKRSGSGATNFSLLKQAGDVEENPGPMWGVFLLAttorney Docket No: 046483-7493WOl(04128)
[0731] YVSMKMGGTTGQNIDQPTEMTATEGAIVQINCTYQTSGFNGLF WYQQHAGEAPTFLSYNVLDGLEEKGRFSSFLSRSKGYSYLLLKE LQMKDSASYLCAGPREEGGGADGLTFGKGTHLIIQP TCR33 beta-alpha MGPGLLCWALLCLLGAGLVDAGVTQSPTHLIKTRGQQVTLRCSP single chain PRT KSGHDTVSWYQQALGQGPQFIFQYYEEEERQRGNFPDRFSGHQF PNYS SELNVN ALLLGD S ALYLC AS SGGDRDYYEQYFGPGTRLTV TRAKRSGSGATNFSLLKQAGDVEENPGPMKKLLAMILWLQLDR LSGELKVEQNPLFLSMQEGKNYTIYCNYSTTSDRLYWYRQDPGK SLESLFVLLSNGAVKQEGRLMASLDTKARLSTLHITAAVHDLSA TPPQFYFGTGTSLTVIP TCR34 beta-alpha MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSP single chain PRT RSGDLSVYWYQQSLDQGLQFLTQYYNGEERAKGNTLERFSAQQF PDLHSELNLSSLELGDSALYFCASSVEGLAGVDGTGELFFGEGSR LTVLRAKRSGSGATNFSLLKQAGDVEENPGPMLLEHLLIILWMQ LTWVSGQQLNQSPQSMFIQEGEDVSMNCTSSSIFNTWLWYKQEP GEGPVLLIALYKAGELTSNGRLTAQFGITRKDSFLNISASIPSDVGI YFCAGPMDSNYQLIWGAGTKLIIKP TCR36 beta-alpha MLSLLLLLLGLGSVFSAVISQKPSRDICQRGTSLTIQCQVDSQVT single chain PRT MMFWYRQQPGQSLTLIATANQGSEATYESGFVIDKFPISRPNLTF STLTVSNMSPEDSSIYLCSARGAEGLNNEQFFGPGTRLTVLRAKR SGSGATNFSLLKQAGDVEENPGPMLLEHLLIILWMQLTWVSGQQ LNQSPQSMFIQEGEDVSMNCTSSSIFNTWLWYKQEPGEGPVLLIA LYKAGELTSNGRLTAQFGITRKDSFLNISASIPSDVGIYFCAGQED TNAGKSTFGDGTTLTVKP TCR37 beta-alpha MSLGLLCCGAFSLLWAGPVNAGVTQTPKFRVLKTGQSMTLLCA single chain PRT QDMNHEYMYWYRQDPGMGLRLIHYSVGEGTTAKGEVPDGYNV SRLKKQNFLLGLESAAPSQTSVYFCASSGTGTEAFFGQGTRLTVV RAKRSGSGATNFSLLKQAGDVEENPGPMLFSSLLCVFVAFSYSGS SVAQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLP SKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDS AKYFCALGELPDYKLSFGAGTTVTVRA TCR38 beta-alpha MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVP single chain PRT ISNHLYFYWYRQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPD GSNFTLKIRSTKLEDSAMYFCASSEEETGGSEAFFGQGTRLTVVR AKRSGSGATNFSLLKQAGDVEENPGPMLFSSLLCVFVAFSYSGSS VAQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPS KEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSA KYFCALGELGNQFYFGTGTSLTVIP TCR39 beta-alpha MLLLLLLLGPGSGLGAVVSQHPSWVICKSGTSVKIECRSLDFQAT single chain PRT TMFWYRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASL TLSTLTVTSAHPEDSSFYICSAYRPGQGDSTEAFFGQGTRLTVVR AKRSGSGATNFSLLKQAGDVEENPGPMRQVARVIVFLTLSTLSL AKTTQPISMDSYEGQEVNITCSHNNIATNDYITWYQQFPSQGPRFI IQGYKTKVTNEVASLFIPADRKSSTLSLPRVSLSDTAVYYCLVVR GAGNMLTFGGGTRLMVKP TCR40 beta-alpha MGTSLLCWMALCLLGADHADTGVSQNPRHKITKRGQNVTFRCD single chain PRT PISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAER PKGSFSTLEIQRTEQGDSAMYLCASSSTLGSYEQYFGPGTRLTVT RAKRSGSGATNFSLLKQAGDVEENPGPMWGVFLLYVSMKMGG
[0732]
[0733] TTGQNIDQPTEMTATEGAIVQINCTYQTSGFNGLFWYQQHAGEAAttorney Docket No: 046483-7493WOl(04128)
[0734] PTFLSYNVLDGLEEKGRFSSFLSRSKGYSYLLLKELQMKDSASYL CAVRDDNAGNMLTFGGGTRLMVKP TCR41 beta-alpha MGPGLLCWALLCLLGAGLVDAGVTQSPTHLIKTRGQQVTLRCSP single chain PRT KSGHDTVSWYQQALGQGPQFIFQYYEEEERQRGNFPDRFSGHQF PNYS SELNVNALLLGDSALYLCAS SGGDRDYYEQYFGPGTRLTV TRAKRSGSGATNFSLLKQAGDVEENPGPMKKLLAMILWLQLDR LSGELKVEQNPLFLSMQEGKNYTIYCNYSTTSDRLYWYRQDPGK SLESLFVLLSNGAVKQEGRLMASLDTKARLSTLHITAAVHDLSA TPPQFYFGTGTSLTVIP TCR42 beta-alpha MLLLLLLLGPGSGLGAVVSQHPSRVICKSGTSVKIECRSLDFQAT single chain PRT TMFWYRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASL TLSTLTVTSAHPEDSSFYICSARDVQPGVSWDEQFFGPGTRLTVL RAKRSGSGATNFSLLKQAGDVEENPGPMWGVFLLYVSMKMGG TTGQNIDQPTEMTATEGAIVQINCTYQTSGFNGLFWYQQHAGEA PTFLSYNVLDGLEEKGRFSSFLSRSKGYSYLLLKELQMKDSASYL CAGPREEGGGADGLTFGKGTHLIIQP TCR43 beta-alpha MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCA single chain PRT QDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNV SRSTTEDFPLRLLSAAPSQTSVYFCASSLAGGFPEQYFGPGTRLTV TRAKRSGSGATNFSLLKQAGDVEENPGPMACPGFLWALVISTCL EFSMAQTVTQSQPEMSVQEAETVTLSCTYDTSESDYYLFWYKQP PSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLG DAAMYFCAYGRRRARLMFGDGTQLVVKP TCR44 beta-alpha MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCA single chain PRT QDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNV SRSTTEDFPLRLLSAAPSQTSVYFCASSPRPPMVSYEQYFGPGTRL TVTRAKRSGSGATNFSLLKQAGDVEENPGPMKSLRVLLVILWLQ LSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYR QYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPS DSATYLCAVNSPGGGADGLTFGKGTHLIIQP TCR45 beta-alpha MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSP single chain PRT RSGDLSVYWYQQSLDQGLQFLIHYYNGEERAKGNILERFSAQQF PDLHSELNLSSLELGDSALYFCASSVEGLAGVDGTGELFFGEGSR LTVLRAKRSGSGATNFSLLKQAGDVEENPGPMLLEHLLIILWMQ LTWVSGQQLNQSPQSMFIQEGEDVSMNCTSSSIFNTWLWYKQDP GGGPVLLIALYKAGELTSNGRLTAQFGITRKDSFLNISASIPSDVGI YFCAGPMDSNYQLIWGAGTKLIIKP TCR46 beta-alpha MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQ single chain PRT NMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVS RKEKRNFPLILESPSPNQTSLYFCASYLTEGGEQFFGPGTRLTVLR AKRSGSGATNFSLLKQAGDVEENPGPMTSIRAVFIFLWLQLDLV NGENVEQHPSTLSVQEGDSAVIKCTYSDSASNYFPWYKQELGKG PQLIIDIRSNVGEKKDQRIAVTLNKTAKHFSLHITETQPEDSAVYF CAATNAGNMLTFGGGTRLMVKP TCR47 beta-alpha MGTRLFFYVALCLLWTGHMDAGITQSPRHKVTETGTPVTLRCH single chain PRT QTENHRYMYWYRQDPGHGLRLIHYSYGVKDTDKGEVSYGYSV SRSKTEDFLLTLESATSSQTSVYFCAISGAGGRVEQYFGPGTRLT VTRAKRSGSGATNFSLLKQAGDVEENPGPMKRILGALLGLLSAQ
[0735]
[0736] VCCVRGIQVEQSPPDLILQEGANSTLRCNFSDSVNNLQWFHQNPAttorney Docket No: 046483-7493WOl(04128)
[0737] WGQLINLFYIPSGTKQNGRLSATTVATERYSLLYISSSQTTDSGV YFCAVEGSSYKLIFGSGTRLLVRP
[0738] 750 TCR48 beta-alpha MLSLLLLLLGLGSVFSAVISQKPSRDICQRGTSLTIQCQVDSQVT single chain PRT MMFWYRQQPGQSLTLIATANQGSEATYESGFVIDKFPISRPNLTF STLTVSNMSPEDSSIYLCSARGAEGLNNEQFFGPGTRLTVLRAKR SGSGATNFSLLKQAGDVEENPGPMLLEHLLIILWMQLTWVSGQQ LNQSPQSMFIQEGEDVSMNCTSSSIFNTWLWYKQDPGEGPVLLIA LYKAGELTSNGRLTAQFGITRKDSFLNISASIPSDVGIYFCAGQED TNAGKSTFGDGTTLTVKP
[0739] 751 TCR49 beta-alpha MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCA single chain PRT QDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNV SRSTTEDFPLRLLSAAPSQTSVYFCASSPDGLPSDEQYFGPGTRLT VTRAKRSGSGATNFSLLKQAGDVEENPGPMLFSSLLCVFVAFSY SGSSVAQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYK QLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQL EDSAKYFCALQTGTASKLTFGTGTRLQVT
[0740] 752 TCR50 beta-alpha MGPGLLCWVLLCLLGAGSVETGVTQSPTHLIKTRGQQVTLRCSS single chain PRT QSGHNTVSWYQQALGQGPQFIFQYYREEENGRGNFPPRFSGLQF PNYSSELNVNALELDDSALYLCASSLGAGPQNEQFFGPGTRLTVL RAKRSGSGATNFSLLKQAGDVEENPGPMETLLGLLILWLQLQW VSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGK GLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATY
[0741]
[0742] LCAGLSYNTDKLIFGTGTRLQVFP
[0743] TCR polypeptides
[0744] Also provided by the invention are recombinant TCR polypeptides, including those encoded by the nucleic acids described above. The TCR polypeptides include the functional portions of any of the TCRs (or functional variants thereof) disclosed herein. With respect to the TCR polypeptides of the invention, the functional portion can be any portion comprising contiguous amino acids of the TCR of which it is a part, provided that the functional portion specifically binds to a cryptic epitope of HDGFL2 or IgL0N5. The term “functional portion” when used in reference to a TCR refers to any part or fragment of the TCR alpha or beta chains of the invention, which retains the biological activity of the original or parent TCR alpha or beta chains of which it is a part.
[0745] Functional portions encompass, for example, those parts of the TCR alpha and / or beta chain(s) that retain the TCR’s ability to specifically bind to a cryptic epitope of HDGFL2 or IgLON5 protein (e.g., as presented on an HLA complex, for example HLA-B*08:01 or HLA-A*03:01), such as those encoded by the polynucleotides described above. In reference to the TCRs of the invention, a functional portion can comprise, for instance, about 10%, 25%, 30%,Attorney Docket No: 046483-7493WOl(04128)
[0746] 50%, 68%, 80%, 90%, 95%, 150%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or more binding activity compared to the original TCR alpha and / or beta chains (or functional variants thereof).
[0747] Tolerable variations of the nucleic acid sequences will be known to those of skill in the art. For example, in some embodiments the nucleic acid(s) encoding the TCRa and TCRp chains of the present invention comprise nucleotide sequence(s) having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity to any of the TCR-encoding nucleotide sequences or TCR encoded amino acid sequences described herein.
[0748] Certain TCRs of the invention have been found to be highly suitable for use in adoptive therapy. Such TCRs may have a KDfor the complex of less than the 100 pM, for example from about 0.05 pM to about 50 pM and / or have a binding half-life (T’A) for the complex in the range of from about 0.5 seconds to about 12 minutes. In some embodiments, TCRs of the invention may have a KDfor the complex of from about 0.25 pM to about 20 pM, from about 0.5 pM to about 15 pM, from about 1 pM to about 10 pM, or from about 2 pM to about 5 pM. Without wishing to be bound by theory, there seems to be an optimum window of affinity for TCRs with therapeutic use in adoptive cell therapy. For example, naturally occurring TCRs recognizing epitopes from tumor antigens are generally of too low affinity (20 pM to 50 pM) and very high affinity TCRs (in the nanomolar range or higher) suffer from cross-reactivity issues (Robbins et al (2008) J. Immunol. 1806116-6131; Zhao et al (2007) J. Immunol. 1795845-5854; Scmid et al (2010) J. Immunol 1844936-4946).
[0749] Techniques for engineering and expressing T cell receptors include, but are not limited to, the production of TCR heterodimers which include the native disulfide bridge which connects the respective subunits (Garboczi, et al., (1996), Nature 384(6605): 134-41; Garboczi, et al., (1996), J Immunol 157(12): 5403-10; Chang et al., (1994), PNAS USA 91: 11408-11412;
[0750] Davodeau et al., (1993), J. Biol. Chem. 268(21): 15455-15460; Golden et al., (1997), J. Imm. Meth. 206: 163-169; U. S. Pat. No. 6,080,840).
[0751] Modified Immune Cells
[0752] In another aspect, the present disclosure provides a population of modified immune cells comprising one or more isolated nucleic acids encoding a cryptic epitope directed TCR, such as a cryptic HDGFL2 epitope-directed TCR or cryptic IgLON5 epitope-directed TCR according to the present disclosure. In some embodiments, the immune cell is selected from the groupAttorney Docket No: 046483-7493WOl(04128)
[0753] consisting of TCR-Treg (CD4+), TCR-Treg / Tsuppressor (CD8+), TCR-TyS, TCR-NK, and TCR-regulatory NK cells. In some embodiments, the immune cell is a T cell. In some embodiments, the T cell is obtained from the group consisting of peripheral blood mononuclear cells, cord blood cells, a purified population of T cells, and a T cell line. In certain embodiments, the T cell is a CD8+T cell, a CD8+Treg cell, a CD8+ suppressor T cell (e.g, KIR+CD8+suppressor T cell), a CD4+T cell, or a CD4+Treg cell.
[0754] In some embodiments, the modified immune cells are isolated from an assay described herein below. The cells may be frozen and expanded for use in other diagnostic or therapeutic methods.
[0755] Techniques for engineering and expressing T cell receptors include, but are not limited to, the production of TCR heterodimers which include the native disulfide bridge connecting the TCRa and TCRp subunits (Garboczi, et al., (1996), Nature 384(6605): 134-41; Garboczi, et al., (1996), J Immunol 157(12): 5403-10; Chang et al., (1994), PNAS USA 91: 11408-11412;
[0756] Davodeau et al., (1993), J. Biol. Chem. 268(21): 15455-15460; Golden et al., (1997), J. Imm. Meth. 206: 163-169; U. S. Pat. No. 6,080,840).
[0757] In some embodiments, a modified T cell of the present invention is genetically edited to disrupt the expression of one or more endogenously expressed genes to reduce the risk of autoreactivity and / or increase the efficacy of the modified cells. In one embodiment, the modified T cell comprises a mutated endogenous genetic locus encoding TCRa chain and / or TCRP chain polynucleotide sequence(s) where the modification(s) reduce or eliminate expression or activity of the endogenous TCR alpha and / or TCR beta chains. In some embodiments, the modified endogenous genetic locus comprises deletion(s) of one or more portions of the TCRa chain and / or TCRP chain polynucleotide sequence(s). In some embodiments, the modifications are designed to ensure that the only TCR expressed by the modified T cell is that encoded by the isolated nucleic acid(s) encoding a cryptic HDGFL2 epitope-directed TCR and / or cryptic IgLON5 epitope-directed TCR according to the present disclosure.
[0758] Alternatively, or in addition, the modified T cell of the present invention may be genetically edited to disrupt the expression of an endogenous Beta-2-microglobulin (B2M) or Class II Major Histocompatibility Complex Transactivator (CIITA).
[0759] In some embodiments, the modification is accomplished using a CRISPR system. TheAttorney Docket No: 046483-7493WOl(04128)
[0760] CRISPR / Cas9 system is a facile and efficient system for inducing targeted genetic alterations. Target recognition by the Cas9 protein requires a ‘seed’ sequence within the guide RNA (gRNA) and a conserved di-nucleotide containing protospacer adjacent motif (PAM) sequence upstream of the gRNA-binding region. The CRISPR / Cas9 system can thereby be engineered to cleave virtually any DNA sequence by redesigning the gRNA in cell lines (such as 293 T cells), primary cells, and CAR T cells. The CRISPR / Cas9 system can simultaneously target multiple genomic loci by co-expressing a single Cas9 protein with two or more gRNAs, making this system uniquely suited for multiple gene editing or synergistic activation of target genes.
[0761] The Cas9 protein and guide RNA form a complex that identifies and cleaves target sequences. Cas9 is comprised of six domains: REC I, REC II, Bridge Helix, PAM interacting, HNH, and RuvC. The Red domain binds the guide RNA, while the Bridge helix binds to target DNA. The HNH and RuvC domains are nuclease domains. Guide RNA is engineered to have a 5’ end that is complementary to the target DNA sequence. Upon binding of the guide RNA to the Cas9 protein, a conformational change occurs activating the protein. Once activated, Cas9 searches for target DNA by binding to sequences that match its protospacer adjacent motif (PAM) sequence. A PAM is a two to six nucleotide base sequence within one nucleotide downstream of the region complementary to the guide RNA. In one non-limiting example, the PAM sequence is 5’-NGG-3’. When the Cas9 protein finds its target sequence with the appropriate PAM, it melts the bases upstream of the PAM and pairs them with the complementary region on the guide RNA. Then the RuvC and HNH nuclease domains cut the target DNA after the third nucleotide base upstream of the PAM.
[0762] One non-limiting example of a CRISPR / Cas system used to inhibit gene expression, CRISPRi, is described in U. S. Publication No. US20140068797. CRISPRi induces permanent gene disruption that utilizes the RNA-guided Cas9 endonuclease to introduce DNA double stranded breaks which trigger error-prone repair pathways to result in frame shift mutations. A catalytically dead Cas9 lacks endonuclease activity. When coexpressed with a guide RNA, a DNA recognition complex is generated that specifically interferes with transcriptional elongation, RNA polymerase binding, or transcription factor binding. This CRISPRi system efficiently represses expression of targeted genes.
[0763] CRISPR / Cas gene disruption occurs when a guide nucleic acid sequence specific for a target gene and a Cas endonuclease are introduced into a cell and form a complex that enablesAttorney Docket No: 046483-7493WOl(04128)
[0764] the Cas endonuclease to introduce a double strand break at the target gene. In certain embodiments, the CRISPR / Cas system comprises an expression vector, such as, but not limited to, an pAd5F35-CRISPR vector. In other embodiments, the Cas expression vector induces expression of Cas9 endonuclease. Other endonucleases may also be used, including but not limited to, T7, Cas3, Cas8a, Cas8b, CaslOd, Csel, Csyl, Csn2, Cas4, CaslO, Csm2, Cmr5, Fokl, other nucleases known in the art, and any combination thereof.
[0765] Sources of Immune Cells
[0766] Typically, the population of immune cells (e.g., T cells) is expanded prior to introduction of the isolated nucleic acid(s). Prior to expansion, a source of immune cells is obtained from a subject for ex vivo manipulation. Sources of target cells for ex vivo manipulation may also include, e.g., autologous or heterologous donor blood, cord blood, or bone marrow. For example, the source of immune cells may be from the subject to be treated with the modified immune cells of the invention, e.g., the subject's blood, the subject's cord blood, or the subject’s bone marrow. Non-limiting examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. In certain exemplary embodiments, the subject is a human.
[0767] T cells can be obtained from a number of sources, including blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, lymph, lymphoid organs, stem cells, such as multipotent and pluripotent stem cells, and induced pluripotent stem cells (iPSCs). In certain embodiments, the cells are human cells. With reference to the subject to be treated, the cells may be allogeneic and / or autologous. The cells typically are primary cells, such as those isolated directly from a subject and / or isolated from a subject and frozen.
[0768] In certain embodiments, the immune cell is selected from the group consisting of TCR-Treg (CD4+), TCR-Treg / Tsuppressor (CD8+), TCR-Tγδ, TCR-NK, and TCR-regulatory NK cells. In some embodiments, the immune cell is a T cell. In some embodiments, the T cell is a CD8+T cell (e.g., a CD8+naive T cell, central memory T cell, or effector memory T cell), a regulatory T cell (e.g., CD4+Treg, a CD8+Treg, or a CD8+ suppressor cell), a CD4+T cell, a natural killer T cell (NKT cells), a gamma-delta T cell, a stem cell memory T cell, a lymphoid progenitor cell, a hematopoietic stem cell, a natural killer cell (NK cell), or a natural killer T cell (NK cell). In some embodiments, the target cell is an induced pluripotent stem (iPS) cell or a cellAttorney Docket No: 046483-7493WOl(04128)
[0769] derived from an iPS cell, e.g., an iPS cell generated from a subject, manipulated to alter (e.g., induce a mutation in) or manipulate the expression of one or more target genes, and differentiated into a T cell, e.g., a CD8+T cell (e.g., a CD8+naive T cell, central memory T cell, or effector memory T cell), a Treg cell (e.g., CD4+Treg, CD8+Treg, or CD8+ suppressor cell), a CD4+T cell, a stem cell memory T cell, a lymphoid progenitor cell or a hematopoietic stem cell. In some embodiments, the CD8+ Treg is HLA-E restricted.
[0770] In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD8+cells, CD4+ / CD8+Treg cells, CD8+ suppressor cells, or CD4+cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and / or persistence capacities, antigen- specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. Among the sub-types and subpopulations of T cells and / or of CD4 and / or of CD8+T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory Treg cells, such as CD4+Treg cells, CD8+Treg cells, CD8+ suppressor cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and gamma / delta T cells. In certain embodiments, any number of T cell lines available in art may be used.
[0771] In some embodiments, the Treg cell expresses the markers CD4, CD25 and FOXP3 (CD4+CD25+FOXP3+). Tregs may also be identified using the cell surface markers CD4 and CD25 in the absence of or in combination with low-level expression of the surface protein CD127(low / -) (CD4+CD25+CD127lo'v / ). Tregs may also express on the cell surface high levels of CTLA-4 (cytotoxic T-lymphocyte associated molecule-4) or GITR (glucocorticoid-induced TNF receptor). Unlike conventional T cells, regulatory T cells do not produce IL-2 and are therefore anergic at baseline. Treg cells include thymus-derived, natural Treg (nTreg) cells and peripherally generated, induced Treg (iTreg) cells.
[0772] In one embodiment, the Treg cell is a CD4+CD25+FOXP3+T cell. In another embodiment, the Treg is a CD4+CD25+CD127~ T cell. In another embodiments, the Treg cell is aAttorney Docket No: 046483-7493WOl(04128)
[0773] CD4+CD25+FOXP3+CD127~ T cell. In some embodiments, the Treg cell is a natural thymus-derived Treg (nTreg) cell that is CD4 CD25 FOXP3 Helios+Neuropilin U. In some embodiments, the Treg cell is an induced Treg (iTreg), which is CD4+CD25+FOXP3+Helios-Neuropilin-T cell which develops from mature CD4+conventional T cells outside of the thymus. For example, iTregs can be induced in vitro from CD4+ CD25-FOXP3- cells in the presence of IL-2 and TGF-b. nTregs have higher expression of PD-1 (programmed cell death-1, pdcdl), neuropilin 1 (Nrpl), Helios (Ikzf2), and CD73. nTregs may be distinguished from iTregs on the basis of the expression of Helios protein or Neuropilin 1 (Nrpl) individually.
[0774] In some embodiments, Treg cells are isolated and transduced with a TCR of interest. Methods for isolating Treg cells are known in the art. See e.g., Mamo et al., Transfusion. 2022 April; 62(4):904-915; US Patent No. 9,801,911 B2; and US Patent Application No.
[0775] 2022 / 0204931 Al, the disclosures of which are expressly incorporated by reference herein. For example, in an exemplary embodiment, PBMCs or CD4 enriched cells are stained with CD4, CD25, and CD 127 antibodies at 4°C for 30 minutes, followed by multiple washes and filtration through a cell strainer. Tregs are sorted via FACS by gating on CD4+, CD25+ and CD127-(negative) and sorting into collection tubes at 4°C and then cryopreserved or put into immediate culture. For culture, Tregs are maintained in RPMI-10 complete media with 300 lU / mL IL-2, refreshed every 2 days, and expanded with ImmunoCult, passaging every 1-2 days. Lentiviral transduction or electroporation is performed on days D1-D3.
[0776] In some embodiments, suppressor CD8+ T cells, such as KIR+CD8+ T cells, are isolated from PBMCs using CD8 microbeads (Miltenyi) per manufacturer’s instructions, stained with CD3, CD56, CD8, and KIR antibodies at 4°C for 30 minutes, followed by multiple washes and filtration through a cell strainer. (See e.g., Li et al., Science 376, 265 (2022)). KIR+ suppressor CD8+ cells are sorted via FACS by gating on CD3+, CD56, CD8+, and KIR+and sorting into collection tubes at 4°C and then cryopreserved or put into immediate culture.
[0777] In some embodiments, the methods include isolating immune cells from the subject, preparing, processing, culturing, and / or engineering them. In some embodiments, preparation of the engineered cells includes one or more culture and / or preparation steps. The cells for engineering as described may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. In some embodiments, the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cellAttorney Docket No: 046483-7493WOl(04128)
[0778] therapy will be administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and / or engineered. Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. The samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g, transduction with viral vector), washing, and / or incubation. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.
[0779] In certain aspects, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and / or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.
[0780] Wang et al., J Immunother Cancer. 2021; 9(12): e003339 and U. S. Patent Publication No. 2024 / 197876 Al are incorporated herein by reference for their disclosures of compositions and methods for isolating gamma-delta T cells from a subject, and preparing, processing, culturing, and / or engineering them.
[0781] In some embodiments, the cells are derived from cell lines, e.g., T cell lines. The cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, nonhuman primate, and pig. In some embodiments, isolation of the cells includes one or more preparation and / or non-affinity-based cell separation steps. In some examples, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, and lyse or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and / or resistance to particular components.Attorney Docket No: 046483-7493WOl(04128)
[0782] In some examples, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples, in certain aspects, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and / or platelets, and in certain aspects contains cells other than red blood cells and platelets. In some embodiments, the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In certain embodiments, the cells are resuspended in a variety of biocompatible buffers after washing. In certain embodiments, components of a blood cell sample are removed, and the cells directly resuspended in culture media. In some embodiments, the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.
[0783] In one embodiment, T cells are obtained from the circulating blood of an individual are obtained by apheresis or leukapheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. The cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media, such as phosphate buffered saline (PBS) or wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations, for subsequent processing steps. As those of ordinary skill in the art would readily appreciate a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca2+-free, Mg2+-free PBS, PlasmaLyte A, or another saline solution with or without buffer. In some embodiments, the undesirable components of the apheresis sample may be removed, and the cells directly resuspended in culture media.
[0784] In some embodiments, the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surfaceAttorney Docket No: 046483-7493WOl(04128)
[0785] markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is affinity- or immunoaffinity-based separation. For example, the isolation in certain aspects includes separation of cells and cell populations based on the cells' expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner. Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and / or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In certain aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population. The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.
[0786] In certain exemplary embodiments, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In certain exemplary embodiments, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.
[0787] In some embodiments, one or more of the T cell populations is enriched for or depleted of cells that are positive for (marker+) or express high levels (markerhigh) of one or moreAttorney Docket No: 046483-7493WOl(04128)
[0788] particular markers, such as surface markers, or that are negative for (marker) or express relatively low levels (markerlow) of one or more markers. For example, in certain embodiments, specific subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, e.g, CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+T cells, are isolated by positive or negative selection techniques. In some cases, such markers are those that are absent or expressed at relatively low levels on certain populations of T cells (such as non-memory cells) but are present or expressed at relatively higher levels on certain other populations of T cells (such as memory cells). In one embodiment, the cells (such as the CD8+cells or the T cells, e.g., CD3+cells) are enriched for (i.e., positively selected for) cells that are positive or expressing high surface levels of CD45RO, CCR7, CD28, CD27, CD44, CD127, and / or CD62L and / or depleted of e.g., negatively selected for) cells that are positive for or express high surface levels of CD45RA. In some embodiments, cells are enriched for or depleted of cells positive or expressing high surface levels of CD122, CD95, CD25, CD27, and / or IL7-Ra (CD 127). In certain exemplary embodiments, CD8 T cells are enriched for cells positive for CD45RO (or negative for CD45RA) and for CD62L. For example, CD3+, CD28+T cells can be positively selected using CD3 / CD28 conjugated magnetic beads (e.g, DYNABEADS® M-450 CD3 / CD28 T Cell Expander).
[0789] In some embodiments, T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD14. In certain aspects, a CD4+or CD8+selection step is used to separate CD4+helper and CD8+cytotoxic T cells. Such CD4+and CD8+populations can be further sorted into subpopulations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and / or effector T cell subpopulations. In some embodiments, CD8+cells are further enriched for or depleted of naive, central memory, effector memory, and / or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, enrichment for central memory T (TCM) cells is carried out to increase efficacy, such as to improve longterm survival, expansion, and / or engraftment following administration, which in certain aspects is particularly robust in such sub-populations. In some embodiments, combining TCM-enriched CD8+T cells and CD4+T cells further enhances efficacy.Attorney Docket No: 046483-7493WOl(04128)
[0790] In some embodiments, memory T cells are present in both CD62L+and CD62L' subsets of CD8+peripheral blood lymphocytes. PBMC can be enriched for or depleted of CD62L-CD8+and / or CD62L+CD8+fractions, such as using anti-CD8 and anti-CD62L antibodies. In some embodiments, a CD4+T cell population and / or a CD8+T population is enriched for central memory (TCM) cells. In some embodiments, the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD 127; in certain aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and / or granzyme B. In certain aspects, isolation of a CD8+population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD 14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment for central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD 14 and CD45RA, and a positive selection based on CD62L. Such selections in certain aspects are carried out simultaneously and in other aspects are carried out sequentially, in either order. In some embodiments, the same CD4 expression-based selection step used in preparing the CD8+cell population or subpopulation, also is used to generate the CD4+cell population or subpopulation, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps.
[0791] CD4+T helper cells can be sorted into naive, central memory, and effector cells by identifying cell populations that have cell surface antigens. CD4+lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+T lymphocytes are CD45RO-, CD45RA+, CD62L+, CD4+T cells. In some embodiments, central memory CD4+cells are CD62L+and CD45RO+. In some embodiments, effector CD4+cells are CD62L" and CD45RO. In one example, to enrich for CD4+cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD1 lb, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and / or negative selection.
[0792] In some embodiments, the cells are incubated and / or cultured prior to or in connection with genetic engineering. The incubation steps can include culture, cultivation, stimulation,Attorney Docket No: 046483-7493WOl(04128)
[0793] activation, and / or propagation. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and / or survival of cells in the population, to mimic antigen exposure, and / or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor. The conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and / or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells. In some embodiments, the stimulating conditions or agents include one or more agent, e.g., ligand, which is capable of activating an intracellular signaling domain of a TCR complex. In certain aspects, the agent turns on or initiates TCR / CD3 intracellular signaling cascade in a T cell. Such agents can include antibodies, such as those specific for a TCR component and / or costimulatory receptor, e.g., anti-CD3, anti-CD28, for example, bound to solid support such as a bead, and / or one or more cytokines. Optionally, the expansion method may further comprise the step of adding anti-CD3 and / or anti CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng / ml). In some embodiments, the stimulating agents include IL-2 and / or IL- 15, for example, an IL-2 concentration of at least about 10 units / mL.
[0794] In another embodiment, T cells are isolated from peripheral blood by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient. Alternatively, T cells can be isolated from an umbilical cord. In any event, a specific subpopulation of T cells can be further isolated by positive or negative selection techniques.
[0795] The cord blood mononuclear cells so isolated can be depleted of cells expressing certain antigens, including, but not limited to, CD34, CD8, CD14, CD19, and CD56. Depletion of these cells can be accomplished using an isolated antibody, a biological sample comprising an antibody, such as ascites, an antibody bound to a physical support, and a cell bound antibody.
[0796] Enrichment of a T cell population by negative selection can be accomplished using a combination of antibodies directed to surface markers unique to the negatively selected cells. An exemplary method is cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+cells by negativeAttorney Docket No: 046483-7493WOl(04128)
[0797] selection, a monoclonal antibody cocktail typically includes antibodies to CD 14, CD20, CD1 lb, CD 16, HLA-DR, and CD8.
[0798] For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, greater than 100 million cells / ml is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet another embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells / ml is used. In further embodiments, concentrations of 125 or 150 million cells / ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion.
[0799] T cells can also be frozen after the washing step, which does not require the monocyteremoval step. While not wishing to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, in a non-limiting example, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or other suitable cell freezing media. The cells are then frozen to -80°C at a rate of 1°C per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20°C or in liquid nitrogen.
[0800] Expansion of T Cells
[0801] Whether prior to or after modification of T cells to express a TCR according to the present invention, the T cells can be activated and expanded in number using methods as described, for example, in U. S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964;
[0802] 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843;
[0803] 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U. S. Publication No. 20060121005.Attorney Docket No: 046483-7493WOl(04128)
[0804] Generally, T cells are expanded by contact with a surface having attached thereto an agent that stimulates a CD3 / TCR complex associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T cells. The present invention comprises a novel method of expanding a population of modified electroporated or transduced T cells comprising culturing the electroporated or transduced population, wherein the modified electroporated or transduced T cells within the population expand at least 10-fold. Expression of the recombinant TCR alpha and beta chains of the invention allows interaction with other cells in the population to stimulate and activate expansion of the modified electroporated or transduced T cells. In one embodiment, at least one cell in the population of cells expresses CD3.
[0805] For example, the T cells of the invention may be expanded by contact with a surface having attached thereto an agent that stimulates a CD3 / TCR complex associated signal and a ligand that stimulates a co- stimulatory molecule on the surface of the T cells. In particular, T cell populations may be stimulated by contact with an anti-CD3 antibody, or an antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For co-stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) and these can be used in the invention, as can other methods and reagents known in the art (see, e.g, ten Berge et al., Transplant Proc. (1998) 30(8): 3975-3977; Haanen et al., J. Exp. Med. (1999) 190(9): 1319-1328; and Garland et al., J. Immunol. Methods (1999) 227(1-2): 53-63).
[0806] Expanding the T cells by the methods disclosed herein can be multiplied by about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700 fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold, or greater, and any and all whole or partial integers therebetween. In one embodiment, the T cells expand in the range of about 20-fold to about 50-fold.
[0807] Following culturing, the T cells can be incubated in cell medium in a culture apparatus for a period of time or until the cells reach confluency or high cell density for optimal passageAttorney Docket No: 046483-7493WOl(04128)
[0808] before passing the cells to another culture apparatus. The culturing apparatus can be of any culture apparatus commonly used for culturing cells in vitro. In certain exemplary embodiments, the level of confluence is 70% or greater before passing the cells to another culture apparatus. In particularly exemplary embodiments, the level of confluence is 90% or greater. A period of time can be any time suitable for the culture of cells in vitro. The T cell medium may be replaced during the culture of the T cells at any time. In certain exemplary embodiments, the T cell medium is replaced about every 2 to 3 days. The T cells are then harvested from the culture apparatus whereupon the T cells can be used immediately or cryopreserved to be stored for use at a later time. In one embodiment, the invention includes cryopreserving the expanded T cells. The cryopreserved T cells are thawed prior to introducing nucleic acids into the T cell.
[0809] In another embodiment, the method comprises isolating T cells and expanding the T cells. In another embodiment, the invention further comprises cryopreserving the T cells prior to expansion. In yet another embodiment, the cryopreserved T cells are thawed for electroporation with nucleic acid(s) (e.g., DNA or RNA) encoding the TCR.
[0810] The culturing step as described herein (contact with agents as described herein or after electroporation) can be very short, for example less than 24 hours such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours. The culturing step as described further herein (contact with agents as described herein) can be longer, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days.
[0811] Various terms are used to describe cells in culture. Cell culture refers generally to cells taken from a living organism and grown under controlled conditions. A primary cell culture is a culture of cells, tissues or organs taken directly from an organism and before the first subculture. Cells are expanded in culture when they are placed in a growth medium under conditions that facilitate cell growth and / or division, resulting in a larger population of the cells. When cells are expanded in culture, the rate of cell proliferation is typically measured by the amount of time required for the cells to double in number, otherwise known as the doubling time.
[0812] Each round of subculturing is referred to as a passage. When cells are subcultured, they are referred to as having been passaged. A specific population of cells, or a cell line, is sometimes referred to or characterized by the number of times it has been passaged. For example, a cultured cell population that has been passaged ten times may be referred to as a P10 culture. The primary culture, i.e., the first culture following the isolation of cells from tissue, isAttorney Docket No: 046483-7493WOl(04128)
[0813] designated P0. Following the first subculture, the cells are described as a secondary culture (Pl or passage 1). After the second subculture, the cells become a tertiary culture (P2 or passage 2), and so on. It will be understood by those of skill in the art that there may be many population doublings during the period of passaging. Therefore, the number of population doublings of a culture is greater than the passage number. The expansion of cells (i.e., the number of population doublings) during the period between passaging depends on many factors, including but is not limited to the seeding density, substrate, medium, and time between passaging.
[0814] In one embodiment, the cells may be cultured for several hours (about 3 hours) to about 14 days or any hourly integer value in between. Conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g, fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-gamma, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF-beta, and TNF-a or any other additives for the growth of cells known to the skilled artisan. Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. Media can include RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or an amount of cytokine(s) sufficient for the growth and expansion of T cells. Antibiotics, e.g., penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells that are to be infused into a subject. The target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (c.g., 37° C) and atmosphere (e.g., air plus 5% CO2).
[0815] The medium used to culture the T cells may include an agent that can co-stimulate the T cells. For example, an agent that can stimulate CD3 is an antibody to CD3, and an agent that can stimulate CD28 is an antibody to CD28. This is because, as demonstrated by the data disclosed herein, a cell isolated by the methods disclosed herein can be expanded approximately 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold, or greater. In one embodiment, the T cells expand in the rangeAttorney Docket No: 046483-7493WOl(04128)
[0816] of about 2-fold to about 50-fold, or more by culturing the electroporated population. In one embodiment, human T regulatory cells are expanded via anti-CD3 antibody coated KT64.86 artificial antigen presenting cells (aAPCs). Methods for expanding and activating immune cells can be found in U. S. Patent Numbers 7,754,482, 8,722,400, and 9,555,105, the contents of which are incorporated herein in their entirety.
[0817] In one embodiment, the method of expanding the immune cells can further comprise isolating the expanded immune cells for further applications. In another embodiment, the method of expanding can further comprise a subsequent electroporation of the expanded immune cells followed by culturing. The subsequent electroporation may include introducing a nucleic acid encoding an agent, such as transducing the expanded immune cells, transfecting the expanded immune cells, or electroporating the expanded immune cells with a nucleic acid, into the expanded population of immune cells, wherein the agent further stimulates the immune cell. The agent may stimulate the immune cells, such as by stimulating further expansion, effector function, or another immune cell function.
[0818] Sources of T Cells
[0819] Prior to expansion, a source of T cells is obtained from a subject. Non-limiting examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. Preferably, the subject is a human. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, and tumors. In certain embodiments, any number of T cell lines available in art may be used. In certain embodiments, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll separation. In one embodiment, cells from the circulating blood of an individual are obtained by apheresis or leukapheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. The cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media, such as phosphate buffered saline (PBS) or wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations, for subsequent processing steps. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS. Alternatively, theAttorney Docket No: 046483-7493WOl(04128)
[0820] undesirable components of the apheresis sample may be removed, and the cells directly resuspended in culture media.
[0821] In another embodiment, T cells are isolated from peripheral blood by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient. Alternatively, T cells can be isolated from umbilical cord. In any event, a specific subpopulation of T cells can be further isolated by positive or negative selection techniques.
[0822] The cord blood mononuclear cells so isolated can be depleted of cells expressing certain antigens, including, but not limited to, CD34, CD8, CD14, CD19 and CD56. Depletion of these cells can be accomplished using an isolated antibody, a biological sample comprising an antibody, such as ascites, an antibody bound to a physical support, and a cell bound antibody.
[0823] Enrichment of a T cell population by negative selection can be accomplished using a combination of antibodies directed to surface markers unique to the negatively selected cells. A preferred method is cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CDllb, CD16, HLA-DR, and CD8.
[0824] For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, greater than 100 million cells / ml is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet another embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells / ml is used. In further embodiments, concentrations of 125 or 150 million cells / ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion.
[0825] T cells can also be frozen after the washing step, which does not require the monocyteremoval step. While not wishing to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in theAttorney Docket No: 046483-7493WOl(04128)
[0826] cell population. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, in a non-limiting example, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or other suitable cell freezing media. The cells are then frozen to -80°C at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20°C or in liquid nitrogen.
[0827] In one embodiment, the population of T cells is comprised within cells such as peripheral blood mononuclear cells, cord blood cells, a purified population of T cells, and a T cell line. In another embodiment, peripheral blood mononuclear cells comprise the population of T cells. In yet another embodiment, purified T cells comprise the population of T cells.
[0828] In another embodiment, the T cells are isolated from cells such as peripheral blood mononuclear cells, cord blood cells, a purified population of T cells, and a T cell line. In another embodiment, the method described herein further comprises isolating a population of T cells from peripheral blood mononuclear cells, cord blood cells, a purified population of T cells, or a T cell line.
[0829] Methods of Generating the Modified TCR Transduced T Cells
[0830] In another aspect, the present invention provides a method for generating a modified TCR transduced T cell as described herein. In an embodiment, the method comprises introducing into the T cell a nucleic acid encoding a TCRa chain and / or TCRβ chain described herein or a recombinant expression vector encoding a TCRα chain and / or TCRβ chain described herein such that the modified T cell expresses a TCR having antigen specificity for a cryptic epitope presented in a complex with a human MHC I polypeptide. The nucleic acid may be introduced by any means, such as transducing the expanded T cells, transfecting the expanded T cells, and electroporating the expanded T cells.
[0831] In some embodiments of the method, the TCR specifically binds a cryptic epitope of HDGFL2. In one embodiment, the HDGFL2 cryptic epitope comprises an amino acid sequence set forth in any one of SEQ ID NOs: 7, 10, 13, or 34.Attorney Docket No: 046483-7493WOl(04128)
[0832] In other embodiments of the method, the TCR specifically binds a cryptic epitope of IgLON5. In one embodiment, the cryptic epitope comprises an amino acid sequence set forth in any one of SEQ ID NOs: 34, 43, or 60.
[0833] In an embodiment of the method, the modified T cell is obtained from the group consisting of peripheral blood mononuclear cells, cord blood cells, a purified population of T cells, and a T cell line. In one embodiment, the T cell is a, a CD8+T cell, a CD8+Treg cell, a CD8+ suppressor cell, a CD4+T cell, or a CD4+Treg cell.
[0834] In some embodiments, the method further comprises the step of modifying expression of endogenous TCRa and / or TCRβ chains. In one embodiment, the step of modifying expression of the endogenous TCRα and / or TCRβ chains is accomplished by use of a CRISPR system as further described herein.
[0835] Methods of introducing nucleic acids into a cell include physical, biological and chemical methods. Physical methods for introducing a polynucleotide, such as RNA, into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. RNA can be introduced into target cells using commercially available methods which include electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg Germany). RNA can also be introduced into cells using cationic liposome mediated transfection using lipofection, using polymer encapsulation, using peptide mediated transfection, or using biolistic particle delivery systems such as “gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001).
[0836] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U. S. Pat. Nos. 5,350,674 and 5,585,362.
[0837] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.Attorney Docket No: 046483-7493WOl(04128)
[0838] An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).
[0839] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, MO; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, NY); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the only solvent since it is more readily evaporated than methanol. “Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10).
[0840] However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic acid complexes.
[0841] Regardless of the method used to introduce nucleic acids into a host cell or otherwise expose a cell to the inhibitor of the present disclosure, in order to confirm the presence of the nucleic acids in the host cell, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention
[0842] Methods for Stimulating a T cell-Mediated Immune Response and Treating a TDP-43 Associated ProteinopathyAttorney Docket No: 046483-7493WOl(04128)
[0843] In another aspect, the invention includes a method for stimulating a T cell-mediated immune response to a target cell or tissue in a subject that expresses a cryptic epitope of HDGFL2 or IgLON5 protein, comprising administering to the subject an effective amount of a modified T cell comprising a nucleic acid encoding a T cell receptor (TCR) specific for a cryptic epitope of HDGFL2 or IgLON5 protein.
[0844] In another aspect, the invention includes a method of treating a TDP-43 associated proteinopathy in a subject exhibiting cryptic epitope expression of HDGFL2 or IgLON5, wherein the subject is administered a therapeutically effective amount of a pharmaceutical composition comprising a modified T cell comprising a nucleic acid encoding a T cell receptor (TCR) specific for a cryptic epitope of HDGFL2 or IgLON5 protein and a pharmaceutically acceptable carrier or excipient.
[0845] Cells of the invention can be administered in dosages and routes and at times to be determined in appropriate pre-clinical and clinical experimentation and trials. Cell compositions may be administered multiple times at dosages within these ranges as further described herein. Administration of the cells of the invention may be combined with other methods useful to treat the desired disease or condition as determined by those of skill in the art.
[0846] The cells of the invention to be administered may be autologous, allogeneic or xenogenic with respect to the subject undergoing therapy.
[0847] In an exemplary embodiment, the TDP-43 proteinopathy is selected from the group consisting of amyotrophic lateral sclerosis (ALS), inclusion body myositis (IBM), frontotemporal dementia (FTD, such as Sporadic or familial with or without motor-neuron disease (MND), frontotemporal lobar degeneration (FTLD), motor neuron disease (MND), Alzheimer’s disease (AD), Parkinson's disease (PD), Huntington’s disease, chronic traumatic encephalopathy (CTE), facial onset sensory and motor neuronopathy (FOSMN), limbic-predominant age-related TDP-43 encephalopathy (LATE), primary progressive aphasia (PPA), Perry disease, Guam parkinsonism-dementia, progressive supranuclear palsy (PSP), Corticobasal degeneration (CBD), Argyrophilic grain disease (AGD) and a Lewy body-related disease.
[0848] In one embodiment, the TDP-43 proteinopathy is ALS. In another embodiment, the TDP-43 proteinopathy is IBM. In another embodiment, the TDP-43 proteinopathy is FTD.
[0849] Administration of the modified T cells of the invention may be carried out in any convenient manner known to those of skill in the art. In some embodiments, the modified T cellsAttorney Docket No: 046483-7493WOl(04128)
[0850] are administered to a subject by injection, transfusion, or intrathecal administration. In some embodiments, the modified T cells described herein are administered to a patient intratumorally, transarterially, subcutaneously, intradermally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In other instances, the modified T cells of the invention are injected directly into a local disease site in the subject, such as muscle or cerebrospinal fluid.
[0851] For the prevention or treatment of disease, the appropriate dosage may depend on the type of disease to be treated, the type of targeting proteins or target proteins, the severity and course of the disease, whether the modified T cells are administered for preventive or therapeutic purposes, previous therapy, the subject's clinical history and response to the modified T cells, and / or the discretion of the attending physician. In some embodiments, the compositions in some embodiments are suitably administered to the subject at one time or over a series of treatments.
[0852] In some embodiments, the modified T cells are administered at a desired dosage, which in some aspects includes a desired dose or number of cells or cell type(s) and / or a desired ratio of cell types. Thus, the dosage of cells in some embodiments is based on a total number of cells (or number per kg body weight) and a desired ratio of the individual populations or sub-types, such as the CD4+to CD8+ratio. In some embodiments, the dosage of cells is based on a desired total number (or number per kg of body weight) of cells in the individual populations or of individual cell types. In some embodiments, the dosage is based on a combination of such features, such as a desired number of total cells, desired ratio, and desired total number of cells in the individual populations.
[0853] In certain embodiments, modified T cells are administered to the subject at a range of about one million to about 100 billion cells, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to aboutAttorney Docket No: 046483-7493WOl(04128)
[0854] 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value in between these ranges.
[0855] In some embodiments, the dose of total cells and / or dose of individual sub-populations of cells is within a range of between at or about IxlO5cells / kg to about IxlO11cells / kg 104and at or about 1011cells / kilograms (kg) body weight, such as between 105and 106cells / kg body weight, for example, at or about 1 x 105cells / kg, 1.5 x 105cells / kg, 2 x 105cells / kg, or 1 x 106cells / kg body weight. For example, in some embodiments, the cells are administered at, or within a certain range of error of, between at or about 104and at or about 109T cells / kilograms (kg) body weight, such as between 105and 106T cells / kg body weight, for example, at or about 1 x 105T cells / kg, 1.5 x 105T cells / kg, 2 x 105T cells / kg, or 1 x 106T cells / kg body weight.
[0856] In other exemplary embodiments, a suitable dosage range of modified cells for use in a method of the present disclosure includes, without limitation, from about IxlO5cells / kg to about IxlO6cells / kg, from about IxlO6cells / kg to about IxlO7cells / kg, from about IxlO7cells / kg to about IxlO8cells / kg, from about IxlO8cells / kg to about IxlO9cells / kg, from about IxlO9cells / kg to about IxlO10cells / kg, from about IxlO10cells / kg to about IxlO11cells / kg. In an exemplary embodiment, a suitable dosage for use in a method of the present disclosure is about IxlO8cells / kg. In an exemplary embodiment, a suitable dosage for use in a method of the present disclosure is about IxlO7cells / kg. In other embodiments, a suitable dosage is from about IxlO7total cells to about 5xl07total cells. In some embodiments, a suitable dosage is from about IxlO8total cells to about 5xl08total cells. In some embodiments, a suitable dosage is from about 1.4x107total cells to about 1.1x109total cells. In an exemplary embodiment, a suitable dosage for use in a method of the present disclosure is about 7xl09total cells.
[0857] In some embodiments, the modified T cells are administered at or within a certain range of error of between at or about 104and at or about 109CD4+and / or CD8+cells / kilograms (kg) body weight, such as between 105and 106Treg, CD8+or CD4+cells / kg body weight, for example, at or about 1 x 105Treg, CD8+or CD4+cells / kg, 1.5 x 105Treg, CD8+or CD4+cells / kg, 2 x 105Treg, CD8+or CD4+cells / kg, or 1 x 106Treg, CD8+or CD4+cells / kg body weight. In some embodiments, the T cells are administered in an amount of at least about 1 x 106, about 2.5 x 106, about 5 x 106, about 7.5 x 106, or about 9 x 106T cells. In someAttorney Docket No: 046483-7493WOl(04128)
[0858] embodiments, the cells are administered at or within a certain range of error of between about 108and 1012or between about 1010and 1011T cells, between about 108and 1012or between about 1010and 1011CD4+cells, and / or between about 108and 1012or between about 1010and 1011CD8+cells.
[0859] In some embodiments, the modified T cells are administered at or within a tolerated range of a desired output ratio of multiple cell populations or sub-types, such as Treg, CD4+and CD8+cells or sub-types. In some aspects, the desired ratio can be a specific ratio or can be a range of ratios, for example, in some embodiments, the desired ratio (e.g., ratio of CD4+to CD8+cells) is between at or about 5: 1 and at or about 5: 1 (or greater than about 1:5 and less than about 5: 1), or between at or about 1:3 and at or about 3: 1 (or greater than about 1:3 and less than about 3: 1), such as between at or about 2: 1 and at or about 1:5 (or greater than about 1:5 and less than about 2: 1, such as at or about 5: 1, 4.5: 1, 4: 1, 3.5: 1, 3: 1, 2.5: 1, 2: 1, 1.9: 1, 1.8: 1, 1.7: 1, 1.6: 1, 1.5: 1, 1.4: 1, 1.3: 1, 1.2: 1, 1.1: 1, 1: 1, 1: 1.1, 1: 1.2, 1: 1.3, 1:1.4, 1: 1.5, 1: 1.6, 1: 1.7, 1: 1.8, 1: 1.9: 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5. In some aspects, the tolerated difference is within about 1%, about 2%, about 3%, about 4% about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50% of the desired ratio, including any value in between these ranges.
[0860] In some embodiments, a dose of modified cells is administered to a subject in need thereof, in a single dose or multiple doses. In some embodiments, a dose of modified cells is administered in multiple doses, e.g., once a week or every 7 days, once every 2 weeks or every 14 days, once every 3 weeks or every 21 days, once every 4 weeks or every 28 days. In an exemplary embodiment, a single dose of modified cells is administered to a subject in need thereof. In an exemplary embodiment, a single dose of modified cells is administered to a subject in need thereof by rapid intravenous infusion.
[0861] In certain embodiments, activated T cells are administered to a subject and then blood is subsequently redrawn (or have an apheresis performed), after which the T cells are activated therefrom according to the present invention, and then reinfused into the subject with these activated and expanded T cells. This process can be carried out multiple times every few weeks. In certain embodiments, T cells can be activated from blood draws of from 10 ml to 400 ml. In certain embodiments, T cells are activated from blood draws of 20 ml, 30 ml, 40 ml, 50 ml, 60 ml, 70 ml, 80 ml, 90 ml, or 100 ml.Attorney Docket No: 046483-7493WOl(04128)
[0862] For the prevention or treatment of disease, the appropriate dosage may depend on the type of disease to be treated, the type of cells or recombinant receptors, the severity and course of the disease, whether the cells are administered for preventive or therapeutic purposes, previous therapy, the subject's clinical history and response to the cells, and the discretion of the attending physician. The compositions and cells are in some embodiments suitably administered to the subject at one time or over a series of treatments.
[0863] In some embodiments, the modified T cells described herein are administered using any number of matrices acting as an artificial lymphoid organ to support, maintain, or modulate the immune system, typically through modulation of T cells. Accordingly, the present invention can utilize those matrix compositions and formulations which have demonstrated utility in tissue engineering. Accordingly, the type of matrix that may be used in the compositions, devices and methods of the invention is virtually limitless and may include both biological and synthetic matrices. In one particular example, the compositions and devices set forth by U. S. Pat. Nos. 5,980,889; 5,913,998; 5,902,745; 5,843,069; 5,787,900; or 5,626,561 are utilized, as such these patents are incorporated herein by reference in their entirety. Matrices comprise features commonly associated with being biocompatible when administered to a mammalian host.
[0864] Matrices may be formed from natural and / or synthetic materials. The matrices may be non-biodegradable in instances where it is desirable to leave permanent structures or removable structures in the body of an animal, such as an implant, or biodegradable. The matrices may take the form of sponges, implants, tubes, telfa pads, fibers, hollow fibers, lyophilized components, gels, powders, porous compositions, or nanoparticles. In addition, matrices can be designed to allow for sustained release of seeded cells or produced cytokine or other active agent. In certain embodiments, the matrix of the present invention is flexible and elastic, and may be described as a semisolid scaffold that is permeable to substances such as inorganic salts, aqueous fluids and dissolved gaseous agents including oxygen.
[0865] As used herein, the matrix is an example of a biocompatible substance. However, the current invention is not limited to matrices and thus, wherever the term matrix or matrices appears these terms should be read to include devices and other substances which allow for cellular retention or cellular traversal, are biocompatible, and are capable of allowing traversal of macromolecules either directly through the substance such that the substance itself is a semi-permeable membrane or used in conjunction with a particular semi-permeable substance.Attorney Docket No: 046483-7493WOl(04128)
[0866] In some embodiments, the cells are administered as part of a combination treatment, such as simultaneously with or sequentially with, in any order, another therapeutic agent or therapeutic intervention. In certain embodiments, the additional therapeutic agent or therapy is selected from the group consisting of chimeric-antigen receptor (CAR)-T cell therapy, monoclonal antibody therapy, biologic therapy or any combination thereof. In some embodiments, the cells are co-administered with another therapy sufficiently close in time such that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cells are administered prior to the one or more additional therapeutic agents. In some embodiments, the cells are administered after the one or more additional therapeutic agents. In some embodiments, the one or more additional agents include a cytokine, such as IL-2 or IL- 10. In some embodiments, the additional agent is an anti-KLRGl antibody (Ulviprubart) capable of selectively depleting highly cytotoxic T cells, while sparing naive, regulatory and central memory T cells.
[0867] Following administration of the modified T cells, the biological activity of the engineered cell populations in some embodiments can be measured, e.g., by any of a number of known methods. Parameters to assess include specific binding of an engineered or natural T cell or other immune cell to antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004). In certain embodiments, the biological activity of the cells is measured by assaying expression and / or secretion of one or more cytokines, such as CD 107a, IFNy, IL-2, and TNF.
[0868] In some embodiments, the subject can be administered a conditioning therapy prior to engineered TCR T cell therapy. In some embodiments, the conditioning therapy comprises administering an effective amount of cyclophosphamide to the subject. In some embodiments, the conditioning therapy comprises administering an effective amount of fludarabine to the subject. In preferred embodiments, the conditioning therapy comprises administering an effective amount of a combination of cyclophosphamide and fludarabine to the subject. Administration of a conditioning therapy prior to TCR T cell therapy may increase the efficacy of the TCR T cellAttorney Docket No: 046483-7493WOl(04128)
[0869] therapy. Methods of conditioning patients for T cell therapy are described in U. S. Patent No. 9,855,298, which is incorporated herein by reference in its entirety.
[0870] In some embodiments, a specific dosage regimen of the present disclosure includes a lymphodepletion step prior to the administration of the modified T cells. In an exemplary embodiment, the lymphodepletion step includes administration of cyclophosphamide and / or fludarabine.
[0871] In some embodiments, the lymphodepletion step includes administration of cyclophosphamide at a dose of between about 200 mg / m2 / day and about 2000 mg / m2 / day (e.g., 200 mg / m2 / day, 300 mg / m2 / day, or 500 mg / m2 / day). In an exemplary embodiment, the dose of cyclophosphamide is about 300 mg / m2 / day. In some embodiments, the lymphodepletion step includes administration of fludarabine at a dose of between about 20 mg / m2 / day and about 900 mg / m2 / day (e.g., 20 mg / m2 / day, 25 mg / m2 / day, 30 mg / m2 / day, or 60 mg / m2 / day). In an exemplary embodiment, the dose of fludarabine is about 30 mg / m2 / day.
[0872] In some embodiments, the lymphodepletion step includes administration of cyclophosphamide at a dose of between about 200 mg / m2 / day and about 2000 mg / m2 / day (e.g., 200 mg / m2 / day, 300 mg / m2 / day, or 500 mg / m2 / day), and fludarabine at a dose of between about 20 mg / m2 / day and about 900 mg / m2 / day (e.g., 20 mg / m2 / day, 25 mg / m2 / day, 30 mg / m2 / day, or 60 mg / m2 / day). In an exemplary embodiment, the lymphodepletion step includes administration of cyclophosphamide (e.g., by intravenous infusion) at a dose of about 300 mg / m2 / day and fludarabine at a dose of about 30 mg / m2 / day (e.g., over three days prior to administration of the modified T cells.
[0873] Dosing of lymphodepletion chemotherapy may be scheduled on Days -6 to -4 (with a -1-day window, i.e., dosing on Days -7 to -5) relative to TCR T cell infusion on Day 0.
[0874] Cells of the invention can be administered in dosages and routes and at times to be determined in appropriate pre-clinical and clinical experimentation and trials. Cell compositions may be administered multiple times at dosages within these ranges. Administration of the cells of the invention may be combined with other methods useful to treat the desired disease or condition as determined by those of skill in the art.
[0875] It is known in the art that one of the adverse effects following infusion of recombinant TCR T cells is the onset of immune activation, known as cytokine release syndrome (CRS). CRS is immune activation resulting in elevated inflammatory cytokines. CRS is a known on-Attorney Docket No: 046483-7493WOl(04128)
[0876] target toxicity, development of which likely correlates with efficacy. Clinical and laboratory measures range from mild CRS (constitutional symptoms and / or grade-2 organ toxicity) to severe CRS (sCRS; grade >3 organ toxicity, aggressive clinical intervention, and / or potentially life threatening). Clinical features include high fever, malaise, fatigue, myalgia, nausea, anorexia, tachycardia / hypotension, capillary leak, cardiac dysfunction, renal impairment, hepatic failure, and disseminated intravascular coagulation. Dramatic elevations of cytokines including interferon-gamma, granulocyte macrophage colony-stimulating factor, IL- 10, and IL-6 have been shown following CAR T-cell infusion. One CRS signature is elevation of cytokines including IL-6 (severe elevation), IFN-gamma, TNF-alpha (moderate), and IL-2 (mild). Elevations in clinically available markers of inflammation including ferritin and C-reactive protein (CRP) have also been observed to correlate with the CRS syndrome. The presence of CRS generally correlates with expansion and progressive immune activation of adoptively transferred cells. It has been demonstrated that the degree of CRS severity is dictated by disease burden at the time of infusion as patients with high tumor burden experience a more sCRS.
[0877] Accordingly, the invention provides for, following the diagnosis of CRS, appropriate CRS management strategies to mitigate the physiological symptoms of uncontrolled inflammation without dampening the antitumor efficacy of the engineered cells (e.g, CAR-T or CAR-NK cells). CRS management strategies are known in the art. For example, systemic corticosteroids may be administered to rapidly reverse symptoms of sCRS (e.g, grade 3 CRS) without compromising initial antitumor response.
[0878] In some embodiments, an anti-IL-6R antibody may be administered. An example of an anti-IL-6R antibody is the Food and Drug Administration-approved monoclonal antibody tocilizumab, also known as atlizumab (marketed as Actemra, or RoActemra). Tocilizumab is a humanized monoclonal antibody against the interleukin-6 receptor (IL-6R). Administration of tocilizumab has demonstrated near-immediate reversal of CRS.
[0879] CRS is generally managed based on the severity of the observed syndrome and interventions are tailored as such. CRS management decisions may be based upon clinical signs and symptoms and response to interventions, not solely on laboratory values alone.
[0880] Mild to moderate cases generally are treated with symptom management with fluid therapy, non-steroidal anti-inflammatory drug (NSAID) and antihistamines as needed for adequate symptom relief More severe cases include patients with any degree of hemodynamicAttorney Docket No: 046483-7493WOl(04128)
[0881] instability; with any hemodynamic instability, the administration of tocilizumab is recommended. The first-line management of CRS may be tocilizumab, in some embodiments, at the labeled dose of 8 mg / kg IV over 60 minutes (not to exceed 800 mg / dose); tocilizumab can be repeated every 8 hours. If suboptimal response to the first dose of tocilizumab, additional doses of tocilizumab may be considered. Tocilizumab can be administered alone or in combination with corticosteroid therapy. Patients with continued or progressive CRS symptoms, inadequate clinical improvement in 12-18 hours or poor response to tocilizumab, may be treated with high-dose corticosteroid therapy, generally hydrocortisone 100 mg IV or methylprednisolone 1-2 mg / kg. In patients with more severe hemodynamic instability or more severe respiratory symptoms, patients may be administered high-dose corticosteroid therapy early in the course of the CRS. CRS management guidance may be based on published standards (Lee et al. (2019) Biol Blood Marrow Transplant, doi.org / 10.1016 / j.bbmt.2018.12.758; Neelapu et al. (2018) Nat Rev Clin Oncology, 15:47; Teachey etal. (2016) Cancer Discov, 6(6):664-679).
[0882] Features consistent with Macrophage Activation Syndrome (MAS) or Hemophagocytic lymphohistiocytosis (HLH) have been observed in patients treated with CAR-T therapy (Henter, 2007), coincident with clinical manifestations of the CRS. MAS appears to be a reaction to immune activation that occurs from the CRS, and should therefore be considered a manifestation of CRS. MAS is similar to HLH (also a reaction to immune stimulation). The clinical syndrome of MAS is characterized by high grade non-remitting fever, cytopenias affecting at least two of three lineages, and hepatosplenomegaly. It is associated with high serum ferritin, soluble interleukin-2 receptor, and triglycerides, and a decrease of circulating natural killer (NK) activity.
[0883] Pharmaceutical compositions and formulations
[0884] Also provided are populations of immune cells of the invention, compositions containing cells and / or enriched for such cells, such as in which cells expressing the TCR make up at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of the total cells in the composition or cells of a certain type such as Treg cells, CD8+T cells, or CD4+T cells. Among the compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy. Also provided are therapeutic methods for administering the cells and compositions to subjects, e.g., patients.Attorney Docket No: 046483-7493WOl(04128)
[0885] Also provided are compositions including the cells for administration, including pharmaceutical compositions and formulations, such as unit dose form compositions including the number of cells for administration in a given dose or fraction thereof. The pharmaceutical compositions and formulations generally include one or more optional pharmaceutically acceptable carrier or excipient. In some embodiments, the composition includes at least one additional therapeutic agent. The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
[0886] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative. In some aspects, the choice of carrier is determined in part by the particular cell and / or by the method of administration. Accordingly, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, e.g., by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol;Attorney Docket No: 046483-7493WOl(04128)
[0887] salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).
[0888] Buffering agents in some aspects are included in the compositions. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).
[0889] The formulations can include aqueous solutions. The formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated with the cells, preferably those with activities complementary to the cells, where the respective activities do not adversely affect one another. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended. Thus, in some embodiments, the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as low-dose IL-2, anti-KLRGl antibodies, and / or anti-CD40L antibodies. The pharmaceutical composition in some embodiments contains the cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. The desired dosage can be delivered by a single bolus administration of the cells, by multiple bolus administrations of the cells, or by continuous infusion administration of the cells.
[0890] Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell populations are administered parenterally. The term "parenteral," as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cells are administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection. Compositions in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, whichAttorney Docket No: 046483-7493WOl(04128)
[0891] may in some aspects be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.
[0892] Sterile injectable solutions can be prepared by incorporating the cells in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, and / or colors, depending upon the route of administration and the preparation desired. Standard texts may in some aspects be consulted to prepare suitable preparations.
[0893] Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, and sorbic acid. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
[0894] The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.
[0895] It should be understood that the method and compositions that would be useful in the present invention are not limited to the particular formulations set forth in the examples. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the cells, expansion and culture methods, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.
[0896] The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology,Attorney Docket No: 046483-7493WOl(04128)
[0897] biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, fourth edition (Sambrook, 2012); “Oligonucleotide Synthesis” (Gait, 1984); “Culture of Animal Cells” (Freshney, 2010); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1997); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Short Protocols in Molecular Biology” (Ausubel, 2002); “Polymerase Chain Reaction: Principles, Applications and Troubleshooting”, (Babar, 2011); “Current Protocols in Immunology” (Coligan, 2002). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention.
[0898] Diagnostic applications
[0899] In another aspect, the present disclosure provides an assay, comprising:
[0900] culturing a biological sample comprising T cells, wherein the T cells are aliquoted into a plurality of wells;
[0901] stimulating the T cells in each of the plurality of wells with (i) a pooled library of overlapping peptides, (ii) one or more sub-pools thereof, and / or (iii) one or more individual peptides encompassing a polypeptide of interest under conditions suitable for expansion of the T cells;
[0902] optionally re-stimulating the T cells in each well with the same pooled library, subpools) thereof, or individual peptide(s) to which the T cells in the well were previously exposed; and
[0903] exposing the T cells in each well to reagents suitable for detecting one or more activation-induced T cell markers,
[0904] wherein detection of the one or more activation-induced T cell markers in a well indicates the presence of a T cell expressing a TCR having antigen specificity for a peptide within the pooled library, sub-pool(s) thereof, or one or more individual peptides thereof to which the T cells in the well were exposed.
[0905] In some embodiments, the pooled library may be tested in one or more wells and, if positive, deconvoluted by testing sub-pools and / or individual peptides.Attorney Docket No: 046483-7493WOl(04128)
[0906] In some embodiments, the assay detects TCR in a subject recognizing a cryptic epitope. In some embodiments, the polypeptide of interest comprises a cryptic epitope that is associated with a TDP-43 proteinopathy. Exemplary polypeptides associated with TDP-43 proteinopathies are encoded by genes selected from the group consisting of AARS, ACTL6B, ARHGAP22, CELSR3, DNM1, EPB41L4A, HDGFL2, IGLON5, MYO18A, NECAB2, PHF2, PTPRZ1, PXDN, SLC24A2, STMN2, SYNJ2, XPO4, and ZNF423.
[0907] In some embodiments, the peptide libraries and / or multimer panel collectively span at least 10, at least 20, at least 30, or at least 50 cryptic-epitope-bearing proteins associated with TDP-43 proteinopathy to address patient heterogeneity.
[0908] In some embodiments, the subject has or is suspected of having a TDP-43 proteinopathy. In some embodiments, the TDP-43 proteinopathy is indicative of the subject having a heterogenous neurodegenerative and inflammatory condition selected from the group consisting of amyotrophic lateral sclerosis (ALS), inclusion body myositis (IBM), frontotemporal dementia (FTD), frontotemporal lobar degeneration (FTLD), motor neuron disease (MND), Alzheimer’s disease (AD), Parkinson's disease (PD), Huntington’s disease, chronic traumatic encephalopathy (CTE), facial onset sensory and motor neuronopathy (FOSMN), limbic-predominant age-related TDP-43 encephalopathy, primary progressive aphasia (PPA), Perry disease, Guam parkinsonismdementia, and a Lewy body-related disease.
[0909] In a more particular embodiment, the TDP-43 proteinopathy is ALS or IBM.
[0910] Biological samples may be obtained from any suitable source, including whole blood, PBMCs, leukapheresis products, buffy coat, or tissue-resident immune cells obtained from biopsy or surgical specimens. In some embodiments, T cells are obtained from cerebrospinal fluid (CSF), muscle biopsy, brain-adjacent fluids, lymph node aspirates / biopsies, spleen, or other inflamed tissues. In some embodiments, the assay is performed using CD8+T cells, CD4+T cells, or regulatory T cells, optionally enriched by magnetic selection or flow sorting. In some embodiments, the sample comprises tumor-infiltrating lymphocytes (TILs) or tissue-infiltrating lymphocytes from neuroinflammatory lesions. In some embodiments, the sample comprises engineered T cells or cell lines expressing TCRs (e.g., primary T cells transduced with candidate TCR sequences or immortalized T cell lines), including TCRs identified from single-cell sequencing of antigen-reactive cells.Attorney Docket No: 046483-7493WOl(04128)
[0911] In some embodiments, the biological sample is from a patient blood collection or healthy patient donation. In some embodiments, the biological sample is selected from whole blood, PBMCs, or cryopreserved PBMCs. In certain embodiments, the PBMCs are enriched for CD8+T cells and / or CD4+ T cells.
[0912] In some embodiments, the overlapping peptide library comprises peptides having a length of about 3 to about 40 amino acids (e.g., 3, 4, 5,..., 40 amino acids). In some embodiments, the peptides are generated using a sliding window step size of 1 to 40 amino acids (e.g., step size 1, 2, 3,..., 40), such that adjacent peptides are offset by the step size across the full-length sequence of the polypeptide of interest. In certain embodiments, the peptide library comprises 8-11-mers, 9-10-mers, 15-mers, 20-mers, 25-mers, 30-mers, 35-mers, and / or 40-mers, optionally provided as pooled libraries, sub-pools, or individual peptides for deconvolution / epitope mapping.
[0913] In some embodiments, adjacent peptides in the pooled library are generated using a sliding window having a step size of between about 1-40 amino acids, and optionally wherein each peptide in the pooled library overlaps an adjoining peptide by between about 1-39 amino acids, optionally between about 3-10 amino acids
[0914] In some embodiments, the length of the peptides in the overlapping pool is between about 10-40 amino acids, between about 15-35 amino acids, between about 20-30 amino acids, or any integer or integer range thereof. In certain embodiments, each peptide in a plurality of the peptides is about 15, 20, 25, 30, 35, or 40 amino acids in length. In one embodiment, the overlapping pool of peptides are 25-mers encompassing the entire length of the protein of interest. In some embodiments, each of the peptides shares an overlap of between about 3, 4, 5, 6, 7, 8, 9, or 10 amino acids with another peptide. In an embodiment, each peptide in the pool comprises an overlap with an adjoining peptide of about 3-10 amino acids, 4-8 amino acids, or 5-6 amino acids, or any integer range thereof. In some embodiments, the assay controls include the vehicle (e.g., DMSO) as negative control; a positive control stimulant (e.g., CEF (cytomegalovirus, Epstein-Barr virus, and influenza virus) control peptide pool for immune cell activation and / or anti-CD3 / anti-CD28 and / or PMA / ionomycin).
[0915] In some embodiments, T cells from any of the foregoing sources are analyzed using one or more of three complementary assays: (1) an AIM assay to measure functional activation in response to cryptic epitope peptide libraries (e.g., AIM or %CD69+CD137+within CD8+TAttorney Docket No: 046483-7493WOl(04128)
[0916] cells); (2) a multimer-based enrichment assay (e.g., TetTCR-SeqHD cell sorting) in which peptide-HLA multimer-positive T cells are isolated and a normalized multimer-positive frequency score is computed (e.g., multimer-positive cells per input T cells, optionally normalized to negative-control multimers); and (3) a sequencing assay performed on multimer-positive or AIM-positive cells to obtain TCRα / β sequences and derive clonality metrics (e.g., clone size, clonal expansion, and / or public / shared clonotypes). In some embodiments, outputs from assays (l)-(3) are combined into a composite “cryptic-reactivity” score for clinical interpretation.
[0917] In some embodiments, the methods are used for (a) early disease diagnosis, (b) monitoring therapeutic effect, and / or (c) patient stratification. For example, a subject may be classified as likely having a TDP-43 proteinopathy-associated immune signature when one or more assay outputs (AAIM, normalized multimer frequency, and / or clonal expansion metrics) exceed a predetermined threshold relative to reference cohorts. In some embodiments, longitudinal sampling is performed, and changes in AAIM, multimer-positive frequency, and / or clone size are used to monitor response to an immunomodulatory therapy or disease progression. In some embodiments, the methods stratify clinically similar syndromes by identifying TDP-43 -associated immune signatures, including distinguishing Alzheimer’s disease without TDP-43 pathology from Alzheimer’s disease with concomitant TDP-43 pathology, and distinguishing ALS, IBM, FTLD / FTD, or mixed neurodegenerative / inflammatory conditions based on cryptic-epitope reactivity patterns across a multi-antigen panel.
[0918] In some embodiments, the assay further comprises the step of isolating T cells from the well in which the one or more activation-induced T cell markers were detected using flow cytometry and cell sorting.
[0919] In some embodiments, culture reagents for the assay include complete T cell media (e.g., RPMI + serum), cytokines as needed for optional expansion (e.g., IL-2, IL-7, IL- 15), as well as suitable plates and tubes for stimulation.
[0920] In some embodiments, detection reagents for the assay may include fluorophore-labeled antibodies for CD8 and AIM markers (e.g., CD69 and CD137 (4-1BB), and may optionally include antibodies and detection reagents for CD 134 (0X40), CD25, CD40L, or CD71; viability dyes; and optional dump channel markers (e.g., CD14 / CD19) to exclude non-T cells.Attorney Docket No: 046483-7493WOl(04128)
[0921] In another aspect, the present invention provides a modified T cell produced by the TCR detection assay described herein.
[0922] Optional reagents for increased specificity may include, for example, secretion inhibitors (e.g., brefeldin A / monensin) to enable intracellular CD69 / CD137 assessment, as well as optional intracellular cytokine stains (e.g., IFNy, TNFa, IL-2) and / or cytotoxic mediators (e.g., GZMB).
[0923] In some embodiments, the foregoing methods facilitate iterative mapping, wherein if a pool of peptides test positive, sub-pools or individual peptides thereof are tested to identify the reactive peptide(s). In some instances, the AIM+CD8+T cells may be isolated for TCR sequencing to identify TCRs recognizing cryptic epitopes.
[0924] In other embodiments, the T cells are further cultured in the presence of an antigen-presenting cell (APC) selected from the group consisting of autologous PBMC APCs, dendritic cells, B cells, and engineered APC cell lines.
[0925] In some embodiments, sequencing comprises single-cell RNA sequencing and / or paired TCR sequencing of antigen-reactive cells to additionally derive phenotype state metrics (e.g., cytotoxic / effector, exhaustion, differentiation, or activation signatures), which may be used for prognosis or treatment monitoring.
[0926] In other embodiments, the methods described herein facilitate alternative readouts, including AIM+frequency, cytokine production, cytotoxicity markers, transcriptional profiling, and combined multiparameter scoring.
[0927] In another aspect, the disclosure provides a method of detecting a TDP-43 proteinopathy-associated immune signature in a subject, comprising:
[0928] (a) contacting T cells obtained from a biological sample from the subject with a panel of peptide–HLA multimers presenting a plurality of cryptic epitopes;
[0929] (b) enriching or isolating multimer-positive T cells by cell sorting;
[0930] (c) determining a normalized multimer-positive frequency score based on multimer-positive T cells relative to input T cells and / or relative to a negative-control multimer; and (d) classifying the subject as positive for the TDP-43 proteinopathy-associated immune signature when the normalized multimer-positive frequency score satisfies a predetermined criterion.
[0931] In an embodiment, the predetermined criterion for classifying a positive subject is based on the relative normalized multimer frequency to a reference distribution generated from anAttorney Docket No: 046483-7493WOl(04128)
[0932] appropriate comparator cohort, healthy and / or disease controls lacking TDP-43 pathology, rather than an absolute event cutoff. For example, after calculating the normalized multimer-positive frequency score, number of multimer-positive cells per input T cells with optional background correction, a subject is classified as positive when its score exceeds a threshold derived from the distribution of scores from comparator cohort. The determined threshold can be the mean plus k=2 or 3 standard deviations of the healthy-control distribution, greater than a specified percentile of the healthy-control distribution (95th or 99th percentile), and / or greater than a cutoff determined from training data (an ROC-derived threshold selected to achieve a desired sensitivity / specificity).
[0933] In some embodiments, where a multimer panel spanning many cryptic targets is used to address patient heterogeneity, the predetermined criterion is applied at the panel level, for example by classifying a subject as positive when the subject exceeds the reference-derived threshold for at least one epitope, for at least m epitopes, and / or when an aggregate panel score (summed or weighted normalized frequencies across the panel) exceeds a reference-derived cutoff.
[0934] In some embodiments, the plurality of cryptic epitopes comprises cryptic epitopes derived from at least 10, at least 20, at least 30, or at least 50 proteins associated with a TDP-43 proteinopathy.
[0935] In some embodiments, the cryptic epitopes are selected from proteins encod...
Claims
Attorney Docket No: 046483-7493WOl(04128)CLAIMSWhat is claimed:
1. An isolated nucleic acid encoding a T cell receptor (TCR), the nucleic acid comprising a first polynucleotide encoding a TCRa (TRA) chain polynucleotide and a second polynucleotide encoding a TCRβ (TRB) chain polypeptide, wherein the TRA and TRB form a paired complex having an antigenic specificity for a cryptic epitope presented in a complex with a human MHC I polypeptide, wherein:a) the epitope is a cryptic epitope of HDGFL2 comprising the amino acid sequence of SEQ ID NO: 7, the MHC I is HLA-B*08:01, the TRA comprises a third complementarity determining region (CDR3) comprising the amino acid sequence of SEQ ID NO: 3, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 6;b) the epitope is a cryptic epitope of HDGFL2 comprising the amino acid sequence of SEQ ID NO: 10, the MHC I is HLA-A*03:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 8, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 9;c) the epitope is a cryptic epitope of HDGFL2 comprising the amino acid sequence of SEQ ID NO: 13, the MHC I is HLA-A*03:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 11, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 12;d) the epitope is a cryptic epitope of HDGFL2 comprising the amino acid sequence of SEQ ID NO: 13, the MHC I is HLA-A*03:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 14, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 15;e) the epitope is a cryptic epitope of HDGFL2 comprising the amino acid sequence of SEQ ID NO: 13, the MHC I is HLA-A*03:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 16, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 17;f) the epitope is a cryptic epitope of HDGFL2 comprising the amino acid sequence of SEQ ID NO: 13, the MHC I is HLA-A*03:01, the TRA comprises a CDR3 comprising theAttorney Docket No: 046483-7493WOl(04128)amino acid sequence of SEQ ID NO: 18, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 19;g) the epitope is a cryptic epitope of HDGFL2 comprising the amino acid sequence of SEQ ID NO: 13, the MHC I is HLA-A*03:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 20, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 21;h) the epitope is a cryptic epitope of HDGFL2 comprising the amino acid sequence of SEQ ID NO: 13, the MHC I is HLA-A*03:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 22, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 23;i) the epitope is a cryptic epitope of HDGFL2 comprising the amino acid sequence of SEQ ID NO: 13, the MHC I is HLA-A*03:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 24, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 25;j) the epitope is a cryptic epitope of HDGFL2 comprising the amino acid sequence of SEQ ID NO: 13, the MHC I is HLA-A*03:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 26, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 27;k) the epitope is a cryptic epitope of HDGFL2 comprising the amino acid sequence of SEQ ID NO: 13, the MHC I is HLA-A*03:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 28, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 29;l) the epitope is a cryptic epitope of HDGFL2 comprising the amino acid sequence of SEQ ID NO: 13, the MHC I is HLA-A*03:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 30, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 31;m) the epitope is a cryptic epitope of IgLON5 comprising the amino acid sequence of SEQ ID NO: 34, the MHC I is HLA-B*35:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 32, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 33;Attorney Docket No: 046483-7493WOl(04128)n) the epitope is a cryptic epitope of IgLON5 comprising the amino acid sequence of SEQ ID NO: 34, the MHC I is HLA-B*35:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 35, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 36;o) the epitope is a cryptic epitope of IgLON5 comprising the amino acid sequence of SEQ ID NO: 43, the MHC I is HLA-A*03:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 39, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 42;p) the epitope is a cryptic epitope of IgLON5 comprising the amino acid sequence of SEQ ID NO: 34, the MHC I is HLA-B*35:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 44, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 45;q) the epitope is a cryptic epitope of IgLON5 comprising the amino acid sequence of SEQ ID NO: 34, the MHC I is HLA-B*35:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 46, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 47;r) the epitope is a cryptic epitope of IgLON5 comprising the amino acid sequence of SEQ ID NO: 34, the MHC I is HLA-B*35:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 48, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 49;s) the epitope is a cryptic epitope of IgLON5 comprising the amino acid sequence of SEQ ID NO: 34, the MHC I is HLA-B*35:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 50, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 51;t) the epitope is a cryptic epitope of IgLON5 comprising the amino acid sequence of SEQ ID NO: 34, the MHC I is HLA-B*35:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 52, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 53;u) the epitope is a cryptic epitope of IgLON5 comprising the amino acid sequence of SEQ ID NO: 34, the MHC I is HLA-B*35:01, the TRA comprises a CDR3 comprising the aminoAttorney Docket No: 046483-7493WOl(04128)acid sequence of SEQ ID NO: 54, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 55;v) the epitope is a cryptic epitope of IgLON5 comprising the amino acid sequence of SEQ ID NO: 34, the MHC I is HLA-B*35:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 56, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 57;w) the epitope is a cryptic epitope of IgLON5 comprising the amino acid sequence of SEQ ID NO: 60, the MHC I is HLA-A*02:01*, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 58, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 59;x) the epitope is a cryptic epitope of IgLON5 comprising the amino acid sequence of SEQ ID NO: 34, the MHC I is HLA-B*35:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 61, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 62;y) the epitope is a cryptic epitope of IgLON5 comprising the amino acid sequence of SEQ ID NO: 362, the MHC I is HLA- B*35:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 588, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 589; orz) the epitope is a cryptic epitope of ZNF423 comprising the amino acid sequence of SEQ ID NO: 491, the MHC I is HLA- A*02:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 562, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 563;aa) the epitope is a cryptic epitope of EPB41L4A comprising the amino acid sequence of SEQ ID NO: 292, the MHC I is HLA- A*01:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 564, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 565;bb)the epitope is a cryptic epitope of ARHGAP22 comprising the amino acid sequence of SEQ ID NO: 246, the MHC I is HLA- B*40:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 580, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 581;Attorney Docket No: 046483-7493WOl(04128)cc) the epitope is a cryptic epitope of PTPRZ1 comprising the amino acid sequence of SEQ ID NO: 418, the MHC I is HLA- A*02:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 582, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 583; ordd)the epitope is a cryptic epitope of PTPRZ1 comprising the amino acid sequence of SEQ ID NO: 418, the MHC I is HLA- A*02:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 584, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 585.
2. The nucleic acid of claim 1, wherein the TRA and TRB have antigenic specificity for a cryptic epitope of HDGFL2.
3. The nucleic acid of claim 2, wherein the epitope comprises the amino acid sequence of SEQ ID NO: 7 and the MHC I is HLA-B*08:01.
4. The nucleic acid of claim 2, wherein the epitope comprises the amino acid sequence of SEQ ID NO: 13 and the MHC I is HLA-B*03:01.
5. The nucleic acid of claim 4, wherein the TRA comprises a CDR3 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 11, 14, 16, 18, 20, 22, 24, 26, 28, and 30, and the TRB comprises a CDR3 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 12, 15, 17, 19, 21, 23, 25, 27, 29, and 31.
6. The nucleic acid of claim 2, wherein the epitope comprises the amino acid sequence of SEQ ID NO: 10 and the MHC I is HLA-B*03:01.
7. The nucleic acid of claim 6, wherein the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 8 and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 9.
8. The nucleic acid of claim 2, wherein the epitope comprises the amino acid sequence of SEQ ID NO: 7, the MHC I is HLA-B*08:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 6.Attorney Docket No: 046483-7493WOl(04128)9. The nucleic acid of claim 8, wherein the TRA further comprises a first complementarity determining region (CDR1) comprising the amino acid sequence of SEQ ID NO: 1 and a second complementarity determining region (CDR2) comprising the amino acid sequence of SEQ ID NO: 2; andwherein the TRB further comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 4 and a CDR2 comprising the amino acid sequence of SEQ ID NO: 5.
10. The nucleic acid of claim 9, wherein the TRA comprises a TCRα chain variable domain (TRAV) comprising at least 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 63.
11. The nucleic acid of claim 10, wherein the TRAV comprises the amino acid sequence of SEQ ID NO: 63.
12. The nucleic acid of any one of claims 8-11, wherein the TRA comprises a TCRα chain comprising at least 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 67.
13. The nucleic acid of 12, wherein the TCRα chain comprises the amino acid sequence of SEQ ID NO: 67.
14. The nucleic acid of any one of claims 8-13, wherein the TRB comprises a TCRP chain variable domain (TRBV) comprising at least 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 64.
15. The nucleic acid of claim 14, wherein the TRBV comprises the amino acid sequence of SEQ ID NO: 64.
16. The nucleic acid of any one of claims 8-15, wherein the TRB comprises a TCRβ chain comprising at least 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 68.
17. The nucleic acid of claim 16, wherein the TCRβ chain comprises the amino acid sequence of SEQ ID NO: 68.Attorney Docket No: 046483-7493WOl(04128)18. The nucleic acid of any one of claims 8-17, wherein the first and second polynucleotides are separated by a third polynucleotide encoding a linker.
19. The nucleic acid of claim 18, wherein the third polynucleotide comprises the nucleotide sequence of SEQ ID NO: 106 and / or encodes an amino acid sequence set forth in SEQ ID NO: 105.
20. The nucleic acid of claim 18 or 19, wherein the first and second polynucleotides are present in a TCRα / β single chain DNA construct.
21. The nucleic acid of claim 18 or 19, wherein the first and second polynucleotides are present in a TCRβ / α single chain DNA construct.
22. The nucleic acid of claim 21, wherein the TCRβ / α single chain DNA construct comprises the nucleotide sequence of SEQ ID NO: 78 and / or encodes the amino acid sequence of SEQ ID NO: 69.
23. The nucleic acid of claim 1, wherein the TRA and TRB have antigenic specificity for an epitope of IgLON5.
24. The nucleic acid of claim 23, wherein the epitope comprises the amino acid sequence of SEQ ID NO: 43 and the MHC I is HLA-A*03:01.
25. The nucleic acid of claim 23, wherein the epitope comprises the amino acid sequence of SEQ ID NO: 34 and the MHC I is HLB-B*35:01.
26. The nucleic acid of claim 25, wherein the TRA comprises a CDR3 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 32, 35, 44, 46, 48, 50, 52, 54, 56, 61, and 588; and the TRB comprises a CDR3 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 33, 36, 45, 47, 49, 51, 53, 55, 57, 62, and 589.
27. The nucleic acid of claim 23, wherein the epitope comprises the amino acid sequence of SEQ ID NO: 60, the MHC I is HLB-A*02:01, and the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 58 and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 59.Attorney Docket No: 046483-7493WOl(04128)28. The nucleic acid of claim 23, wherein the epitope comprises the amino acid sequence of SEQ ID NO: 43, the MHC I is HLA-A*03:01, the TRA comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 39, and the TRB comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 42.
29. The nucleic acid of claim 28, wherein the TRA further comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 37 and a CDR2 comprising the amino acid sequence of SEQ ID NO: 38; andwherein the TRB further comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 40 and a CDR2 comprising the amino acid sequence of SEQ ID NO: 41.
30. The nucleic acid of claim 29, wherein the TRA comprises a TRAV comprising at least 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 79.
31. The nucleic acid of claim 30, wherein the TRAV comprises the amino acid sequence of SEQ ID NO: 79.
32. The nucleic acid of any one of claims 28-31, wherein the TRA comprises a TCRα chain comprising at least 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 83.
33. The nucleic acid of claim 32, wherein the TCRα chain comprises the amino acid sequence of SEQ ID NO: 83.
34. The nucleic acid of any one of claims 28-33, wherein the TRB comprises a TRBV comprising at least 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 80.
35. The nucleic acid of claim 34, wherein the TRBV comprises the amino acid sequence of SEQ ID NO: 80.
36. The nucleic acid of any one of claims 28-35, wherein the TRB comprises a TCRβ chain comprising at least 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 84.Attorney Docket No: 046483-7493WOl(04128)37. The nucleic acid of claim 36, wherein the TCRβ chain comprises the amino acid sequence of SEQ ID NO: 84.
38. The nucleic acid of any one of claims 28-37, wherein the first and second polynucleotides are separated by a third polynucleotide encoding a linker.
39. The nucleic acid of claim 38, wherein the third polynucleotide comprises the nucleotide sequence of SEQ ID NO: 106 and / or encodes the amino acid sequence of SEQ ID NO: 105.
40. The nucleic acid of any one of claims 28-39, wherein the first and second polynucleotides are present in a TCRα / β single chain DNA construct.
41. The nucleic acid of any one of claims 28-39, wherein the first and second polynucleotides are present in a TCRβ / α single chain DNA construct.
42. The nucleic acid of claim 41, wherein the TCRβ-α single chain construct comprises the nucleotide sequence of SEQ ID NO: 94 and / or encodes the amino acid sequence of SEQ ID NO: 85.
43. A recombinant expression construct encoding the first and second polynucleotides of the nucleic acid of any one of claims 1-42.
44. A T cell receptor encoded by the recombinant expression vector of claim 43.
45. A modified immune cell comprising the nucleic acid of any one of claims 1-42 or the recombinant expression vector of claim 43.
46. The modified immune cell of claim 45, wherein the immune cell is selected from the group consisting of TCR-Treg (CD4Q, TCR-Treg / T suppressor (CD8+), TCR-TyS, TCR-NK, and TCR-regulatory NK cells.
47. The modified immune cell of claim 46, wherein the immune cell is a T cell.Attorney Docket No: 046483-7493WOl(04128)48. The modified immune cell of claim 47, wherein the T cell is obtained from the group consisting of peripheral blood mononuclear cells, cord blood cells, a purified population of T cells, and a T cell line.
49. The modified immune cell of claim 47 or 48, wherein the T cell is a CD4+T cell, a CD8+T cell, a CD4+Treg cell, a CD8+Treg cell, or a CD8+ suppressor cell.
50. The modified immune cell of any one of claims 45-49, wherein the T cell further comprises a modified endogenous genetic locus.
51. The modified immune cell of claim 50, wherein the modified endogenous genetic locus comprises TCRα chain and / or TCRβ chain polynucleotide sequence(s).
52. The modified immune cell of claim 50, wherein the modified endogenous genetic locus comprises deletion(s) of the TCRa chain and / or TCRp chain polynucleotide sequence(s).
53. The modified immune cell of claim 51 or 52, wherein expression from the modified endogenous genetic locus reduces or eliminates expression of the TCRa chain and / or TCRp chains.
54. The modified immune cell of any one of claims 50-53, wherein the modification is accomplished by use of a CRISPR system.
55. A method for generating the modified immune cell of any one of claims 45-54, comprising introducing into the immune cell the isolated nucleic acid of any one of claims 1-42 or the recombinant expression construct of claim 43, wherein the modified T cell expresses the TCR.
56. The method of claim 55, wherein the TCR specifically binds a cryptic epitope of HDGFL2.
57. The method of claim 56, wherein the cryptic epitope comprises an amino acid sequence set forth in any one of SEQ ID NOs: 7, 10, or 13.Attorney Docket No: 046483-7493WOl(04128)58. The method of claim 55, wherein the TCR specifically binds a cryptic epitope of IgL0N5.
59. The method of claim 58, wherein the cryptic epitope comprises an amino acid sequence set forth in any one of SEQ ID NOs: 34, 43, 60, or 362.
60. The method of any one of claims 55-59, wherein the immune cell is obtained from the group consisting of peripheral blood mononuclear cells, cord blood cells, a purified population of T cells, and a T cell line.
61. The method of claim 60, wherein the immune cell is a T cell.
62. The method of claim 61, wherein the T cell is a CD8+cell, a CD8+Treg cell, a CD8+ suppressor cell, a CD4+T cell, or a CD4+Treg cell.
63. The method of any one of claims 55-62, further comprising the step of modifying expression of endogenous TCRα and / or TCRβ chains.
64. The method of claim 63, wherein the step of modifying expression of the endogenous TCRα and / or TCRβ chains is accomplished by use of a CRISPR system.
65. A method for stimulating a cell-mediated immune response in a subject in need thereof, comprising administering to the subject an effective amount of the modified immune cell of any one of claims 45-54.
66. A method of treating a subject with a TDP-43 proteinopathy, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the modified immune cell of any one of claims 45-54 and a pharmaceutically acceptable carrier or excipient.
67. The method of claim 66, wherein the TDP-43 proteinopathy is selected from the group consisting of amyotrophic lateral sclerosis (ALS), inclusion body myositis (IBM), frontotemporal dementia (FTD), frontotemporal lobar degeneration (FTLD), motor neuron disease (MND), Alzheimer’s disease (AD), Parkinson's disease (PD), Huntington’s disease, chronic traumatic encephalopathy (CTE), facial onset sensory and motor neuronopathyAttorney Docket No: 046483-7493WOl(04128)(FOSMN), limbic-predominant age-related TDP-43 encephalopathy, primary progressive aphasia (PPA), Perry disease, Guam parkinsonism-dementia, and a Lewy body-related disease.
68. The method of claim 67, wherein the TDP-43 proteinopathy is ALS or IBM.
69. The method of any one of claims 66-68, comprising administering to the subject one or more additional therapeutic agents.
70. The method of claim 69, wherein the additional therapeutic agent is selected from the group consisting of chemotherapy, chimeric-antigen receptor (CAR)-T cell therapy, monoclonal antibody therapy, biologic therapy, allogeneic stem cell transplant, radiologic therapy, and any combination thereof.
71. A modified T cell comprising a T cell receptor that specifically binds a cryptic epitope set forth in Table 3 or Table 4.
72. A modified T cell comprising a T cell receptor set forth in Table 6.
73. The modified T cell of claim 71 or 72, wherein the T cell is a CD8+cell, a CD8+Treg cell, a CD8+suppressor cell, a CD4+T cell, or a CD4+Treg cell.
74. A pharmaceutical composition comprising the modified immune cell of any one of claims 45-54, or the modified T cell of any one of claims 71-73; and a pharmaceutically acceptable carrier.
75. A method for stimulating a cell-mediated immune response in a subject in need thereof, comprising administering to the subject an effective amount of the modified immune cell of any one of claims 45-54 or the modified T cell of claim 71 or 72.
76. A method of treating a subject with a TDP-43 proteinopathy, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 73.
77. The method of claim 76, wherein the TDP-43 proteinopathy is selected from the group consisting of amyotrophic lateral sclerosis (ALS), inclusion body myositis (IBM),Attorney Docket No: 046483-7493WOl(04128)frontotemporal dementia (FTD), frontotemporal lobar degeneration (FTLD), motor neuron disease (MND), Alzheimer’s disease (AD), Parkinson's disease (PD), Huntington’s disease, chronic traumatic encephalopathy (CTE), facial onset sensory and motor neuronopathy (FOSMN), limbic-predominant age-related TDP-43 encephalopathy, primary progressive aphasia (PPA), Perry disease, Guam parkinsonism-dementia, and a Lewy body-related disease.
78. The method of claim 77, wherein the TDP-43 proteinopathy is ALS or IBM.
79. An assay, comprising:culturing a biological sample comprising T cells from a subject, wherein the T cells are aliquoted into a plurality of wells;stimulating the T cells in each of the plurality of wells with (i) a pooled library of overlapping peptides, (ii) one or more sub-pools thereof, or (iii) one or more individual peptides encompassing a polypeptide of interest under conditions suitable for expansion of the T cells; optionally re-stimulating the T cells in each well with the same pooled library, subpools) thereof, or individual peptide(s) to which the T cells in the well were previously exposed; andexposing the T cells in each well to one or more detection reagents suitable for detecting one or more activation-induced T cell markers,wherein detection of the one or more activation-induced T cell markers in a well indicates the presence of a T cell expressing a TCR having antigen specificity for a peptide within the pooled library, sub-pool(s) thereof, or one or more individual peptides thereof to which the T cells in the well were exposed.
80. The assay of claim 79, wherein the protein of interest is encoded by a gene selected from the group consisting of AARS, ACTL6B, ARHGAP22, CELSR3, DNM1, EPB41L4A, HDGFL2, IGLON5, MYO18A, NECAB2, PHF2, PTPRZ1, PXDN, SLC24A2, STMN2, SYNJ2, XPO4, and ZNF423.
81. The assay of claim 79 or 80, wherein the subject has or is suspected of having a TDP-43 proteinopathy.Attorney Docket No: 046483-7493WOl(04128)82. The assay of claim 81, wherein the TDP-43 proteinopathy is indicative of the subject having a heterogenous neurodegenerative and inflammatory condition selected from the group consisting of amyotrophic lateral sclerosis (ALS), inclusion body myositis (IBM), frontotemporal dementia (FTD), frontotemporal lobar degeneration (FTLD), motor neuron disease (MND), Alzheimer’s disease (AD), Parkinson's disease (PD), Huntington’s disease, chronic traumatic encephalopathy (CTE), facial onset sensory and motor neuronopathy (FOSMN), limbic-predominant age-related TDP-43 encephalopathy, primary progressive aphasia (PPA), Perry disease, Guam parkinsonism-dementia, and a Lewy body-related disease.
83. The assay of claim 82, wherein the TDP-43 proteinopathy is ALS or IBM.
84. The assay of any one of claims 79-83, wherein the biological sample is from a patient blood collection or healthy patient donation.
85. The assay of any one of claims 79-84, wherein the biological sample is selected from the group consisting of whole blood, PBMCs, cryopreserved PBMCs, leukapheresis products, buffy coat preparations, cerebrospinal fluid (CSF), lymph node or lymph node biopsy, muscle biopsy, tissue-resident immune cells obtained from biopsy or surgical specimens, and engineered or transduced T cells or cell lines expressing one or more TCRs.
86. The assay of any one of claims 9, wherein the biological sample comprises PBMCs enriched for CD8+ T cells and / or CD4+ T cells.
87. The assay of any one of claims 79-86, wherein the pooled library of overlapping peptides, sub-pools thereof, or individual peptide(s) comprise peptides between about 3-40 amino acids in length, optionally between about 10-40 amino acids in length, optionally between about 15-35 amino acids in length, optionally between about 20-30 amino acids in length.
88. The assay of any one of claims 79-87, wherein adjacent peptides in the pooled library are generated using a sliding window having a step size of between about 1-40 amino acids, and optionally wherein each peptide in the pooled library overlaps an adjoining peptide by between about 1-39 amino acids, optionally between about 3-10 amino acids.Attorney Docket No: 046483-7493WOl(04128)89. The assay of any one of claims 79-88, wherein the one or more detection reagents are suitable for detecting CD69 and CD 137.
90. The assay of any one of claims 79-89, wherein the T cells are further cultured in the presence of an antigen-presenting cell (APC) selected from the group consisting of autologous PBMC APCs, dendritic cells, B cells, and engineered APC cell lines.
91. The assay of any one of claims 79-90, further comprising the step of isolating T cells from the well in which the one or more activation-induced T cell markers were detected.
92. A modified T cell produced by the assay of claim 91.
93. A method of detecting a TDP-43 proteinopathy-associated immune signature in a subject, comprising:(a) contacting T cells obtained from a biological sample from the subject with a panel of peptide–HLA multimers presenting a plurality of cryptic epitopes;(b) enriching or isolating multimer-positive T cells by cell sorting;(c) determining a normalized multimer-positive frequency score based on multimer-positive T cells relative to input T cells and / or relative to a negative-control multimer; and (d) classifying the subject as positive for the TDP-43 proteinopathy-associated immune signature when the normalized multimer-positive frequency score satisfies a predetermined criterion.
94. The method of claim 93, wherein the plurality of cryptic epitopes comprises cryptic epitopes derived from at least 10, at least 20, at least 30, or at least 50 proteins associated with a TDP-43 proteinopathy.
95. The method of claim 93 or 94, wherein the cryptic epitopes are selected from proteins encoded by genes selected from the group consisting of AARS, ACTL6B, ARHGAP22, CELSR3, DNM1, EPB41L4A, HDGFL2, IGLON5, MYO18A, NECAB2, PHF2, PTPRZ1, PXDN, SLC24A2, STMN2, SYNJ2, XPO4, and ZNF423.
96. The method of any one of claims 93-95, wherein the multimers comprise tetramers, dextramers, pentamers, or streptamers.Attorney Docket No: 046483-7493WOl(04128)97. The method of any one of claims 93-96, further comprising sequencing nucleic acids from the multimer-positive T cells to obtain TCRα and / or TCRβ sequences.
98. The method of claim 97, further comprising determining a clonality metric selected from clone size, clonal expansion, clonal enrichment, repertoire diversity, and / or presence of public / shared clonotypes.
99. The method of claim 97 or 98, wherein sequencing comprises single-cell RNA sequencing and the method further comprises determining a phenotype metric of the multimer-positive T cells comprising an effector / cytotoxic state, exhaustion state, activation state, memory / differentiation state, and / or expression of cytotoxic mediators and inflammatory cytokines.
100. The method of any one of claims 93-99, further comprising performing an activation-induced marker (AIM) assay by contacting T cells from the subject with an overlapping peptide library spanning one or more proteins comprising cryptic epitopes and measuring a frequency of AIM-positive T cells.
101. The method of claim 100, wherein AIM-positive T cells are CD69+CD137+T cells.
102. The method of claim 100 or 101, wherein the AIM assay output comprises ΔAIM relative to a matched negative control condition.
103. The method of any one of claims 100-102, further comprising isolating AIM-positive T cells and determining TCRα and / or TCRβ sequences from the isolated AIM-positive T cells.
104. The method of any one of claims 100-103, wherein the overlapping peptide library comprises peptides having a length of about 3 to about 40 amino acids.
105. The method of claim 104, wherein adjacent peptides in the library are offset by a step size of about 1 to about 40 amino acids.
106. The method of any one of claims 93-105, wherein the biological sample is selected from the group consisting of whole blood, PBMCs, cryopreserved PBMCs, leukapheresisAttorney Docket No: 046483-7493WOl(04128)products, buffy coat, cerebrospinal fluid (CSF), lymph node aspirate or biopsy, muscle biopsy, tissue-resident immune cells from a biopsy or surgical specimen, and engineered or transduced T cells or cell lines expressing one or more TCRs.
107. The method of any one of claims 93-106, wherein classifying comprises performing early disease diagnosis.
108. The method of any one of claims 93-106, further comprising repeating steps (a)-(d) at a subsequent time point and using a change in the normalized multimer-positive frequency score, clonality metric, phenotype metric, AIM output, or any combination thereof to monitor disease progression and / or monitor therapeutic effect.
109. The method of any one of claims 93-106, wherein classifying comprises patient stratification.
110. The method of claim 109, wherein patient stratification comprises distinguishing (i) Alzheimer’s disease without TDP-43 pathology from (ii) Alzheimer’s disease with concomitant TDP-43 pathology based on one or more of the normalized multimer-positive frequency score, clonality metric, phenotype metric, and / or AIM output.