Humanized antibodies and chimeric antigen receptors targeted to EGFR

Abstract

This disclosure relates, inter alia, to compositions comprising and methods making and using T cells expressing a chimeric antigen receptor (CAR) targeted to human EGFR and certain human EGFR mutants as well as antibodies and other antibody formats (e.g., Fab, scFv, scFab) targeted to the same.

Description

[0001] Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0002] Humanized Antibodies and Chimeric Antigen Receptors Targeted to EGFR

[0003] CLAIM OF PRIORITY

[0004] This application claims the benefit of U. S. Provisional Application Serial No. 63 / 746,501, filed on January 17, 2025. The entire contents of the foregoing are incorporated herein by reference.

[0005] TECHNICAL FIELD

[0006] This disclosure concerns humanized antibodies and chimeric antigen receptors targeted to human epidermal growth factor receptor and various mutants thereof.

[0007] BACKGROUND

[0008] Human epidermal growth factor receptor (EGFR) is a transmembrane receptor tyrosine kinase composed of four extracellular domains (I, II, III and IV). EGFR is a dynamic protein that has a variety of conformations, including a monomeric compact or “tethered” formation in which Domains I and IV form an intramolecular interaction and an extended or “untethered” conformation which permits binding of EGF, TGFA and other ligands. Ligand binding stabilizes the extended conformation in which Domain II is exposed, leading to dimerization and activation. Human epidermal growth factor receptor VIII (EGFRVIII) is a constitutively active mutant form of human EGFR, lacking exons 2-7 (amino acids 6-273) of human EGFR. The loss of exons 2-7 eliminates the ligand binding domain and creates a glycine residue between amino acids 5 and 274 of EGFR. EGFRVIII is detectable in about 23-33% of human glioblastomas. EGFRVIII is present in certain other tumors as well.

[0009] The signaling pathways activated by EGFR include: the RAS / mitogen activated protein kinase (MAPK) / extracellular signal-regulated kinase (ERK) pathway, the PI3K / protein kinase B (PKB / AKT) pathway, the Janus Kinase (JAK) / STAT pathway, and the protein kinase C (PKC) pathway. Certain of these signaling pathways are frequently mutated in glioblastoma.

[0010] Murine mAb806 (PMID: 11454674, 15770208) recognizes an epitope within the extracellular Domain II of human EGFR (UniProt P00533), which becomes exposed during EGFR higher level expression and / or its constitutive activation, for example in the constitutively active EGFRVIII mutant (Ng et al. 2015 Proteins 83:153; Gan et al. 2012 Cancer Res 72 2924, 2012). Thus, mAb806 can bind sufficiently expressed human EGFR, EGFRVIII and other variants in which the mAb806 epitope is accessible. Better treatments are needed for use in humans.Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0011] SUMMARY

[0012] The present disclosure concerns antibodies and chimeric antigen receptors (CAR) having a domain (e.g., an scFv) that bind both sufficiently expressed human EGFR as well as EGFRVIII and other EGFR variants in which the epitope (amino acids 287 - 302 of EGFR) is accessible. This disclosure also concerns methods comprising the use thereof to treat subjects suffering from a cancer (e.g., a cancer comprising cells expressing EGFR and / or EGFRVIII; e.g., a glioma, glioblastoma). Three humanized variants of mAb806 - h6T, h6O and h3T - were constructed as Fabs. The stability and affinity of the Fabs were shown to be comparable or even superior to Fab806, the Fab from which mAb806 was derived. Each Fab was converted to an scFv and used to construct CAR. In addition, certain variants of the humanized antibodies were designed.

[0013] Described herein are nucleic acid molecules comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) comprising:

[0014] (i) an anti-EGFR domain comprising an scFv selected from:

[0015] a scFv comprising a VH comprising SEQ ID NO: 5 and a VL comprising SEQ ID NO: 6;

[0016] a scFv comprising a VH comprising SEQ ID NO: 8 and a VL comprising SEQ ID NO: 9;

[0017] a scFv comprising a VH comprising SEQ ID NO: 11 and a VL comprising SEQ ID NO: 12;

[0018] a scFv comprising SEQ ID NO: 4;

[0019] a scFv comprising SEQ ID NO: 7;

[0020] a scFv comprising SEQ ID NO: 10;

[0021] a scFv comprising SEQ ID NO: 131;

[0022] a scFv comprising SEQ ID NO: 132;

[0023] a scFv comprising SEQ ID NO: 133;

[0024] (ii) a spacer domain;

[0025] (iii) a transmembrane domain;

[0026] (iv) a costimulatory domain; and

[0027] (v) a CD3ζ signaling domain.

[0028] In some embodiments, described herein are nucleic acid molecules comprising a nucleotide sequence encoding a CAR comprising:

[0029] (i) an scFv selected from:Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0030] a scFv comprising a VH comprising amino acids 21-105 of SEQ ID NO: 5 and a VL comprising amino acids 11-104 of SEQ ID NO: 6;

[0031] a scFv comprising a VH comprising amino acids 5-101 of SEQ ID NO: 8 and a VL comprising amino acids 11-104 of SEQ ID NO: 9;

[0032] a scFv comprising a VH comprising amino acids 17-105 of SEQ ID NO: 11 and a VL comprising amino acids 11-97 of SEQ ID NO: 12;

[0033] (ii) a spacer domain;

[0034] (iii) a transmembrane domain;

[0035] (iv) a costimulatory domain; and

[0036] (v) a CD3ζ signaling domain.

[0037] The targeting domain (also called the extracellular binding domain and anti-EGFR domain) of any of the nucleic acid molecules, polypeptides, and CAR described herein can comprise any one of:

[0038] a VH comprising amino acids 21-105 of SEQ ID NO: 5 and a VL comprising amino acids 11-104 of SEQ ID NO: 6;

[0039] a VH comprising amino acids 5-101 of SEQ ID NO: 8 and a VL comprising amino acids 11-104 of SEQ ID NO: 9;

[0040] a VH comprising amino acids 17-105 of SEQ ID NO: 11 and a VL comprising amino acids 11-97 of SEQ ID NO: 12;

[0041] a VH comprising SEQ ID NO: 5 or a variant thereof having 1-5 (e.g., 3 or 4) single amino acid modifications (e.g., substitutions), and a VL comprising SEQ ID NO: 6 or a variant thereof having 1-5 (e.g., 3 or 4) single amino acid modifications (e.g., substitutions), wherein the amino acid modifications are not in the CDRs;

[0042] a VH comprising SEQ ID NO: 8 or a variant thereof having 1-5 (e.g., 3 or 4) single amino acid modifications (e.g., substitutions) and a VL comprising SEQ ID NO: 9 or a variant thereof having 1-5 (e.g., 3 or 4) single amino acid modifications (e.g., substitutions), wherein the amino acid modifications are not in the CDRs;

[0043] a VH comprising SEQ ID NO: 11 and a VL comprising amino acids 11-97 of SEQ ID NO: 12 or a variant thereof having 1-5 (e.g., 3 or 4) single amino acid modifications (e.g., substitutions), wherein the amino acid modifications are not in the CDRs;

[0044] a VH comprising SEQ ID NO: 5 or a variant thereof having 1-5 (e.g., 3 or 4) single amino acid modifications (e.g., substitutions), wherein the modifications are not in amino acids 21-105 of SEQ ID NO: 5, and a VL comprising SEQ ID NO:6 or a variant thereofAttorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0045] having 1-5 (e.g., 3 or 4) single amino acid modifications (e.g., substitutions), wherein the modifications are not in amino acids 11-104 of SEQ ID NO: 6;

[0046] a VH comprising SEQ ID NO:8 or a variant thereof having 1-5 (e.g., 3 or 4) single amino acid modifications (e.g., substitutions), wherein the modifications are not in amino acids 5-101 of SEQ ID NO: 8, and a VL comprising SEQ ID NO:9 or a variant thereof having 1-5 (e.g., 3 or 4) single amino acid modifications (e.g., substitutions), wherein the modifications are not in amino acids 11-104 of SEQ ID NO: 9; and

[0047] a VH comprising SEQ ID NO:11 or a variant thereof having 1-5 (e.g., 3 or 4) single amino acid modifications (e.g., substitutions), wherein the modifications are not in amino acids 17-105 of SEQ ID NO: 11, and a VL comprising SEQ ID NO: 12 or a variant thereof having 1-5 (e.g., 3 or 4) single amino acid modifications (e.g., substitutions), wherein the modifications are not in amino acids 11-97 of SEQ ID NO: 12.

[0048] In various cases, the spacer comprises or consists of a sequence selected from the group consisting of: SEQ ID NOs: 24-34 and 134; the transmembrane domain comprises or consists of a sequence selected from the group consisting of SEQ ID NOs: 15-23; the costimulatory domain comprises or consists of a sequence selected from the group consisting of SEQ ID NOs: 36-40; and the CD3ζ signaling domain comprises or consists of a sequence selected from the group consisting of SEQ ID NOs: 35 and 47-53.

[0049] In various cases, the transmembrane domain selected from the group consisting of:

[0050] a CD4 transmembrane domain,

[0051] a CD8 transmembrane domain,

[0052] a CD28 transmembrane domain, and

[0053] a CD3ζ transmembrane domain.

[0054] In various cases, the costimulatory domain selected from the group consisting of

[0055] a CD28 costimulatory domain,

[0056] a 41-BB costimulatory domain,

[0057] an 0X40 costimulatory domain, and

[0058] a 2B4 costimulatory domain.

[0059] In various cases, the CAR further comprising a linker consisting of 1 - 5 amino acids between one or more of: the scFV and the spacer domain, the spacer domain and the transmembrane domain, the transmembrane domain and the co-stimulatory domain, and the costimulatory domain and the CD3ζ signaling domain.Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0060] In various cases: the transmembrane domain comprises or consists of the amino acid sequence of any of SEQ ID NO: 15-23; the co-stimulatory domain comprises or consists of the amino acid sequence of any of SEQ ID NO: 36-40; the CD3ζ signaling domain comprises the amino acid sequence of any of SEQ ID NO: 35 and 47-53; the chimeric antigen receptor comprises the amino acid sequence of any of SEQ ID NOs: 54-100 and 127-129.

[0061] In some embodiments, the CAR comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 54-100 and 127-129 or a variant thereof having 1-10 (e.g., 3 or 8) or 1-5 single amino acid substitutions, wherein the amino acid modifications are not in the CDRs. In some embodiments, the amino acid modifications are not the VH or the VL. In some embodiments, the amino acid sequences are not in the scFv.

[0062] In various cases, the chimeric antigen receptor comprises or consists of an amino acid sequence depicted in any of FIGs 7-30 and 32.

[0063] Also described herein is nucleic acid molecule comprising a nucleotide sequence encoding a chimeric antigen receptor comprising (scFab CAR): (i) A single chain Fab comprising an amino acid sequence selected from: SEQ ID NOs: 105, 106, 109, 110, 119, 120, 123 and 124, a single chain Fab comprising SEQ ID NOs: 135 and 136, a single chain Fab comprising SEQ ID NOs: 107 and 108, a single chain Fab comprising SEQ ID NOs: 137 and 138, and a single chain Fab comprising SEQ ID NOs: 121 and 122, (ii) a spacer domain; (iii) a transmembrane domain; (iv) a costimulatory domain; and (v) a CD3ζ signaling domain.

[0064] In various cases of scFab CAR: the spacer comprises a sequence selected from the group consisting of: SEQ ID NOs: 24-34 and 134; the transmembrane domain comprises a sequence selected from the group consisting of SEQ ID NOs: 15-23; the costimulatory domain comprises a sequence selected from the group consisting of SEQ ID NOs: 36-40, and the CD3ζ signaling domain comprises a sequence selected from the group consisting of SEQ ID NOs: 35-40; the transmembrane domain selected from the group consisting of: a CD4 transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, and a CD3 transmembrane domain; the costimulatory domain selected from the group consisting of: a CD28 costimulatory domain, a 41 -BB costimulatory domain, an 0X40 costimulatory domain, and a 2B4 costimulatory domain.Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0065] In various cases, the scFab CAR further comprises: a linker consisting of 1 - 5 amino acids between one or more of: the scFV and the spacer domain, the spacer domain and the transmembrane domain, the transmembrane domain and the co-stimulatory domain, and the costimulatory domain and the CD3 signaling domain; the transmembrane domain comprises the amino acid sequence of any of SEQ ID NO: 15-23; the co-stimulatory domain comprises the amino acid sequence of any of SEQ ID NO: 36-40;

[0066] the CD3ζ signaling domain comprises the amino acid sequence of any of SEQ ID NO: 35 and 47-53; the chimeric antigen receptor comprises the amino acid sequence of any of SEQ ID NOs: 47-101.

[0067] Also described are CAR encoded by any of the forgoing nucleic acid molecules.

[0068] Also described are populations of human cells (e.g., human T cells, human NK cells) harboring any of the forgoing nucleic acid molecules.

[0069] Also described herein are polypeptides comprising any of:

[0070] a VH comprising amino acids 21-105 of SEQ ID NO: 5 and a VL comprising amino acids 11-104 of SEQ ID NO: 6;

[0071] a VH comprising amino acids 5-101 of SEQ ID NO: 8 and a VL comprising amino acids 11-104 of SEQ ID NO: 9;

[0072] a VH comprising amino acids 17-105 of SEQ ID NO: 11 and a VL comprising amino acids 11-97 of SEQ ID NO: 12;

[0073] VH comprising SEQ ID NO: 5 and a VL comprising SEQ ID NO: 6;

[0074] VH comprising SEQ ID NO: 8 and a VL comprising SEQ ID NO: 9; or

[0075] VH comprising SEQ ID NO: 11 and a VL comprising SEQ ID NO: 12.

[0076] SEQ ID NO: 4;

[0077] SEQ ID NO: 7;

[0078] SEQ ID NO: 10;

[0079] SEQ ID NO: 131;

[0080] SEQ ID NO: 132; and

[0081] SEQ ID NO: 133.

[0082] Also described are scFv comprising or consisting of any of: a VH comprising amino acids 21-105 of SEQ ID NO: 5 and a VL comprising amino acids 11-104 of SEQ ID NO: 6; a VH comprising amino acids 5-101 of SEQ ID NO: 8 and a VL comprising amino acids 11-104 of SEQ ID NO: 9; a VH comprising amino acids 17-105 of SEQ ID NO: 11 and a VL comprisingAttorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0083] amino acids 11-97 of SEQ ID NO: 12; a VH comprising SEQ ID NO: 5 and a VL comprising SEQ ID NO: 6; a VH comprising SEQ ID NO: 8 and a VL comprising SEQ ID NO: 9; a VH comprising SEQ ID NO: 11 and a VL comprising SEQ ID NO: 12; SEQ ID NO: 4; SEQ ID NO: 7; SEQ ID NO: 10; SEQ ID NO: 131; SEQ ID NO: 132; and SEQ ID NO: 133;

[0084] Also described are single chain Fab comprising an amino acid sequence selected from: SEQ ID NOs: 105, 106, 109, 110, 119, 120, 123 and 124.

[0085] Also described are antibodies having:

[0086] a) a heavy chain comprising SEQ ID NO: 13 and a light chain comprising SEQ ID NO: 14;

[0087] b) a heavy chain comprising SEQ ID NO: 101 and a light chain comprising SEQ ID NO: 102;

[0088] c) a heavy chain comprising SEQ ID NO: 103 and a light chain comprising SEQ ID NO: 104;

[0089] d) a heavy chain comprising SEQ ID NO: 111 and a light chain comprising SEQ ID NO: 112;

[0090] e) a heavy chain comprising SEQ ID NO: 113 and a light chain comprising SEQ ID NO: 114;

[0091] f) a heavy chain comprising SEQ ID NO: 115 and a light chain comprising SEQ ID NO: 116;

[0092] g) a heavy chain comprising SEQ ID NO: 117 and a light chain comprising SEQ ID NO: 118; or

[0093] h) a heavy chain comprising SEQ ID NO: 125 and a light chain comprising SEQ ID NO: 126;

[0094] Also described is an antibody having: (a) a heavy chain VH-CH1 comprising SEQ ID NO: 107 and a light chain VL-CL comprising SEQ ID NO: 108; or (b) a heavy chain VH-CH1 comprising SEQ ID NO: 121 and a light chain VL-CL comprising SEQ ID NO: 122.

[0095] Also described are single chain Fab comprising: SEQ ID NOs: 107 and 108, SEQ ID NOs: 137 and 138, or SEQ ID NOs: 121 and 122.

[0096] In some embodiments, any of the antibodies and other antibody formats (e.g., scFv, Fab, single chain Fab, IgG antibodies (including IgG antibodies with variant Fc regions), and antibodies having a meditope-enabled Fab) can comprise any one of:Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0097] a VH comprising amino acids 21-105 of SEQ ID NO: 5 and a VL comprising amino acids 11-104 of SEQ ID NO: 6;

[0098] a VH comprising amino acids 5-101 of SEQ ID NO: 8 and a VL comprising amino acids 11-104 of SEQ ID NO: 9;

[0099] a VH comprising amino acids 17-105 of SEQ ID NO: 11 and a VL comprising amino acids 11-97 of SEQ ID NO: 12;

[0100] a VH comprising SEQ ID NO: 5 or a variant thereof having 1-5 (e.g., 3 or 4) single amino acid modifications (e.g., substitutions), and a VL comprising SEQ ID NO: 6 or a variant thereof having 1-5 (e.g., 3 or 4) single amino acid modifications (e.g., substitutions), wherein the amino acid modifications are not in the CDRs;

[0101] a VH comprising SEQ ID NO: 8 or a variant thereof having 1-5 (e.g., 3 or 4) single amino acid modifications (e.g., substitutions) and a VL comprising SEQ ID NO: 9 or a variant thereof having 1-5 (e.g., 3 or 4) single amino acid modifications (e.g., substitutions), wherein the amino acid modifications are not in the CDRs;

[0102] a VH comprising SEQ ID NO: 11 and a VL comprising amino acids 11-97 of SEQ ID NO: 12 or a variant thereof having 1-5 (e.g., 3 or 4) single amino acid modifications (e.g., substitutions), wherein the amino acid modifications are not in the CDRs;

[0103] a VH comprising SEQ ID NO: 5 or a variant thereof having 1-5 (e.g., 3 or 4) single amino acid modifications (e.g., substitutions), wherein the modifications are not in amino acids 21-105 of SEQ ID NO: 5, and a VL comprising SEQ ID NO:6 or a variant thereof having 1-5 (e.g., 3 or 4) single amino acid modifications (e.g., substitutions), wherein the modifications are not in amino acids 11-104 of SEQ ID NO: 6;

[0104] a VH comprising SEQ ID NO:8 or a variant thereof having 1-5 (e.g., 3 or 4) single amino acid modifications (e.g., substitutions), wherein the modifications are not in amino acids 5-101 of SEQ ID NO: 8, and a VL comprising SEQ ID NO:9 or a variant thereof having 1-5 (e.g., 3 or 4) single amino acid modifications (e.g., substitutions), wherein the modifications are not in amino acids 11-104 of SEQ ID NO: 9; and

[0105] a VH comprising SEQ ID NO:11 or a variant thereof having 1-5 (e.g., 3 or 4) single amino acid modifications (e.g., substitutions), wherein the modifications are not in amino acids 17-105 of SEQ I D NO: 11, and a VL comprising SEQ I D NO: 12 or a variant thereof having 1 -5 (e.g., 3 or 4) single amino acid modifications (e.g., substitutions), wherein the modifications are not in amino acids 11-97 of SEQ ID NO: 12.

[0106] Also described herein are methods of treating a cancer (e.g., a cancer expressing a constitutively active EGFR or overexpressing EGFR) by administering a cell expressing a CAR described herein or administering an antibody described herein (or other antibodyAttorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0107] format described herein). In some embodiments, the cancer is any one of: Glioma, Glioblastoma, Lung Cancer, Colorectal Cancer, Genitourinary Cancers, Breast Cancer, NonSmall Cell Lung Cancer (NSCLC), Head and Neck Squamous Cell Carcinoma (HNSCC), Glioblastoma Multiforme (GBM), Triple-Negative Breast Cancer (TNBC), Esophageal Cancer, Gastric Cancer, Pancreatic Cancer, Cervical Cancer, Bladder Cancer, Ovarian Cancer, Prostate Cancer, Hepatocellular Carcinoma (HCC), Cholangiocarcinoma (Bile Duct Cancer), a Soft Tissue Sarcoma, Thyroid Cancer, Mesothelioma, Renal Cell Carcinoma, a Skin Cancer.

[0108] In various embodiments of any of the nucleic acid molecules and / or CAR herein: the spacer region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 24-34 and 134 or a variant thereof having 1, 2, 3, 4, or 5 single amino acid substitutions; the transmembrane domain selected from the group consisting of: a CD4 transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, and a CD3 transmembrane domain; the costimulatory domain selected from the group consisting of: a CD28 costimulatory domain, a 41-BB costimulatory domain, an 0X40 costimulatory domain, and a 2B4 costimulatory domain. In some embodiments, the nucleic acid molecule further comprises an interdomain linker consisting of 1 - 5 amino acids between one or more of: the scFv and the spacer domain, the spacer domain and the transmembrane domain, the transmembrane domain and the co-stimulatory domain, and / or the costimulatory domain and the CD3 signaling domain; the interdomain linker can consist of 1-5 glycine; in some embodiments, the CAR further comprises an interdomain linker consisting of the sequence GGG that is located between the costimulatory domain and the CD3ζ signaling domain.

[0109] In some embodiments, a CAR comprises: an scFv comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 4, 7, 10, 131, 132, and 133 or a variant thereof having 1, 2, 3, 4, or 5 single amino acid substitutions (e.g., conservative amino acid substitutions) that are not in a CDR; a spacer comprising a sequence selected from the group consisting of: SEQ ID NOs: 24-34 and 134 or a variant thereof having 1, 2, 3, 4, or 5 single amino acid substitutions; a transmembrane domain comprising a sequence selected from the group consisting of SEQ ID NOs: 15-23 or a variant thereof having 1, 2, 3, 4, or 5 single amino acid substitutions; a costimulatory domain comprising a sequence selected from the group consisting of SEQ ID NOs: 36-40 or a variant thereof having 1, 2, 3, 4, or 5 single amino acid substitutions; and a CD3 signaling domain comprising SEQ ID NO: 35 orAttorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0110] a variant thereof having 1, 2, 3, 4, or 5 single amino acid substitutions. In some embodiments, the amino acid substitutions are conservative.

[0111] In some embodiments, a CAR can be expressed in a form that includes a signal sequence, e.g., a human GM-CSF receptor alpha signal sequence (MLLLVTSLLLCELPHPAFLLIP; SEQ ID NO:44). Any CAR described herein can be expressed with additional sequences that are useful for monitoring expression or inhibiting CAR expression via an inducible suicide switch or facilitating co-expression, for example, a T2A skip sequence and a truncated EGFR or truncated CD19. In other cases, the signal sequence in an IL-2 signal sequence comprising or consisting of the sequence: MYRMQLLSCIALSLALVTNS (SEQ ID NO: 45) or an Ig kappa signal sequence comprising or consisting of the sequence:

[0112] MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 46).

[0113] Also disclosed herein are vectors (e.g., viral vectors) comprising a nucleic acid molecule described herein; a population of immune cells comprising a vector comprising a nucleotide sequence described here; a population of immune cells expressing a CAR described here; a population of human T cells (e.g., a population comprising central memory T cells) or of human NK cells transduced by a vector comprising a nucleic acid molecule described herein. In some embodiments, the T cells comprise PBMC, dPBMC (PBMC with depletion of CD14+ and CD25+ cells), Tn / mem (naive and memory T cells, CD62L+ enriched from dPBMC), or Tern (central memory T cells).

[0114] In various embodiments: a population of immune cell can comprise one or more of helper T cells, cytotoxic T cells, memory T cells, naive T cells, regulatory T cells, natural killer T cells, or combinations thereof. In various embodiments: at least 20%, 30%, 40%, or 50% of the CAR T cells are CD8+ T cells. In various embodiments: at least 20%, 30%, 40%, or 50% of the CAR T cells are CD4+ T cells. A method for preparing central memory T cells (Tern) is described in Wang et al. (2016) Blood 127:2980. A cell population composed of central memory, naive and stem cell memory T cells can be prepared by a modification of the method for preparing Tern by omitting the CD45RA depletion step.

[0115] Described herein, inter alia, are populations of human immune cells (e.g., human T cells, human NK cells) harboring any of nucleic acid molecules described herein, populations of human immune cells (e.g., human T cells, human NK cells) expressing any of the CAR described herein, and populations of human immune cells (e.g., human T cells, human NK cells) expressing any of the polypeptides described herein.Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0116] Also described herein are compositions comprising any of the populations of human immune cells (e.g., human T cells, human NK cells) described herein, any of the antibodies described herein, and any of the antibody fragments and formats, including but not limited to scFv, Fab, single chain Fab, IgG antibodies (including IgG antibodies with variant Fc regions), and antibodies having a meditope-enabled targeting domains, e.g., Fab.

[0117] In some embodiments, methods of treating a subject having a cancer comprising cells that express EGFR and / or EGFRVIII on the cancer cell surface, the methods comprising administering to the subject (i) any population of immune cells described herein (e.g., anti-EGFR CAR T cells), or (ii) any antibody, polypeptide, or other antibody format (e.g., scFv, scFab) described herein. In some embodiments, described herein are methods of treating a cancer in a patient comprising administering a composition comprising a population of immune cells that express a CAR that targets EGFR or EGFRVIII (e.g., overexpressed EGFR or EGFRVIII). In some embodiments, described herein methods of reducing or ameliorating a symptom associated with a cancer in a patient comprising administering a composition comprising a population of immune cells described herein (e.g., a population of CAR T cells that target cells overexpressing EGFR or expressing EGFRVIII. In some embodiments, described herein is a method of reducing cancer cells overexpressing EGFR or expressing EGFRVIII in a patient comprising administering a composition comprising a population of immune cells described herein (e.g., a population of CAR T cells).

[0118] In some embodiments, a composition comprising CAR T cells or CAR NK cells described herein is administered locally or systemically. In some embodiments, a composition comprising CAR T cells or CAR NK cells described herein is administered by single or repeat dosing. In some embodiments, a composition comprising CAR T cells or CAR NK cells described herein is administered to a patient having a cancer (e.g., a cancer overexpressed EGFR or EGFRVIII). In some embodiments, the cancer can be Glioblastoma, and other EGFR mutated or over-expressed tumors, including but not limited to Lung Cancer, Colorectal Cancer, Genitourinary Cancers, Breast Cancer, Non-Small Cell Lung Cancer (NSCLC), Head and Neck Squamous Cell Carcinoma (HNSCC), Glioblastoma Multiforme (GBM), Triple-Negative Breast Cancer (TNBC), Esophageal Cancer, Gastric Cancer, Pancreatic Cancer, Cervical Cancer, Bladder Cancer, Ovarian Cancer, Prostate Cancer, Hepatocellular Carcinoma (HCC), Cholangiocarcinoma (Bile Duct Cancer), Soft Tissue Sarcomas, Thyroid Cancer, Mesothelioma, Renal Cell Carcinoma, and Skin Cancers (e.g., Squamous Cell Carcinoma).Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0119] An amino acid modification refers to an amino acid substitution, insertion, and / or deletion in a protein or peptide sequence. An “amino acid substitution” or “substitution” refers to replacement of an amino acid at a particular position in a parent peptide or protein sequence with another amino acid. A substitution can be made to change an amino acid in the resulting protein in a non-conservative manner ( / .e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping) or in a conservative manner ( / .e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping). Such a conservative change generally leads to less change in the structure and function of the resulting protein. The following are examples of various groupings of amino acids: 1) Amino acids with nonpolar R groups: Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine, Tryptophan, Methionine; 2) Amino acids with uncharged polar R groups: Glycine, Serine, Threonine, Cysteine, Tyrosine, Asparagine, Glutamine; 3) Amino acids with charged polar R groups (negatively charged at pH 6.0): Aspartic acid, Glutamic acid; 4) Basic amino acids (positively charged at pH 6.0): Lysine, Arginine, Histidine (at pH 6.0). Another grouping may be those amino acids with phenyl groups: Phenylalanine, Tryptophan, and Tyrosine.

[0120] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0121] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0122] DESCRIPTION OF DRAWINGS

[0123] FIGS. 1A-1D. Engineering and characterization of anti-EGFR CAR T Cells. (A) Total T cell counts (M, in million) were measured on 7 and 14 days after CD3 / CD28 beads activation. Mock T cells without lenti-virus transduction are included as Ctrl. (B) anti-EGFR BBζ T cells derived from healthy donor were stained with either anti-CD19-PECy7 or anti-Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0124] G4S-PE to detect the percentage of cells that express CD19t or CAR transgenes.

[0125] Immunoreactive cells are calculated based on untransduced MockT cells (control). (C) Dot plot indicates the geometric mean (GM) of each CAR that was calculated based on immune staining of G4S. (D) anti-EGFR BB T cells were stained with CD4 and CD8. Percentage of CD4+ and CD8+ cells are calculated in total CAR T cells.

[0126] FIGS. 2A-2E. Humanized anti-EGFR CAR T Cells mediate EGFR-selective cytotoxicity in vitro. (A) Flow cytometric analysis of EGFR expression on various patient-derived glioblastoma (GBM) cells. The percentage of immunoreactive cells (red line) compared to isotype antibody staining Ctrl (grey line) are depicted in each histogram. (B) Cell lytic activity was compared by impedance-based assay. Mock T cells or anti-EGFR BBζ T Cells (effector cells) and the indicated tumor cells (target cells) were seeded at an E: T ratio of 1:5 or 1:20. The detachment and death of adherent tumor cells was monitored as a decrease in normalized cell index upon the addition of effector cells. The time of effector cell addition was indicated by a dotted line. (C) Flow cytometric analysis of EGFR and EGFRvlll expression on engineered GBM cells. The percentage of immunoreactive cells (red line) compared to isotype antibody staining Ctrl (grey line) are depicted in each histogram. (D) Geometric Mean (GM) of EGFR expression in each engineered cell as in (C). (E) Cell lytic activity was analyzed as in (B). Mock T cells or anti-EGFR BBζ T Cells and the indicated tumor cells were seeded at an E: T ratio of 1:80.

[0127] FIGS. 3A-3B. Humanized anti-EGFR CAR T Cells induce comparable cytotoxicity against EGFRvlll-expressing tumor cells as 806-CART. (A) Cell lytic activity was compared by flow cytometry. Mock T cells or anti-EGFR BBζ T cells and the indicated tumor cells were cocultured at an E: T ratio of 1:20 for 72 hrs, then the number of CAR T cells and tumor cells were counted by flow cytometry. Percentage of cytotoxicity (% Cytotoxicity) was calculated by dividing the tumor cell count in each treatment group by the average count from the tumor-alone control. Error bars were calculated using Welch’s unpaired t test: *p < 0.05; ****p < 0.0001. (B) Mock T cells or anti-EGFR BBζ T cells were stimulated with the same tumor lines as in Fig. 2C at an E: T ratio of 2:1. IFN-y and IL2 levels in supernatant were collected and measured after 18 hrs co-culture using human cytokine bead array.

[0128] FIGS. 4A-4B. Humanized anti-EGFR CAR T cells retain cytotoxicity through multiple rounds of tumor challenge. (A) Schema of a tumor line repetitive rechallenge assay. Anti-EGFR BBζ T cells were cocultured with PBT030-EGFRvlllmed(2000 CAR T cells and 0.01 M tumor cells), repetitively challenged with either 0.01 M or 0.02M tumor cells every one or twoAttorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0129] days (time points indicated in red). Numbers of CAR T cells and tumor cells were counted by flow cytometry at the indicated time points (arrows at day 3 or day 10). (B) Total CAR T cell and remaining viable tumor cell counts after tumor challenge cycle- 1 and cycle-6.

[0130] Percentage of cytotoxicity in cycle- 1 was calculated by dividing the tumor cell count in each treatment group by the average count from the tumor-alone control. Using Welth’s unpaired t test: *p < 0.05; **p < 0.01; ***p < 0.001.

[0131] FIGS. 5A-5E. Humanized anti-EGFR(6T) CAR T cells exhibit comparable antitumor activity as 806-CART in vivo. (A) Schema of animal study using orthotopic xenograft GBM tumor model. NSG mice were inoculated intracranial (i.c. or IC) with 0.1M ffLuc+ PBT030-EGFRvlllmedand then treated with 0.1M CAR T cells by intraventricular (i.c.v. or ICV) injection 3 weeks after tumor implantation. Tumor growth and survival were monitored by bioluminescence imaging in mice. (B) Bioluminescent flux plot quantifying tumor volume in response to different CAR T cells treatment over time. Median flux in each group is indicated in the red line. Last flux data is shown as mean ± SD. Using two-way ANOVA test: *p < 0.05; ***p < 0.01; ***p < 0.001. (C) Mice from each group showing relative tumor burden over time. T cell infusion on day 23 is indicated by the vertical dotted line. (D) Kaplan-Meier survival analysis demonstrating comparable survival for mice treated with humanized anti-EGFR(6T) BB T cells as 806 BB T cells. Using log-rank (Mantel-Cox) test: **p < 0.01; ****p < 0.0001.] (E) Survival analysis following tumor rechallenge. Cured mice after treatment with either 806 BBζ T cells or 6T BBζ T cells or 6O BBζ T cells were rechallenged with 0.05M ffLuc+ PBT030-EGFRvlllmedi.c. Naive mice without prior treatment were included as Ctrl. Using logrank (Mantel-Cox) test: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0132] FIGS. 6A-6B. Humanized anti-EGFR(6T) BBC T cells exhibit comparable antitumor activity with various spacer and transmembrane domain. (A) anti-EGFR BBC T cells derived from healthy donor were stained with anti-G4S-PE to detect the percentage of cells that express CAR transgenes. Immunoreactive cells are calculated based on untransduced MockT control cells. (B) Cell lytic activity was compared by impedance-based assay. Mock T cells or anti-EGFR BBζ T Cells (effector cells) and the indicated tumor cells (target cells) were seeded at an E: T ratio of 1:40. The detachment and death of adherent tumor cells was monitored as a decrease in normalized cell index upon the addition of effector cells. The time of effector cell addition was indicated by a dotted line.

[0133] FIG. 7A depicts the amino acid sequence of scFv6T-lgG4(S228P, L235E, N297Q)-CD28tm-CD28gg-CD3Zeta (SEQ ID NO: 54).Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0134] FIG. 7B depicts the amino acid sequence of scFv6T-lgG4(HL-CH3)-CD28tm-CD28gg-CD3Zeta (SEQ ID NO: 55).

[0135] FIG. 8A depicts the amino acid sequence of scFv6T-CD8h-CD28tm-CD28gg-CD3Zeta (SEQ ID NO: 56).

[0136] FIG. 8B depicts the amino acid sequence of scFv6T-lgG4H-CD28tm-CD28gg-CD3Zeta (SEQ ID NO: 57).

[0137] FIG. 9A depicts the amino acid sequence of scFv6T-L-CD28tm-CD28gg-CD3Zeta (SEQ ID NO: 58).

[0138] FIG. 9B depicts the amino acid sequence of scFv6T-lgG4(S228P, L235E, N297Q)-CD28tm-CD28gg-41BB-CD3Zeta (SEQ ID NO: 59).

[0139] FIG. 10A depicts the amino acid sequence of scFv6T-lgG4(HL-CH3)-CD28tm-CD28gg-41BB-CD3Zeta (SEQ ID NO: 60).

[0140] FIG. 10B depicts the amino acid sequence of scFv6T-CD8h-CD28tm-CD28gg-41BB-CD3Zeta (SEQ ID NO: 61).

[0141] FIG. 11 A depicts the amino acid sequence of scFv6T-lgG4H-CD28tm-CD28gg-41BB-CD3Zeta (SEQ ID NO: 62).

[0142] FIG. 11B depicts the amino acid sequence of scFv6T-L-CD28tm-CD28gg-41BB-CD3Zeta (SEQ ID NO: 63).

[0143] FIG. 12A depicts the amino acid sequence of scFv6T-lgG4(L235E, N297Q)-CD4tm-41BB-CD3Zeta (SEQ ID NO: 64).

[0144] FIG. 12B depicts the amino acid sequence of scFv6T-lgG4(HL-CH3)-CD4tm-41BB-CD3Zeta (SEQ ID NO: 65).

[0145] FIG. 13A depicts the amino acid sequence of scFv6T-CD8h-CD28tm-41BB-CD3Zeta (SEQ ID NO: 66).

[0146] FIG. 13B depicts the amino acid sequence of scFv6T-lgG4H-CD28tm-41BB-CD3Zeta (SEQ ID NO: 67).

[0147] FIG. 14A depicts the amino acid sequence of scFv6T-L-CD28tm-41BB-CD3Zeta (SEQ ID NO: 68).Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0148] FIG. 14B depicts the amino acid sequence of scFv6T-CD8h(45aa)-CD8tm3-41BB-CD3Zeta (SEQ ID NO: 69).

[0149] FIG. 15A depicts the amino acid sequence of scFv6O-lgG4(S228P, L235E, N297Q)-CD28tm-CD28gg-CD3Zeta (SEQ ID NO: 70).

[0150] FIG. 15B depicts the amino acid sequence of scFv6O-lgG4(HL-CH3)-CD28tm-CD28gg-CD3Zeta (SEQ ID NO: 71).

[0151] FIG. 16A depicts the amino acid sequence of scFv6O-CD8h-CD28tm-CD28gg-CD3Zeta (SEQ ID NO: 72).

[0152] FIG. 16B depicts the amino acid sequence of scFv6O-lgG4H-CD28tm-CD28gg-CD3Zeta (SEQ ID NO: 73).

[0153] FIG. 17A depicts the amino acid sequence of scFv6O-L-CD28tm-CD28gg-CD3Zeta (SEQ ID NO: 74).

[0154] FIG. 17B depicts the amino acid sequence of scFv6O-lgG4(S228P, L235E, N297Q)-CD28tm-CD28gg-41BB-CD3Zeta (SEQ ID NO: 75).

[0155] FIG. 18A depicts the amino acid sequence of scFv6O-lgG4(HL-CH3)-CD28tm-CD28gg-41BB-CD3Zeta (SEQ ID NO: 76).

[0156] FIG. 18B depicts the amino acid sequence of scFv6O-CD8h-CD28tm-CD28gg-41BB-CD3Zeta (SEQ ID NO: 127).

[0157] FIG. 19A depicts the amino acid sequence of scFv6O-lgG4H-CD28tm-CD28gg-41BB-CD3Zeta (SEQ ID NO: 77).

[0158] FIG. 19B depicts the amino acid sequence of scFv6O-L-CD28tm-CD28gg-41BB-CD3Zeta (SEQ ID NO: 78).

[0159] FIG. 20A depicts the amino acid sequence of scFv6O-lgG4(L235E, N297Q)-CD4tm-41BB-CD3Zeta (SEQ ID NO:79).

[0160] FIG. 20B depicts the amino acid sequence of scFv6O-lgG4(HL-CH3)-CD4tm-41BB-CD3Zeta (SEQ ID NO: 80).

[0161] FIG. 21 A depicts the amino acid sequence of scFv6O-CD8h-CD28tm-41 BB-CD3Zeta (SEQ ID NO: 81).Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0162] FIG. 21 B depicts the amino acid sequence of scFv6O-lgG4H-CD28tm-41BB-CD3Zeta (SEQ ID NO: 82).

[0163] FIG. 22A depicts the amino acid sequence of scFv6O-L-CD28tm-41BB-CD3Zeta (SEQ ID NO: 83).

[0164] FIG. 22B depicts the amino acid sequence of scFv6O-CD8h(45aa)-CD8tm3-41BB-CD3Zeta (SEQ ID NO: 84).

[0165] FIG. 23A depicts the amino acid sequence of scFv3H-lgG4(S228P, L235E, N297Q)-CD28tm-CD28gg-CD3Zeta (SEQ ID NO: 85).

[0166] FIG. 23B depicts the amino acid sequence of ScFv3H-lgG4(HL-CH3)-CD28tm-CD28gg-CD3Zeta (SEQ ID NO: 86).

[0167] FIG. 24A depicts the amino acid sequence of ScFv3H-CD8h-CD28tm-CD28gg-CD3Zeta (SEQ ID NO: 87).

[0168] FIG. 24B depicts the amino acid sequence of ScFv3H-lgG4H-CD28tm-CD28gg-CD3Zeta (SEQ ID NO: 88).

[0169] FIG. 25A depicts the amino acid sequence of ScFv3H-L-CD28tm-CD28gg-CD3Zeta (SEQ ID NO: 89).

[0170] FIG. 25B depicts the amino acid sequence of ScFv3H-lgG4(S228P, L235E, N297Q)-CD28tm-CD28gg-41BB-CD3Zeta (SEQ ID NO: 90).

[0171] FIG. 26A A depicts the amino acid sequence of ScFv3H-lgG4(HL-CH3)-CD28tm-CD28gg-41BB-CD3Zeta (SEQ ID NO: 91).

[0172] FIG. 26B depicts the amino acid sequence of ScFv3H-CD8h-CD28tm-CD28gg-41BB-CD3Zeta (SEQ ID NO: 92).

[0173] FIG. 27A depicts the amino acid sequence of ScFv3H-lgG4H-CD28tm-CD28gg-41BB-CD3Zeta (SEQ ID NO: 93).

[0174] FIG. 27B depicts the amino acid sequence of ScFv3H-L-CD28tm-CD28gg-41BB-CD3Zeta (SEQ ID NO: 94).

[0175] FIG. 28A depicts the amino acid sequence of ScFv3H-lgG4(L235E, N297Q)-CD4tm-41BB-CD3Zeta (SEQ ID NO: 95).Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0176] FIG. 28B depicts the amino acid sequence of ScFv3H-lgG4(HL-CH3)-CD4tm-41BB-CD3Zeta (SEQ ID NO: 96).

[0177] FIG. 29A depicts the amino acid sequence of ScFv3H-CD8h-CD28tm-41BB-CD3Zeta (SEQ ID NO: 97).

[0178] FIG. 29B depicts the amino acid sequence of ScFv3H-lgG4H-CD28tm-41BB-CD3Zeta (SEQ ID NO: 98).

[0179] FIG. 30A depicts the amino acid sequence of ScFv3H-L-CD28tm-41BB-CD3Zeta (SEQ ID NO: 99).

[0180] FIG. 30B depicts the amino acid sequence of scFv6O-CD8h(45aa)-CD8tm3-41BB-CD3Zeta (SEQ ID NO: 100).

[0181] FIGS. 31A-31C depict the nucleotide sequence of vector used for expression of scFv6T-lgG4(SmP)-HL-CD4tm-41BB-CD3Zeta (SEQ ID NO:139)

[0182] FIG. 32A depicts the amino acid sequence of ScFv6T-lgG4(S228P)H-L-CD4tm-41BB-CD3Zeta (SEQ ID NO: 128)

[0183] FIG. 32B depicts the amino acid sequence of ScFv6T-lgG4H-L-CD4tm-41BB-CD3Zeta (SEQ ID NO: 129)

[0184] FIGS. 33A-33C depict the nucleotide sequence of vector used for expression of scFv6T-CD8h(45aa)-CD8tm3-41BB-CD3Zeta (SEQ ID NO:140).

[0185] FIGS. 34A-34C depict humanized anti-EGFR(6T) BB T cells induce low cytotoxicity against WT-EGFR expressing normal astrocytes. (A) Flow cytometric analysis of EGFR expression (red) on normal human astrocytes, negative control Nalm6 cells, or positive control Nalm6-EGFRvlll cells as compared to control staining (grey). (B) T cells that were either mock-transduced (Mock), or transduced to express the parental EGFR-CAR (806), the humanized 6T EGFR-CAR (h806-6T) or the Cetuximab-based EGFR-CAR (Cetux) were used as effectors in a flow cytometry based 48-hour astrocyte killing assay at the indicated E: T ratios. % Astrocyte killing was calculated by comparing the viable numbers of the indicated target line co-cultured with CAR+ T cells to that of astrocytes only. Mean ± S. E. M. values are depicted. (C) Mock T cells or the anti-EGFR BBζ T cells were co-cultured with each cell line depicted in (A) at an E: T ratio of 2:1 for 19 hours. Harvested supernatants were evaluated for IFNy levels by ELISA. Bars indicate Means with SD of triplicate wells.Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0186] FIG. 35 depicts humanized anti-EGFR(6T) CAR shows selective recognition of EGFRvlll over WT EGFR. Flow cytometric analysis of WT EGFR (left) and EGFRvlll (right) binding to Jurkat cells that did not express a CAR (None), or Jurkat cells transduced to express either 6T-CAR (h806-6T) or the Cetuximab-based EGFR-CAR (Cetux). Mean flourescence intensity (MFI) values for the binding of titrated concentrations of biotinylated WT EGFR or EGFRvlll proteins as detected using APC-conjugated streptavidin are depicted.

[0187] DETAILED DESCRIPTION

[0188] I. Chimeric Antigen Receptors

[0189] A chimeric antigen receptor (CAR) refers to an artificial immune cell receptor that is engineered to recognize and bind to a surface antigen. A T cell that expresses a CAR polypeptide is referred to as a CAR T cell. CAR can redirect T-cell specificity and reactivity toward a selected target in a non-MHC-restricted manner. The non-MHC-restricted antigen recognition gives CAR T cells the ability to recognize an antigen independent of antigen processing, thereby bypassing a major mechanism of tumor escape. A CAR can also be expressed by other immune effector cells, including but not limited to natural killer CAR (“NK CAR”) and directed NK cell killing to cells expressing the target of the CAR.

[0190] There are various generations of CARs, each of which contains different components. First generation CARs join an antibody-derived scFv to the CD3ζ intracellular signaling domain of the T cell receptor through a spacer region (also called a hinge domain) and a transmembrane domain. Second generation CARs incorporate an additional co-stimulatory domain (e.g., CD28, 4-BB, ICOS or OX40) to supply a co-stimulatory signal. Third generation CARs contain two co-stimulatory domains (e.g., a combination of CD27, CD28, 4-1BB, ICOS, or OX40) fused with the TCR CD3ζ chain.

[0191] There can be a spacer between the co-stimulatory domain and the CD3ζ domain, but this is optional. A CAR is often fused to a signal peptide at the N-terminus for surface expression. In some cases, the CAR can be co-expressed with a polypeptide that can serve as marker, for example a truncated EGFR receptor lacking signaling function or a truncated CD19 receptor lacking signaling function.

[0192] (a) Extracellular Binding DomainAttorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0193] Useful CAR described herein have an extracellular domain that binds overexpressed human EGFR and the human EGFR variant EGFRVIII. The scFv can include any of:

[0194] H6T scFv (SEQ ID NO: 4);

[0195] VH (SEQ ID NO: 5) followed by VL (SEQ ID NO: 6) of H6T scFv joined by a linker;

[0196] H6O scFv (SEQ ID NO: 7);

[0197] VH (SEQ ID NO: 8) followed by VL (SEQ ID NO: 9) of H6) scFv joined by a linker;

[0198] H3t scFv (SEQ ID NO: 10); or

[0199] VH (SEQ ID NO: 11) followed by VL (SEQ ID NO: 12) of H3t scFv joined by a linker.

[0200] The extracellular domain that binds EGFR can be an scFv, Fab, a single chain Fab, a meditope enabled Fab, or a meditope enabled single chain Fab.

[0201] The linker joining the VH and VL is a flexible linker of, e.g., 5-25 amino acids. In some embodiments, a useful flexible linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats of the sequence GGGS. In some embodiments, a useful flexible linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats of the sequence GGGGS (SEQ ID NO: 141). In some embodiments, the VL domain is amino terminal to the VH domain in other cases it is carboxy terminal to the VH domain. In some cases, the linker comprises the sequence SSGGGGSGGGGSGGGGS (SEQ ID NO: 142). Other useful linkers include SGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 143) and GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 144).

[0202] (b) Transmembrane Domain

[0203] The CAR disclosed herein can contain a transmembrane domain, which can be a hydrophobic alpha helix that spans the membrane. As used herein, a transmembrane domain refers to any protein structure that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane.

[0204] Useful transmembrane domains include those shown below.

[0205] Table 1: Examples of Transmembrane Domains

[0206] Name Accession Length Sequence

[0207] CD3z J04132.1 21 aa LCYLLDGILFIYGVILTALFL (SEQ ID NO: 15)

[0208]

[0209] Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0210] CD28 NM_006139 27aa FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 16)

[0211] CD28(M) NM_006139 28aa M FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 17)

[0212] CD4 M35160 22aa MALIVLGGVAGLLLFIGLGIFF (SEQ ID NO: 18)

[0213] CD8tm NM_001768 21aa IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 19)

[0214] CD8tm2 NM_001768 23aa IYIWAPLAGTCGVLLLSLVITLY (SEQ ID NO: 20)

[0215] CD8tm3 NM_001768 24aa IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO:

[0216] 21)

[0217] 4-1 BB NM_001561 27aa IISFFLALTSTALLFLLFF LTLRFSVV (SEQ ID NO:

[0218] 22)

[0219] NKG2D NM_007360 21aa PFFFCCFIAVAMGIRFIIMVA (SEQ ID NO: 23)

[0220]

[0221] (c) Spacer Domain

[0222] The CAR described herein can include a spacer domain located between the targeting domain and the transmembrane domain. The spacer region can function to provide flexibility to the CAR, or domains thereof, or to prevent steric hindrance of the CAR, or domains thereof. A variety of different spacers can be used. Some of them include at least portion of a human Fc region, for example a hinge portion of a human Fc region or a CH3 domain or variants thereof. Table 2 below provides various spacer domains that can be used in the CARs described herein.

[0223] Table 2: Examples of Spacer Domains

[0224] Name Length Sequence

[0225] a3 3 aa AAA

[0226] linker 10 aa GGGSSGGGSG (SEQ ID NO: 24)

[0227] lgG4 hinge (S→P) 12 aa ESKYGPPCPPCP (SEQ ID NO: 25)

[0228] (S228P)

[0229]

[0230] Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0231] lgG4 hinge 12 aa ESKYGPPCPSCP (SEQ ID NO: 26)

[0232] lgG4 hinge (S228P) + 22 aa ESKYGPPCPPCPGGGSSGGGSG (SEQ ID NO: linker 27)

[0233] CD28 hinge 39 aa IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGP SKP (SEQ ID NO: 28)

[0234] CD8 hinge-48aa 48 aa AKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAG GAVHTRGLDFACD (SEQ ID NO: 29)

[0235] CD8 hinge-45aa 45aa TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV HTRGLDFACD (SEQ ID NO: 30) lgG4(H(S228P)L- 129 aa ESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTL CH3) PPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQE

[0236] Also called

[0237] GNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID lgG4(H(S228P)L- NO: 31)

[0238] ACH2)

[0239] (includes S228P in

[0240] hinge)

[0241] lgG4(HL-CH3) 129 aa ESKYGPPCPSCPGGGSSGGGSGGQPREPQVYTL PPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNG

[0242] Also called lgG4(HL- QPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQE ACH2)

[0243] GNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 134)

[0244] lgG4(L235E, N297Q) 229 aa ESKYGPPCPSCPAPEFEGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHN AKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQ EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 32)

[0245] lgG4(S228P, 229 aa ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMI L235E. N297Q) SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHN AKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQ EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 33)

[0246]

[0247] Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0248] lgG4(CH3) 107 aa GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS

[0249] Also called

[0250] RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL

[0251] lgG4(ACH2)

[0252] SLSLGK (SEQ ID NO: 34)

[0253]

[0254] Some spacer domains include all or partof an immunoglobulin (e.g., lgG1, lgG2, lgG3, lgG4) hinge region, i.e., the sequence that falls between the CH1 and CH2 domains of an immunoglobulin, e.g., an lgG4 Fc hinge or a CD8 hinge. Some spacer domains include an immunoglobulin CH3 domain (called CH3 or ACH2) or both a CH3 domain and a CH2 domain. The immunoglobulin derived sequences can include one or more amino acid modifications, for example, 1, 2, 3, 4 or 5 substitutions, e.g., substitutions that reduce off-target binding.

[0255] The spacer domain can also comprise an lgG4 hinge region having the sequence ESKYGPPCPSCP (SEQ ID NO: 26) or ESKYGPPCPPCP (SEQ ID NO: 25). The hinge / linger region can also comprise the sequence ESKYGPPCPPCP (SEQ ID NO: 3) followed by the linker sequence GGGSSGGGSG (SEQ ID NO: 24) followed by lgG4 CH3 sequence:

[0256] GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 34). Thus, the spacer domain can comprise the sequence:

[0257] ESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY TQKSLSLSLGK (SEQ ID NO: 31). In some cases, the spacer has 1, 2, 3, 4, or 5 single amino acid changes (e.g., conservative changes) compared to SEQ ID NO: 31. In some cases, the lgG4 Fc hinge / linker region that is mutated at two positions (L235E; N297Q) in a manner that reduces binding by Fc receptors (FcRs).

[0258] (d) Intracellular Signaling Domains

[0259] Any of the CAR constructs described herein contain one or more intracellular signaling domains (e.g., CD3, and optionally one or more co-stimulatory domains), which are the functional end of the receptor. Following antigen recognition, receptors cluster and a signal is transmitted to the cell.

[0260] CD3 is the cytoplasmic signaling domain of the T cell receptor complex. CD3 contains three immunoreceptor tyrosine-based activation motifs (ITAMs), which transmit an activation signal to the T cell after the T cell is engaged with a cognate antigen. In some cases, CD3Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0261] provides a primary T cell activation signal but not a fully competent activation signal, which requires a co-stimulatory signal.

[0262] Accordingly, in some examples, the CAR polypeptides disclosed herein may further comprise one or more co-stimulatory signaling domains in addition to CD3. For example, the co-stimulatory domain CD28 and / or 4-1 BB can be used to transmit a proliferative / survival signal together with the primary signaling mediated by CD3

[0263] The co-stimulatory domain(s) are located between the transmembrane domain and the CD3ζ signaling domain. Table 3 includes examples of suitable co-stimulatory domains together with the sequence of the CD3ζ signaling domain.

[0264] Table 3: CD3 Domain and Examples of Co-stimulatory Domains

[0265] Name Accession Lengt Sequence

[0266] h

[0267] CD3C J04132.1 113 aa RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV LDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY DALHMQALPPR (SEQ ID NO: 35)

[0268] ITAMS 1-3 underlined

[0269] CD3C 113 aa RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV LDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKM

[0270] variant

[0271] AEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTF DALHMQALPPR (SEQ ID NO:47)

[0272] CD3C 113 aa RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV LDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKM

[0273] variant

[0274] AEAFSEIGMKGERRRGKGHDGLYQGLSTATKDTY DALHMQALPPR (SEQ ID NO: 48)

[0275] CD3C 113 aa RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV LDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM

[0276] variant

[0277] AEAYSEIGMKGERRRGKGHDGLFQGLSTATKDTF DALHMQALPPR (SEQ ID NO: 49)

[0278] CD3C 113 aa RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV LDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM

[0279] variant

[0280] AEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTF DALHMQALPPR (SEQ ID NO:50)

[0281] CD3C 113 aa RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV LDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKM

[0282] variant

[0283] AEAYSEIGMKGERRRGKGHDGLFQGLSTATKDTF DALHMQALPPR (SEQ ID NO:51)

[0284]

[0285] Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0286] CD3C 113 aa RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV LDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKM

[0287] variant

[0288] AEAFSEIGMKGERRRGKGHDGLYQGLSTATKDTF DALHMQALPPR (SEQ ID NO:52)

[0289] CD3C 113 aa RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV LDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKM

[0290] variant

[0291] AEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTY DALHMQALPPR (SEQ ID NO:53)

[0292] CD28 NM_006139 42 aa RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPR DFAAYRS (SEQ ID NO: 36)

[0293] CD28gg NM_006139 42 aa RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPP RDFAAYRS (SEQ ID NO: 37)

[0294] 41BB NM_001561 42 aa KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPE EEEGGCEL (SEQ ID NO: 38)

[0295] 0X40 NM_003327 42 aa ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADA HSTLAKI (SEQ ID NO:39)

[0296] 2B4 NM_016382 120 aa WRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQ EQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQ PSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPA RLSRKELENFDVYS (SEQ ID NO: 40)

[0297]

[0298] In some examples, the CD3ζ signaling domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 98% identical to SEQ ID NO: 35. In such instances, the CD3 signaling domain has 1, 2, 3, 4, or 5 amino acid changes (preferably conservative substitutions) compared to SEQ ID NO: 35. In other examples, the CD3 signaling domain is SEQ ID NO: 35.

[0299] In various embodiments: the co-stimulatory domain is selected from the group consisting of: a co-stimulatory domain depicted in Table 3 or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications, a CD28 co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications, a 4-1 BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications and an 0X40 co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications. In certain embodiments, a 4-1 BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications is present in the CAR polypeptides described herein.

[0300] In some embodiments, there are two co-stimulatory domains, for example, a CD28 co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modificationsAttorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0301] (e.g., substitutions) and a 4-1 BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions). In various embodiments the 1-5 (e.g., 1 or 2) amino acid modification are substitutions. In various embodiments, the co-stimulatory domain is amino terminal to the CD3 signaling domain and a short linker consisting of 2 -10, e.g., 3 amino acids (e.g., GGG) can be positioned between the co-stimulatory domain and the CD3ζ signaling domain.

[0302] In some cases, the CAR can be produced using a vector in which the CAR open reading frame is followed by a T2A ribosome skip sequence and a truncated EGFR (EGFRt), which lacks the cytoplasmic signaling tail, or a truncated CD19R (also called CD19t). In this arrangement, co-expression of EGFRt or CD19t provides an inert, non-immunogenic surface marker that allows for accurate measurement of gene modified cells, and enables positive selection of gene-modified cells, as well as efficient cell tracking of the therapeutic T cells in vivo following adoptive transfer. Efficiently controlling proliferation to avoid cytokine storm and off-target toxicity is an important hurdle for the success of T cell immunotherapy. The EGFRt or the CD19t incorporated in the CAR lentiviral vector can act as suicide gene to ablate the CAR+ T cells in cases of treatment-related toxicity.

[0303] The CD3 signaling domain can be followed by a ribosomal skip sequence (e.g., LEGGGEGRGSLLTCGDVEENPGPR; SEQ ID NO: 41) and a truncated EGFR having a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to:

[0304] LVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGD SFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAWSL NITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVC HALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQ AMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCT YGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM (SEQ ID NO: 42). In some cases, the truncated EGFR has 1, 2, 3, 4 of 5 amino acid changes (preferably conservative) compared to SEQ ID NO: 42.

[0305] Alternatively the CD3 signaling domain can be followed by a ribosomal skip sequence (e.g., LEGGGEGRGSLLTCGDVEENPGPR; SEQ ID NO: 41) and a truncated CD19R (also called CD19t) having a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to:

[0306] MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPF LKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGEL FRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCVPPRDSL NQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMAttorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0307] WVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKVSAVTLAY LIFCLCSLVGILHLQRALVLRRKR (SEQ ID NO: 43).

[0308] The CAR described herein can be produced by any means known in the art, though preferably it is produced using recombinant DNA techniques. Nucleic acids encoding the several regions of the chimeric receptor can be prepared and assembled into a complete coding sequence by standard techniques of molecular cloning known in the art (genomic library screening, overlapping PCR, primer-assisted ligation, site-directed mutagenesis, etc.) as is convenient. The resulting coding region is preferably inserted into an expression vector and used to transform a suitable expression host cell line, preferably a T lymphocyte, and most preferably an autologous T lymphocyte.

[0309] Various T cell subsets isolated from the patient can be transduced with a vector for CAR or polypeptide expression. Central memory T cells are one useful T cell subset. Central memory T cell can be isolated from peripheral blood mononuclear cells (PBMC) by selecting for CD45RO+ / CD62L+ cells, using, for example, the CliniMACS® device to immunomagnetically select cells expressing the desired receptors. The cells enriched for central memory T cells can be activated with anti-CD3 / CD28, transduced with, for example, a lentiviral vector that directs the expression of a CAR or as well as a non-immunogenic surface marker for in vivo detection, ablation, and potential ex vivo selection. The activated / genetically modified central memory T cells can be expanded in vitro with IL-2 / IL-15 and then cryopreserved. Additional methods of preparing CAR T cells can be found in PCT / US2016 / 043392.

[0310] Methods for preparing useful T cell populations are described in, for example, WO 2017 / 015490 and WO 2018 / 102761. In some cases, it may be useful to use natural killer (NK) cells, e.g., allogenic NK cells derived from peripheral blood or cord blood. In other cases, NK cells can be derived from human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs).

[0311] In some embodiments, described herein is a composition comprising the iPSC-derived CAR T cells or CAR NK cells. In some embodiments, a composition comprising iPSC-derived CAR T cells or CAR NK cells has enhanced therapeutic properties. In some embodiments, the iPSC-derived CAR T cells or CAR NK cells demonstrate enhanced functional activity including potent cytokine production, cytotoxicity and cytostatic inhibition of tumor growth, e.g., as activity that reduces the amount of tumor load.Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0312] The CAR can be transiently expressed in a T cell population by an mRNA encoding the CAR. The mRNA can be introduced into the T cells by electroporation (Wiesinger et al. 2019 Cancers (Basel) 11:1198).

[0313] In some embodiments, a composition comprising the CAR T cells comprise one or more of helper T cells, cytotoxic T cells, memory T cells, naive T cells, regulatory T cells, natural killer T cells, or combinations thereof.

[0314] Various CAR targeted to EGFR are depicted in FIGs. 7A-33C that include an scFv comprising the amino acid sequence of any one of SEQ ID NO: 4, 7 or 10.

[0315] The present disclosure includes variants of the EGFR CAR in FIGs 7A-33C. For example, in each construct having an scFv comprising SEQ ID NO: 7, it could be replaced by an scFv comprising SEQ ID NO: 132. In each construct having an scFv comprising SEQ ID NO: 10, it could be replaced by an scFv comprising SEQ ID NO: 133.

[0316] The present disclosure includes variants of the EGFR CAR in FIGs 7A-33C. For example, in each construct having a CD28 co-stimulatory domain comprising RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 37), it could be replaced by a CD28 co-stimulatory domain comprising RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 36).

[0317] The present disclosure includes variants of the EGFR CAR in FIGs 7A-33C. For example, in each construct having a spacer that includes an lgG4 hinge comprising the sequence ESKYGPPCPPCP (SEQ ID NO: 25), it could be replaced by an lgG4 hinge comprising the sequence ESKYGPPCPSCP (SEQ ID NO: 26).

[0318] The following CAR targeted to EGFR are used in the some of the Examples below.

[0319] EGFRscFv(h6T)-lgG4(HL-CH3)-CD4tm-41BB-CD3Zeta EVQLVESGGGLVQPGGSLRLSCAVTGYSITSDFAWNWIRQAPGKGLEWMGYISYSGN TRYNPSLKSRITISRDTSKNTFYLQMNSLRAEDTAVYYCVTAGRGFPYWGQGTLVTVSS GGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHSSQDINSNIGWLQQKPGK AFKGLIYHGTNLDDEVPSRFSGSGSGADYTLTISSLQPEDFATYYCVQYAQFPWTFGQ GTKVEIKESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSC SVMHEALHNHYTQKSLSLSLGKMALIVLGGVAGLLLFIGLGIFFKRGRKKLLYIFKQPFMR PVQTTQEEDGCSCRFPEEEEGGCELGGGRVKFSRSADAPAYQQGQNQLYNELNLGRAttorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0320] REEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 65, also depicted in FIG. 12B)

[0321] EGFRscFv(h6O)-lgG4(HL-CH3)-CD4tm-41BB-CD3Zeta QVQLEESGPGLVKPSETLSLTCSVTGYSITSDFAWNWIRQPPGKGLEWMGYISYSGNT RYNPSLKSRITISRDTSKNEFSLRLTSVTAADTAVYYCVTAGRGFPYWGQGTLVTVSSGG GGSGGGGSGGGGSDIVMTQSPSSLSASIGDRVTITCHSSQDINSNIGWLQQKPGKAFK GLIYHGTNLDDEVPSRFSGSGSGAEYTLTIRSLQPEDFATYYCVQYAQFPWTFGQGTKV EIKESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGKMALIVLGGVAGLLLFIGLGIFFKRGRKKLLYIFKQPFMRPVQTT QEEDGCSCRFPEEEEGGCELGGGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYD VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL YQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 80, also depicted in FIG. 20B)

[0322] EGFRscFv(h3T)-lgG4(HL-CH3)-CD4tm-41BB-CD3Zeta

[0323] QVQLQESGPGLVKPSQTLSLTCTVSGYSITSDFAWNWIRQPPGKGLEWIGYISYSGNT RYNPSLKSRVSISVDTSKNQFSLKLNSVTAADTAVYYCVTAGRGFPYWGQGTLVTVSS GGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHSSQDINSNIGWYQQRPGK APKLLIYHGTNLDDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCVQYAQFPWTFGQG TKVEIKESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS VMHEALHNHYTQKSLSLSLGKMALIVLGGVAGLLLFIGLGIFFKRGRKKLLYIFKQPFMR PVQTTQEEDGCSCRFPEEEEGGCELGGGRVKFSRSADAPAYQQGQNQLYNELNLGR REEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 96, also depicted in FIG. 28B)

[0324] EGFRscFv(806)-lgG4(HL-CH3)-CD4tm-41BB-CD3Zeta

[0325] DVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYISYSGNT RYNPSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCVTAGRGFPYWGQGTLVTVSSGG GGSGGGGSGGGGSDILMTQSPSSMSVSLGDTVSITCHSSQDINSNIGWLQQRPGKSF KGLIYHGTNLDDEVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPWTFGGGTK LEIKESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVM HEALHNHYTQKSLSLSLGKMALIVLGGVAGLLLFIGLGIFFKRGRKKLLYIFKQPFMRPV QTTQEEDGCSCRFPEEEEGGCELGGGRVKFSRSADAPAYQQGQNQLYNELNLGRRE EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 130)Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0326] II. ANTIBODIES

[0327] The VL and VH domains of H6T, H6O and H3T can be incorporated into a variety of antibody formats, including, among others, scFv, Fab, single chain Fab, IgG antibodies (including IgG antibodies with variant Fc regions) and antibodies having a meditope-enabled Fab. Examples of such antibodies include the following:

[0328] H6T Antibodies

[0329] VH EVQLVESGGGLVQPGGSLRLSCAVTGYSITSDFAWNWIRQAPGKGLEWMGYISYSGNTRY NPSLKSRITISRDTSKNTFYLQMNSLRAEDTAVYYCVTAGRGFPYWGQGTLVTVSS (SEQ ID NO: 5)

[0330] VL DIQMTQSPSSLSASVGDRVTITCHSSQDINSNIGWLQQKPGKAFKGLIYHGTNLDDEVPSRF SGSGSGADYTLTISSLQPEDFATYYCVQYAQFPWTFGQGTKVEIK (SEQ ID NO: 6)

[0331] H6O Antibodies

[0332] VH QVQLEESGPGLVKPSETLSLTCSVTGYSITSDFAWNWIRQPPGKGLEWMGYISYSGNTRYN PSLKSRITISRDTSKNEFSLRLTSVTAADTAVYYCVTAGRGFPYWGQGTLVTVSS (SEQ ID NO: 8)

[0333] VL DIVMTQSPSSLSASIGDRVTITCHSSQDINSNIGWLQQKPGKAFKGLIYHGTNLDDEVPSRF SGSGSGAEYTLTIRSLQPEDFATYYCVQYAQFPWTFGQGTKVEIK (SEQ ID NO: 9)

[0334] H3T Antibodies

[0335] VHAttorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0336] QVQLQESGPGLVKPSQTLSLTCTVSGYSITSDFAWNWIRQPPGKGLEWIGYISYSGNTRYN PSLKSRVSISVDTSKNQFSLKLNSVTAADTAVYYCVTAGRGFPYWGQGTLVTV (SEQ ID NO: 11)

[0337] VL DIQMTQSPSSLSASVGDRVTITCHSSQDINSNIGWYQQRPGKAPKLLIYHGTNLDDGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCVQYAQFPWTFGQGTKVEI K (SEQ ID NO: 12)

[0338] In some embodiments, the antibodies and other antibody formats can include:

[0339] a VH comprising the amino acid sequence of any one of SEQ ID NOs:5, 8, and 11 or a variant thereof having 1, 2, or 3 single amino acid modifications (e.g., 2 conservative amino acid substitutions), wherein the amino acid modifications are not in the CDRs; and a VL comprising the amino acid sequence of any one of SEQ ID NOs:6, 9, and 12 or a variant thereof having 1, 2, or 3 single amino acid substitutions (e.g., 3 conservative amino acid substitutions), wherein the amino acid substitutions are not in the CDRs.

[0340] For example, a H3T antibody, Fab, scFv, or other antibody format can comprise: a VH domain comprising or consisting of SEQ ID NO: 11 having 0, 1, or 2 singe amino acid substitutions, wherein the substitutions are not within amino acids 16-105 of SEQ ID NO:11; and a VL domain comprising or consisting of SEQ ID NO: 12 having 0, 1, or 2 singe amino acid substitutions, wherein the substitutions are not within amino acids 20-97 of SEQ ID NO:12.

[0341] A variety of additional antibodies can include the VL and VH domains of H6T, H6O and H3T. These include the following.

[0342] H6T Antibodies

[0343] H6T scFv (H-L) EVQLVESGGGLVQPGGSLRLSCAVTGYSITSDFAWNWIRQAPGKGLEWMGYISYSGNTRY NPSLKSRITISRDTSKNTFYLQMNSLRAEDTAVYYCVTAGRGFPYWGQGTLVTVSSGGGGS GGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHSSQDINSNIGWLQQKPGKAFKGLIYH GTNLDDEVPSRFSGSGSGADYTLTISSLQPEDFATYYCVQYAQFPWTFGQGTKVEIK (SEQ ID NO: 4)

[0344] H6T scFv (L-H)Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0345] DIQMTQSPSSLSASVGDRVTITCHSSQDINSNIGWLQQKPGKAFKGLIYHGTNLDDEVPSRF SGSGSGADYTLTISSLQPEDFATYYCVQYAQFPWTFGQGTKVEIKGGGGSGGGGSGGGG SEVQLVESGGGLVQPGGSLRLSCAVTGYSITSDFAWNWIRQAPGKGLEWMGYISYSGNTR YNPSLKSRITISRDTSKNTFYLQMNSLRAEDTAVYYCVTAGRGFPYWGQGTLVTVSS (SEQ ID NO: 131)

[0346] An H6T IgG can be composed of a heavy chain and a light chain having the following sequences.

[0347] H6T IgG heavy chain:

[0348] EVQLVESGGGLVQPGGSLRLSCAVTGYSITSDFAWNWIRQAPGKGLEWMGYISYSGNTRY NPSLKSRITISRDTSKNTFYLQMNSLRAEDTAVYYCVTAGRGFPYWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS WTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK (SEQ ID NO: 13)

[0349] H6T IgG light chain:

[0350] DIQMTQSPSSLSASVGDRVTITCHSSQDINSNIGWLQQKPGKAFKGLIYHGTNLDDEVPSRF SGSGSGADYTLTISSLQPEDFATYYCVQYAQFPWTFGQGTKVEI KRTVAAPSVFI FPPSDEQ LKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 14)

[0351] H6T FcEN

[0352] The Fc portion of an H6T IgG antibody can be altered to reduce Fc receptor binding. This is variant is referred to as an FcEN antibody.

[0353] H6T Heavy Chain FcEN

[0354] EVQLVESGGGLVQPGGSLRLSCAVTGYSITSDFAWNWIRQAPGKGLEWMGYISYSGNTRY NPSLKSRITISRDTSKNTFYLQMNSLRAEDTAVYYCVTAGRGFPYWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSAttorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0355] VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK (SEQ ID NO: 101)

[0356] H6T Light Chain FcEN

[0357] DIQMTQSPSSLSASVGDRVTITCHSSQDINSNIGWLQQKPGKAFKGLIYHGTNLDDEVPSRF SGSGSGADYTLTISSLQPEDFATYYCVQYAQFPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 102)

[0358] H6T FcSTR

[0359] The Fc portion of an H6T IgG antibody can be altered to reduce Fc receptor binding. This is variant is referred to as an FcSTR antibody.

[0360] H6T Heavy Chain FcSTR EVQLVESGGGLVQPGGSLRLSCAVTGYSITSDFAWNWIRQAPGKGLEWMGYISYSGNTRY NPSLKSRITISRDTSKNTFYLQMNSLRAEDTAVYYCVTAGRGFPYWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPESTRGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 103)

[0361] H6T Light Chain FcSTR DIQMTQSPSSLSASVGDRVTITCHSSQDINSNIGWLQQKPGKAFKGLIYHGTNLDDEVPSRF SGSGSGADYTLTISSLQPEDFATYYCVQYAQFPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 104)

[0362] H6T can also be in form of a single chain Fab (scFab). The light chain can precede the heavy chain (LH version) or the heavy chain can precede the light chain (HL version). In either case, a linker is present between the two chains.Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0363] H6T VH-CH1 EVQLVESGGGLVQPGGSLRLSCAVTGYSITSDFAWNWIRQAPGKGLEWMGYISYSGNTRY NPSLKSRITISRDTSKNTFYLQMNSLRAEDTAVYYCVTAGRGFPYWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 135)

[0364] H6T VL-CL

[0365] DIQMTQSPSSLSASVGDRVTITCHSSQDINSNIGWLQQKPGKAFKGLIYHGTNLDDEVPSRF SGSGSGADYTLTISSLQPEDFATYYCVQYAQFPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 136)

[0366] H6T scFab-LH

[0367] DIQMTQSPSSLSASVGDRVTITCHSSQDINSNIGWLQQKPGKAFKGLIYHGTNLDDEVPSRF SGSGSGADYTLTISSLQPEDFATYYCVQYAQFPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGECGGSSGSGSGSTGTSSSGTGTSAGTTGTSA STSGSGSGGGGGSGGGGSAGGTATAGASSGSEVQLVESGGGLVQPGGSLRLSCAVTGY SITSDFAWNWIRQAPGKGLEWMGYISYSGNTRYNPSLKSRITISRDTSKNTFYLQMNSLRA EDTAVYYCVTAGRGFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSC (SEQ ID NO: 105)

[0368] H6T scFab-HL

[0369] EVQLVESGGGLVQPGGSLRLSCAVTGYSITSDFAWNWIRQAPGKGLEWMGYISYSGNTRY NPSLKSRITISRDTSKNTFYLQMNSLRAEDTAVYYCVTAGRGFPYWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGGSSGSGSGSTGTSSSGTGTSAG TTGTSASTSGSGSGGGGGSGGGGSAGGTATAGASSGSDIQMTQSPSSLSASVGDRVTIT CHSSQDINSNIGWLQQKPGKAFKGLIYHGTNLDDEVPSRFSGSGSGADYTLTISSLQPEDF ATYYCVQYAQFPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAAttorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0370] KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC (SEQ ID NO: 106)

[0371] H6T antibodies can be in the form of a Meditope-enabled antibody.

[0372] HT6-Meditope VH-CH1 EVQLVESGGGLVQPGGSLRLSCAVTGYSITSDFAWNWIRQAPGKGLEWMGYISYSGNTRY NPSLKSRITISRDTSKNTFYLQMNSLRAEDTAIYYCVTAGRGFPYWGQGTLVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS WTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 107)

[0373] HT6-Meditope VL-CL DIQMTQSPILLSASVGDRVTITCHSSQDINSNIGWLQQRTNGSPRGLIYHGTNLDDEVPSRF SGSGSGADYTLTISSLQPEDIADYYCVQYAQFPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 108)

[0374] H6T-Meditope scFab-HL

[0375] EVQLVESGGGLVQPGGSLRLSCAVTGYSITSDFAWNWIRQAPGKGLEWMGYISYSGNTRY NPSLKSRITISRDTSKNTFYLQMNSLRAEDTAIYYCVTAGRGFPYWGQGTLVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGGSSGSGSGSTGTSSSGTGTSAGT TGTSASTSGSGSGGGGGSGGGGSAGGTATAGASSGSDIQMTQSPILLSASVGDRVTITCH SSQDINSNIGWLQQRTNGSPRGLIYHGTNLDDEVPSRFSGSGSGADYTLTISSLQPEDIADY YCVQYAQFPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC (SEQ ID NO: 109)

[0376] H6T-Meditope scFab-LH

[0377] DIQMTQSPILLSASVGDRVTITCHSSQDINSNIGWLQQRTNGSPRGLIYHGTNLDDEVPSRF SGSGSGADYTLTISSLQPEDIADYYCVQYAQFPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGECGGSSGSGSGSTGTSSSGTGTSAGTTGTSAAttorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0378] STSGSGSGGGGGSGGGGSAGGTATAGASSGSEVQLVESGGGLVQPGGSLRLSCAVTGY SITSDFAWNWIRQAPGKGLEWMGYISYSGNTRYNPSLKSRITISRDTSKNTFYLQMNSLRA EDTAIYYCVTAGRGFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSC (SEQ ID NO: 110)

[0379]

[0380] H6T-

[0381] H6T-Meditope IgG heavy chain:

[0382] EVQLVESGGGLVQPGGSLRLSCAVTGYSITSDFAWNWIRQAPGKGLEWMGYISYSGNTRY NPSLKSRITISRDTSKNTFYLQMNSLRAEDTAIYYCVTAGRGFPYWGQGTLVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS WTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK (SEQ ID NO: 111)

[0383] H6T-Meditope IgG light chain:

[0384] DIQMTQSPILLSASVGDRVTITCHSSQDINSNIGWLQQRTNGSPRGLIYHGTNLDDEVPSRF SGSGSGADYTLTISSLQPEDIADYYCVQYAQFPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 112)

[0385] H6O Antibodies

[0386] H6O scFv (H-L) QVQLEESGPGLVKPSETLSLTCSVTGYSITSDFAWNWIRQPPGKGLEWMGYISYSGNTRYN PSLKSRITISRDTSKNEFSLRLTSVTAADTAVYYCVTAGRGFPYWGQGTLVTVSSGGGGSG GGGSGGGGSDIVMTQSPSSLSASIGDRVTITCHSSQDINSNIGWLQQKPGKAFKGLIYHGT NLDDEVPSRFSGSGSGAEYTLTIRSLQPEDFATYYCVQYAQFPWTFGQGTKVEIK (SEQ ID NO: 7)

[0387]

[0388] H6O scFvAttorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0389] DIVMTQSPSSLSASIGDRVTITCHSSQDINSNIGWLQQKPGKAFKGLIYHGTNLDDEVPSRF SGSGSGAEYTLTI RSLQPEDFATYYCVQYAQFPWTFGQGTKVEI KGGGGSGGGGSGGGG SQVQLEESGPGLVKPSETLSLTCSVTGYSITSDFAWNWIRQPPGKGLEWMGYISYSGNTRY NPSLKSRITISRDTSKNEFSLRLTSVTAADTAVYYCVTAGRGFPYWGQGTLVTVSS (SEQ ID NO: 132)

[0390] An H6O IgG can be composed of a heavy chain and a light chain having the following sequences.

[0391] H6O IgG heavy chain:

[0392] QVQLEESGPGLVKPSETLSLTCSVTGYSITSDFAWNWIRQPPGKGLEWMGYISYSGNTRYN PSLKSRITISRDTSKNEFSLRLTSVTAADTAVYYCVTAGRGFPYWGQGTLVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK (SEQ ID NO: 113)

[0393] H6O IgG light chain:

[0394] DIVMTQSPSSLSASIGDRVTITCHSSQDINSNIGWLQQKPGKAFKGLIYHGTNLDDEVPSRF SGSGSGAEYTLTI RSLQPEDFATYYCVQYAQFPWTFGQGTKVEI KRTVAAPSVFI FPPSDEQ LKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYA_CEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 114)

[0395] H6O FcEN

[0396] The Fc portion of an H6T IgG antibody can be altered to reduce Fc receptor binding. This is variant is referred to as an FcEN antibody.

[0397] H6O Heavy Chain FcEN QVQLEESGPGLVKPSETLSLTCSVTGYSITSDFAWNWIRQPPGKGLEWMGYISYSGNTRY NPSLKSRITISRDTSKNEFSLRLTSVTAADTAIYYCVTAGRGFPYWGQGTLVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 115)Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0398] H6O Light Chain FcEN DIVMTQSPILLSASIGDRVTITCHSSQDINSNIGWLQQRTNGSPRGLIYHGTNLDDEVPSRFS GSGSGAETLTI RSLQPEDI ADYYCVQYAQFPWTFGQGTKVEI KRTVAAPSVFI FPPSDEQLK SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 116)

[0399] H6O FcSTR

[0400] The Fc portion of an H6T IgG antibody can be altered to reduce Fc receptor binding. This is variant is referred to as an FcSTR antibody.

[0401] H6O Heavy Chain FcSTR QVQLEESGPGLVKPSETLSLTCSVTGYSITSDFAWNWIRQPPGKGLEWMGYISYSGNTRYN PSLKSRITISRDTSKNEFSLRLTSVTAADTAVYYCVTAGRGFPYWGQGTLVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPESTRGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK (SEQ ID NO: 117)

[0402] H6O Light Chain FcSTR DIVMTQSPSSLSASIGDRVTITCHSSQDINSNIGWLQQKPGKAFKGLIYHGTNLDDEVPSRF SGSGSGAEYTLTI RSLQPEDFATYYCVQYAQFPWTFGQGTKVEI KRTVAAPSVFI FPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 118)

[0403] H6O can also be in form of a single chain Fab (scFab). The light chain can precede the heavy chain (LH version) or the heavy chain can precede the light chain (HL version). In either case, a linker is present between the two chains.

[0404] H6O LV-CL

[0405] DIVMTQSPSSLSASIGDRVTITCHSSQDINSNIGWLQQKPGKAFKGLIYHGTNLDDEVPSRF SGSGSGAEYTLTI RSLQPEDFATYYCVQYAQFPWTFGQGTKVEI KRTVAAPSVFI FPPSDEQAttorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0406] LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 137)

[0407] H6O HV-CH1

[0408] QVQLEESGPGLVKPSETLSLTCSVTGYSITSDFAWNWIRQPPGKGLEWMGYISYSGNTRYN PSLKSRITISRDTSKNEFSLRLTSVTAADTAVYYCVTAGRGFPYWGQGTLVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 138)

[0409] H6O scFab-LH

[0410] DIVMTQSPSSLSASIGDRVTITCHSSQDINSNIGWLQQKPGKAFKGLIYHGTNLDDEVPSRF SGSGSGAEYTLTI RSLQPEDFATYYCVQYAQFPWTFGQGTKVEI KRTVAAPSVFI FPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGECGGSSGSGSGSTGTSSSGTGTSAGTTGTSAST SGSGSGGGGGSGGGGSAGGTATAGASSGSQVQLEESGPGLVKPSETLSLTCSVTGYSITS DFAWNWIRQPPGKGLEWMGYISYSGNTRYNPSLKSRITISRDTSKNEFSLRLTSVTAADTAV YYCVTAGRGFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE PKSC (SEQ ID NO: 119)

[0411] H6O scFab-HL

[0412] QVQLEESGPGLVKPSETLSLTCSVTGYSITSDFAWNWIRQPPGKGLEWMGYISYSGNTRYN PSLKSRITISRDTSKNEFSLRLTSVTAADTAVYYCVTAGRGFPYWGQGTLVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGGSSGSGSGSTGTSSSGTGTSAGTTG TSASTSGSGSGGGGGSGGGGSAGGTATAGASSGSDIVMTQSPILLSASIGDRVTITCHSSQ DINSNIGWLQQRTNGSPRGLIYHGTNLDDEVPSRFSGSGSGAEYTLTIRSLQPEDIADYYCV QYAQFPWTFGQGTKVEI KRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC (SEQ ID NO: 120)Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0413] H6O antibodies can be in the form of a Meditope-enabled antibody.

[0414] H6O-Meditope VH-CH1 QVQLEESGPGLVKPSETLSLTCSVTGYSITSDFAWNWIRQPPGKGLEWMGYISYSGNTRYN PSLKSRITISRDTSKNEFSLRLTSVTAADTAVYYCVTAGRGFPYWGQGTLVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 121)

[0415] H6O-Meditope-VL-CL

[0416] DIVMTQSPILLSASIGDRVTITCHSSQDINSNIGWLQQRTNGSPRGLIYHGTNLDDEVPSRFS GSGSGAEYTLTI RSLQPEDI ADYYCVQYAQFPWTFGQGTKVEI KRTVAAPSVFI FPPSDEQL KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 122)

[0417] H6O antibodies can be in the form of a meditope-enabled scFabs. The light chain can precede the heavy chain (LH version) or the heavy chain can precede the light chain (HL version). In either case, a linker is present between the two chains.

[0418] H6O-Meditope scFab-HL QVQLEESGPGLVKPSETLSLTCSVTGYSITSDFAWNWIRQPPGKGLEWMGYISYSGNTRYN PSLKSRITISRDTSKNEFSLRLTSVTAADTAIYYCVTAGRGFPYWGQGTLVTVSSASTKGPSV FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCCGGSSGSGSGSTGTSSSGTGTSAGTTG TSASTSGSGSGGGGGSGGGGSAGGTATAGASSGSDIVMTQSPILLSASIGDRVTITCHSSQ DINSNIGWLQQRTNGSPRGLIYHGTNLDDEVPSRFSGSGSGAEYTLTIRSLQPEDIADYYCV QYAQFPWTFGQGTKVEI KRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC (SEQ ID NO: 123)

[0419] H6O-Meditope scFab-LH DIVMTQSPILLSASIGDRVTITCHSSQDINSNIGWLQQRTNGSPRGLIYHGTNLDDEVPSRFS GSGSGAEYTLTI RSLQPEDI ADYYCVQYAQFPWTFGQGTKVEI KRTVAAPSVFI FPPSDEQL KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGECCGGSSGSGSGSTGTSSSGTGTSAGTTGTSA STSGSGSGGGGGSGGGGSAGGTATAGASSGSQVQLEESGPGLVKPSETLSLTCSVTGYSAttorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0420] ITSDFAWNWIRQPPGKGLEWMYISYSGNTRYNPSLKSRITISRDTSKNEFSLRLTSVTAADT AIYYCVTAGRGFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSC (SEQ ID NO: 124)

[0421] H6O Meditope IgG

[0422] H6O-Meditope IgG heavy chain:

[0423] QVQLEESGPGLVKPSETLSLTCSVTGYSITSDFAWNWIRQPPGKGLEWMGYISYSGNTRYN PSLKSRITISRDTSKNEFSLRLTSVTAADTAIYYCVTAGRGFPYWGQGTLVTVSSASTKGPSV FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK (SEQ ID NO: 125)

[0424] H6O-Meditope IgG light chain:

[0425] DIVMTQSPILLSASIGDRVTITCHSSQDINSNIGWLQQRTNGSPRGLIYHGTNLDDEVPSRFS GSGSGAEYTLTI RSLQPEDI ADYYCVQYAQFPWTFGQGTKVEI KRTVAAPSVFI FPPSDEQL KSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 126)

[0426] H3T Antibodies

[0427] H3T scFv (H-L) QVQLQESGPGLVKPSQTLSLTCTVSGYSITSDFAWNWIRQPPGKGLEWIGYISYSGNTRYN PSLKSRVSISVDTSKNQFSLKLNSVTAADTAVYYCVTAGRGFPYWGQGTLVTVSSGGGGSG GGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHSSQDINSNIGWYQQRPGKAPKLLIYHGT NLDDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCVQYAQFPWTFGQGTKVEIK (SEQ ID NO: 10)

[0428] H3T scFv (L-H)Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0429] DIQMTQSPSSLSASVGDRVTITCHSSQDINSNIGWYQQRPGKAPKLLIYHGTNLDDGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCVQYAQFPWTFGQGTKVEIKSSGGGGSGGGGSGGG GSQVQLQESGPGLVKPSQTLSLTCTVSGYSITSDFAWNWIRQPPGKGLEWIGYISYSGNTR YNPSLKSRVSISVDTSKNQFSLKLNSVTAADTAVYYCVTAGRGFPYWGQGTLVTV (SEQ ID NO: 133)

[0430] Meditope-enabled antibodies

[0431] In Fab, single chain FABS antibodies and certain other antibody formats, the heavy chain variable (VH) region, the light chain variable (VL) region, the heavy chain constant region (CH) and the light chain constant region (CL) define a central cavity. Framework region amino acids in the cavity can be modified to form a peptide binding site. Such modified antibody formats are referred to as meditope-enabled antibodies. Meditope-enabled antibodies can bind certain peptides and peptidyl moieties.

[0432] The central cavity with respect to the three-dimensional structure of a Fab, refers to the internal cavity of the Fab lined by portions of the heavy and light chain variable and constant regions and including amino acids lining a hole within the cavity. In embodiments, where the antibody region includes a Fab, the central cavity thus is lined by residues of the VH, VL, CHI, and CL regions. The central cavity does not include the antigen binding site. Thus, the compound that binds to the central cavity does not meaningfully impact the binding of the antibody region to the epitope. In other words, in embodiments, occupancy of this site does not meaningfully impact antigen binding. In embodiments, the central cavity is lined by amino acid residues capable of interacting with a compound including a peptidyl moiety (e.g., a meditope) provided herein including embodiments thereof (e.g., a peptide of formula (I) or (II)). The amino acids residues capable of interacting with the compound including a peptidyl moiety (e.g. a meditope) may from part of the peptide binding site (also referred to herein as a meditope binding site).

[0433] In some embodiments, the meditope binding site includes residues 40, 41, 83, and 85 of the light chain of a meditope-enabled antibody, according to Kabat numbering, and / or residues 39, 89, 105, and 108 of the heavy chain of the meditope-enabled antibody, according to Kabat numbering.Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0434] In some embodiments, the meditope binding site is located within a cavity formed by residues 8, 9, 10, 38, 39, 40, 41 42, 43, 44, 45, 82, 83, 84, 85, 86, 87, 99, 100, 101, 102, 103, 104, 105, 142, 162, 163, 164, 165, 166, 167, 168, and 173 of the light chain and 6, 9, 38, 39, 40, 41, 42, 43, 44, 45, 84, 86, 87, 88, 89, 90, 91, 103, 104, 105, 106, 107, 108, 111, 110, 147, 150, 151,152,173, 174, 175, 176, 177, 185, 186, and 187 of the heavy chain of the antibody, according to Kabat numbering.

[0435] With respect to a Fab portion of a meditope-enabled antibody, the meditope binding site includes residues within the central cavity. The meditope-binding site typically further includes constant region residues.

[0436] A peptide or peptides that binds to a meditope-binding site of a meditope-enabled antibody, which antibody has a threonine at position 40, an asparagine at position 41, and an aspartatic acid at position 85 of its light chain, according to Kabat numbering can be referred to as a meditope. Exemplary meditopes include, but are not limited to, the cQFD and cQYN peptides and variants as well as multivalent and labeled meditopes. Other molecules may also bind to meditope binding sites of meditope-enabled antibodies, with functional characteristics similar to those of a meditope.

[0437] In embodiments, the amino acid residues lining the central cavity include a residue at a position corresponding to Kabat position 83, a residue at a position corresponding to Kabat position 30 or a residue at a position corresponding to Kabat position 52. In embodiments, the amino acid residues lining the central cavity include a residue at a position corresponding to Kabat position 40, a residue at a position corresponding to Kabat position 41, a residue at a position corresponding to Kabat position 30, a residue at a position corresponding to Kabat position 52, a residue at a position corresponding to Kabat position 83, or a residue at a position corresponding to Kabat position 85. In embodiments, the amino acid residues lining the central cavity include a residue at a position corresponding to Kabat position 40. In embodiments, the amino acid residues lining the central cavity include a residue at a position corresponding to Kabat position 41. In embodiments, the amino acid residues lining the central cavity include a residue at a position corresponding to Kabat position 30. In embodiments, the amino acid residues lining the central cavity include a residue at a position corresponding to Kabat position 52. In embodiments, the amino acid residues lining the central cavity include a residue at a position corresponding to Kabat 83. In embodiments, the amino acid residues lining the central cavity include a residue at a position corresponding to Kabat position 85.Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0438] In embodiments, the amino acid residues lining the central cavity include a residue at a position corresponding to Kabat position 30. In embodiments, the residue at a position corresponding to Kabat position 30 is a negatively charged amino acid residue. In embodiments, the residue at a position corresponding to Kabat position 30 is aspartic acid. In embodiments, the amino acid residues lining the central cavity include a residue at a position corresponding to Kabat position 52. In embodiments, the residue at a position corresponding to Kabat position 52 is a negatively charged amino acid residue. In embodiments, the residue at a position corresponding to Kabat position 52 is aspartic acid. In embodiments, the amino acid residues lining the central cavity include a residue at a position corresponding to Kabat position 83. In embodiments, the residue at a position corresponding to Kabat position 83 is a negatively charged amino acid residue. In embodiments, the residue at a position corresponding to Kabat position 83 is glutamic acid. In embodiments, the residue at a position corresponding to Kabat position 83 is isoleucine. In embodiments, the amino acid residues lining the central cavity include a residue at a position corresponding to Kabat position 85.

[0439] In embodiments, the central cavity is lined by (formed by) a light chain residue at a position corresponding to Kabat position Gln38, Thr40, Gln41, Gly42, Ser43, Asp 52, Asp85, Ile83, Tyr87, Lys103, Val163, Thr164, orGlu165. In embodiments, the central cavity is lined (e.g., formed) by a light chain residue at a position corresponding to Kabat position Gln38. In embodiments, the central cavity is lined (e.g., formed) by a light chain residue at a position corresponding to Kabat position Thr40 In embodiments, the central cavity is lined (e.g., formed) by a light chain residue at a position corresponding to Kabat position Gln41. In embodiments, the central cavity is lined (e g., formed) by a light chain residue at a position corresponding to Kabat position Gly42. In embodiments, the central cavity is lined (e.g., formed) by a light chain residue at a position corresponding to Kabat position to Ser43. In embodiments, the central cavity is lined (e.g., formed) by a light chain residue at a position corresponding to Kabat position Asp85. In embodiments, the central cavity is lined (e.g., formed) by a light chain residue at a position corresponding to Kabat position Tyr87. In embodiments, the central cavity is lined (e.g., formed) by a light chain residue at a position corresponding to Kabat position Lys103. In embodiments, the central cavity is lined (e g., formed) by a light chain residue at a position corresponding to Kabat position Val163. In embodiments, the central cavity is lined (e.g., formed) by a light chain residue at a position corresponding to Kabat position Thr164 In embodiments, the central cavity is lined (e.g., formed) by a light chain residue at a position corresponding to Kabat position Glu165.Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0440] In embodiments, the central cavity is lined by (formed by) a heavy chain residue at a position corresponding to Kabat position Asp 30, Gln39, Pro40, Thr91, Ala92, Ile93, Tyr95, Gln112, Leu115, Glu155, Pro156, Pro174, Ala175, or Tyr183. In embodiments, the central cavity is lined (e.g., formed) by a heavy chain residue at a position corresponding to Kabat position Gln39. In embodiments, the central cavity is lined (e.g., formed) by a heavy chain residue at a position corresponding to Kabat position. In embodiments, the central cavity is lined (e.g., formed) by a heavy chain residue at a position corresponding to Kabat position Pro40. In embodiments, the central cavity is lined (e.g., formed) by a heavy chain residue at a position corresponding to Kabat position Thr91. In embodiments, the central cavity is lined (e.g., formed) by a heavy chain residue at a position corresponding to Kabat position Ala92. In embodiments, the central cavity is lined (e.g., formed) by a heavy chain residue at a position corresponding to Kabat position Ile93. In embodiments, the central cavity is lined (e.g., formed) by a heavy chain residue at a position corresponding to Kabat position Tyr95. In embodiments, the central cavity is lined (e.g., formed) by a heavy chain residue at a position corresponding to Kabat position Gln112. In embodiments, the central cavity is lined (e.g., formed) by a heavy chain residue at a position corresponding to Kabat position Leu115. In embodiments, the central cavity is lined (e.g., formed) by a heavy chain residue at a position corresponding to Kabat position Glu155. In embodiments, the central cavity is lined (e g., formed) by a heavy chain residue at a position corresponding to Kabat position Pro156. In embodiments, the central cavity is lined (e.g., formed) by a heavy chain residue at a position corresponding to Kabat position Pro174. In embodiments, the central cavity is lined (e.g., formed) by a heavy chain residue at a position corresponding to Kabat position Ala175. In embodiments, the central cavity is lined (e.g., formed) by a heavy chain residue at a position corresponding to Kabat position Tyr183.

[0441] The central cavity provided herein includes a peptide binding site (also referred to herein as a meditope binding site) including framework region amino acid (FR) residues. In embodiments, the peptide binding site does not include CDR residues of the heavy chain or the light chain. In embodiments, the peptide binding site includes FR residues of the heavy chain or the light chain. In embodiments, the peptide binding site includes FR residues of the heavy chain and the light chain. In embodiments, the peptide binding site includes a residue at a position corresponding to Kabat position 83, a residue at a position corresponding to Kabat position 30 or a residue at a position corresponding to Kabat position 52. In embodiments, the peptide binding site includes a residue at a position corresponding to Kabat position 40, a residue at a position corresponding to Kabat position 41, a residue at aAttorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0442] position corresponding to Kabat position 30, a residue at a position corresponding to Kabat position 52, a residue at a position corresponding to Kabat position 83, or a residue at a position corresponding to Kabat position 85. In embodiments, the peptide binding site includes a residue at a position corresponding to Kabat position 40. In embodiments, the peptide binding site includes a residue at a position corresponding to Kabat position 41. In embodiments, the peptide binding site includes a residue at a position corresponding to Kabat position 30. In embodiments, the peptide binding site includes a residue at a position corresponding to Kabat position 52. In embodiments, the peptide binding site includes a residue at a position corresponding to Kabat position 83. In embodiments, the peptide binding site includes a residue at a position corresponding to Kabat position 85. In embodiments, residues forming a peptide binding site are described in Donaldson et al. (“Identification and grafting of a unique peptide-binding site in the Fab framework of monoclonal antibodies,” PNAS 110:17456-17461, 2013) and in published US application US 2012 / 0301400, both of which are hereby incorporated by reference in their entireties and for all purposes.

[0443] In embodiments, the central cavity is lined by amino acid residues capable of binding a compound including a peptidyl moiety. Thus, in embodiments, the peptide binding site provided herein is capable of binding a compound including a peptidyl moiety. In embodiments, the peptide binding site is capable of binding the peptidyl moiety. In embodiments, the peptide binding site provided herein is bound to a compound including a peptidyl moiety. In embodiments, the peptide binding site is bound to the peptidyl moiety. In embodiments, the peptidyl moiety is a moiety as described in published US application US 2012 / 0301400 and Avery et al. (Scientific Reports 5:7817, 2015) which are hereby incorporated by reference in their entirety and for all purposes.

[0444] In embodiments, the compound that binds to the peptide binding site is a peptide or includes a peptidyl moiety. In embodiments, the compound is a substituted peptide. In embodiments, the peptide is between 5 and 16 amino acids in length. In embodiments, the compound includes a substituted peptidyl moiety. In embodiments, the peptidyl moiety is between 5 and 16 amino acids in length. The peptide or peptidyl moiety provided herein may also be referred to as a "meditope." In embodiments, the peptide or peptidyl moiety has the formula:

[0445] X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12 (I).Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0446] Where the sequence of Formula (I) is a peptidyl moiety, a person having ordinary skill in the art will immediately understand that the peptidyl moiety is attached to the remainder of the compound at one or more attachments points. In formula (I), X1 is Cys, Gly, p -alanine, 2,3-diaminopropionic acid, p -azidoalanine, or null; X2 is Gin or null; X3 is Phe, Tyr, p-p’-diphenyLAIa, His, Asp, 2- bromo-L-phenylalanine, 3-bromo-L-phenylalanine, 4-bromo-L-phenylalanine, Asn, Gin, a modified Phe, a hydratable carbonyl-containing residue or a boronic acid-containing residue; X4 is Asp or Asn; X5 is Leu; P-P’-diphenyl-Ala, Phe, a nonnatural analog of phenylalanine, tryptophan, tyrosine, a hydratable carbonyl-containing residue or a boronic acid-containing residue; X6 is Ser or Cys; X7 is Thr, Ser or Cys; X8 is Arg, a modified (substituted) Arg, a hydratable carbonyl or a boronic acid containing residue; X9 is Arg or Ala; X10 is Leu, Gin, Glu, P-P’-diphenyl-Ala, Phe, a non-natural analog of phenylalanine, tryptophan, tyrosine, a hydratable carbonyl-containing residue ora boronic acid-containing residue; X11 is Lys; and X12 is Cys, Gly, 7-aminoheptanoic acid, p-alanine, diaminopropionic acid, propargylglycine, isoaspartic acid or null; wherein the modified Phe is a Phe with one or more halogen incorporated into the phenyl ring and wherein the modified Arg has a structure of the formula:

[0447] 0 M-i

[0448]

[0449] In formula (I A), R, R’ and R” are independently substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or NHR’” and R’” is substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl.

[0450] In embodiments, the peptide is a cyclic peptide. In embodiments, the peptidyl moiety is a cyclic peptidyl moiety. In embodiments, the peptide or peptidyl moiety includes a disulfide bridge, a thioether bridge, a lactam linkage, cycloaddition. In embodiments, the cyclic portion of the cyclic peptide or cyclic peptidyl moiety is formed through binding between X1 and X12, X1 and X11, X3 and X11, X4 and X11, orX2 and X12. In embodiments, the non-natural amino acid is P-P’-diphenyl-Ala, branched alkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl. In embodiments, each of the one or more halogen is an ortho-, meta-, or para-bromo phenyl substituent.Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0451] In embodiments, the peptide or peptidyl moiety has the formula:

[0452]

[0453] In formula (II), R3is hydrogen, R3A-substituted or unsubstituted aryl, wherein R3Ais hydrogen, halogen or C1-4 unsubstituted alkyl. R3is hydrogen, R3A’-substituted or unsubstituted aryl, wherein R3A’is hydrogen, halogen or C1-4 unsubstituted alkyl. R5is R5A-substituted or unsubstituted C1-8 (e.g., C1-4) alkyl. R5Ais oxo, acetal, ketal, -B(OH)2, boronic ester, phosphonate ester, ortho ester, -CO2CI-4 alkyl, -CH=CH-CHO, -CH=CH-C(O)R5A’, -CH=CH-CO2R5A’-CC>2H, -CONH2, or R5A”-substituted or unsubstituted aryl, R5A”-substituted or unsubstituted heteroaryl (e.g., naphthyl, imidazole, indole), wherein R5A’is substituted or unsubstituted C1-4 alkyl and R5A”is -OH, fluoro, chloro, bromo or iodo. R6is -L6OH or-L6’SH, wherein L6is substituted or unsubstituted C1-4 alkylene. R7is -L7’OH or-L7’SH, wherein L7’is substituted or unsubstituted C1-4 alkyl. The symbol m is 0, 1, 2, 3, 4, or 5.

[0454] In formula (II), R8is -OH, -NRaRb, -N(Rc)C(O)Re, or -N(Rc)C(=NRd)Re. Rais H. Rbis H or C1-8 alkyl optionally substituted with one or more substituents selected from the group consisting of oxo, acetal, and ketal, -B(OH)2, -SH, boronic ester, phosphonate ester, ortho ester, -CH=CH- CHO, -CH=CH-C(O)C1-4alkyl, -CH=CH-CO2C1-4 alkyl, -CO2H, or -CO2C1-4alkyl group. Rcis H, C1-8 alkyl, C3-8 cycloalkyl, branched alkyl, or aryl. Rdis H or a C1-8 alkyl, C2-8 alkenyl, C2-8 alkynyl, C3-8 cycloalkyl, branched alkyl, or aryl group, each optionally substituted with one or more substituents selected from the group consisting of -N3, -NH2, -OH, -SH, halogen, oxo, acetal, ketal, -B(OH)2, boronic ester, phosphonate ester, ortho ester, -CH=CH-CHO, -CH=CH-C(O)C1-4alkyl, -CH=CH-CO2C1-4alkyl, -CO2H, and-CO2C1-4alkyl group. Reis H, -NHRd; or a C1-12 alkyl, C3-8 cycloalkyl, C2-12 alkenyl, C2-8 alkynyl, or aryl group, each optionally substituted with one or more substituents selected from the group consisting of -N3, -NH2, -OH, -SH, oxo, C2-4acetal, C2-4ketal, -B(OH)2, boronic ester,Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0455] phosphonate ester, ortho ester, -CH=CH-CHO, -CH=CH-C(O)C1-4alkyl, -CH=CH-CO₂C1-4alkyl, and -CO₂C1-4alkyl group.

[0456] In formula (II), R9is substituted or unsubstituted C1-4alkyl. R10is R10A- substituted or unsubstituted C1-8 alkyl, wherein R10Ais oxo, acetal, ketal, -B(OH)₂, boronic ester, phosphonate ester, ortho ester, -CH=CH-CHO, -CH=CH-C(O)C1-4alkyl, -CH=CH-CO₂C1-4alkyl, -CO₂C1-4alkyl, -CO₂H, -CONH₂, R10B-substituted or unsubstituted phenyl, R10B-substituted or unsubstituted naphthyl, R10B-substituted or unsubstituted imidazolyl, or R10B-substituted or unsubstituted indolyl, wherein R10Bis -OH or halogen. The symbol n is 0 or 1. The symbol p is 0 or 1.

[0457] In formula (II), X is Rx-substituted or unsubstituted C1-8 alkylene, Rx-substituted or unsubstituted C2-8 alkenylene, Rxis oxo, -C(O), -NH2, -NHC(O) or-NHC(O)Ry, wherein one carbon of the alkenylene is optionally replaced with -C(O)NH, a 5-membered heteroarylene, or -S-S, and Ryis -C1-4alkyl, -CH(RZ)C(O) or -CH(RZ)CO2H, wherein Rzis -H or Rz-substituted or unsubstituted -C1-4alkyl, wherein Rzis -OH, -SH, or-NH2. Formula (I) or (II) includes all appropriate pharmaceutically acceptable salts. More information regarding the concepts of peptide binding sites (meditope binding sites) and peptides (meditopes) can be found in PCT / US2011 / 055656, PCT / US2015 / 053880, PCT / US2012 / 032938 and US 9,669,108, which are hereby incorporated in their entirety and for all purposes.

[0458] II. Treatment of Patients

[0459] Aspects of the present disclosure provide methods for treating a subject with cancer (e.g., a glioma, a glioblastoma, a cancer expressing EGFRVIII) by administering a population of immune cells described herein, e.g., anti-EGFR CAR T cells, and / or an antibody or antibody fragment described herein, e.g., an H6T, H6O, H3T Ab, Fab, scFab, scFv. The cancer can include cells that overexpress human EGFR or express human EGFRVIII.

[0460] (a) Subjects

[0461] The terms “subject” and “patient” are used interchangeably throughout. The subject to be treated by the methods described herein can be a human subject having a cancer comprising cells that overexpress human EGFR or express human EGFRVIII. In some embodiments, a subject has Glioma, Glioblastoma, Lung Cancer, Colorectal Cancer, Genitourinary Cancers, Breast Cancer, Non-Small Cell Lung Cancer (NSCLC), Head and Neck Squamous Cell Carcinoma (HNSCC), Glioblastoma Multiforme (GBM), Triple-NegativeAttorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0462] Breast Cancer (TNBC), Esophageal Cancer, Gastric Cancer, Pancreatic Cancer, Cervical Cancer, Bladder Cancer, Ovarian Cancer, Prostate Cancer, Hepatocellular Carcinoma (HCC), Cholangiocarcinoma (Bile Duct Cancer), a Soft Tissue Sarcoma, Thyroid Cancer, Mesothelioma, Renal Cell Carcinoma, and / or a Skin Cancer (e.g., Squamous Cell Carcinoma).

[0463] (b) Administration

[0464] An effective amount of a therapy (e.g., immune cells expressing a CAR described herein or an antibody or fragment thereof described herein) can be administered to a subject (e.g., a human) via any suitable route (e.g., administered locally or systemically to a subject).

[0465] Suitable modes of administration include injection, infusion, instillation, or ingestion. Injection includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intraventricular, intradermal, intraperitoneal, subcutaneous injection, and infusion.

[0466] An effective amount generally refers to the amount of each active agent required to confer therapeutic effect on the subject, either alone or in combination with one or more other active agents. Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of treatment, the nature of concurrent therapy, if any, the specific route of administration and like factors. A therapeutic effect on the subject can be determined as the amelioration of one symptom associated with the condition being treated or a reduction of size or number of cancer cells in the subject (e.g., 1%, 2%, 3%, 4%, or 5% decrease in size or number of cancer cells).

[0467] EXAMPLES

[0468] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0469] EXAMPLE 1: Humanized scFv that bind EGFRand EGFRVIII

[0470] Described below are scFv that have the same CDR sequences as mAb806.

[0471] mAb806 Antibodies

[0472] mAb806 scFvAttorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0473] DVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYISYSGNTRYN PSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCVTAGRGFPYWGQGTLVTVSSGGGGSGG GGSGGGGSDILMTQSPSSMSVSLGDTVSITCHSSQDINSNIGWLQQRPGKSFKGLIYHGTN LDDEVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPWTFGGGTKLEIK (SEQ ID NO:1)

[0474] VH DVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYISYSGNTRYN PSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCVTAGRGFPYWGQGTLVTV (SEQ ID NO: 2)

[0475] VL DILMTQSPSSMSVSLGDTVSITCHSSQDINSNIGWLQQRPGKSFKGLIYHGTNLDDEVPSRF SGSGSGADYSLTISSLESEDFADYYCVQYAQFPWTFGGGTKLEIK (SEQ ID NO: 3)

[0476] First, three humanized Fab were prepared (FabH6T, FabH6O and FabH3T) and characterized. Surface plasmon resonance (SPR) was used to assess the binding characteristics of the new Fab and Fab806. The thermal stability of each Fab was also measured. The results of this analysis are presented below.

[0477] Fab806

[0478] ka (1 / Ms): 2.40E+05

[0479] kd (1 / s): 2.84E-03

[0480] KD (M): 1.18E-08

[0481] Tm (°C): 79.3±0.07

[0482] FabH6T

[0483] ka (1 / Ms): 2.32E+05

[0484] kd (1 / s): 1.88E-03

[0485] KD (M): 8.13E-09

[0486] Tm (°C): 82.8±0.03

[0487] FabH6O

[0488] ka (1 / Ms): 2.82E+05

[0489] kd (1 / s): 3.06E-03

[0490] KD (M): 1.08E-08Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0491] Tm (°C): 82.3±0.09

[0492] FabH3T

[0493] ka (1 / Ms): 2.80E+04

[0494] kd (1 / s): 1.88E-03

[0495] KD (M): 6.70E-08

[0496] Tm (oC): 82.2±0.1

[0497] Fab H6T was crystallized by sitting drop vapor diffusion at 293 K from 36 mM NaCI, 0.1 M MES pH 6.5, 21% (w / v) PEG 4000. Diffraction data were collected using NSLS II beam line 17-ID1 (AMX). Fab H6O was crystallized by sitting drop vapor diffusion at 293 K from 0.1M Tris HCI pH 7, 20% (w / v) PEG 2000 MME. Diffraction data were collected using NSLS II beam line 17-ID2 (FMX). Data were processed with Autoproc and structures refined using Phenix suite of programs. Structural alignment of the variable region of the Fab806 results in an RMSD of 0.37 Angstroms with Fab H6T and 0.40 Angstroms with H6O.

[0498] The H6T, H6O and H3T Fab were used to create scFv comprising a VH, a spacer and a VL. The sequences of each of the scFv are presented below along with alignments between each of H6T, H6O and H3T and mAb806 scFv.

[0499] H6T scFv EVQLVESGGGLVQPGGSLRLSCAVTGYSITSDFAWNWIRQAPGKGLEWMGYISYSGNTRY NPSLKSRITISRDTSKNTFYLQMNSLRAEDTAVYYCVTAGRGFPYWGQGTLVTVSSGGGGS GGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHSSQDINSNIGWLQQKPGKAFKGLIYH GTNLDDEVPSRFSGSGSGADYTLTISSLQPEDFATYYCVQYAQFPWTFGQGTKVEIK (SEQ ID NO: 4)

[0500] VH EVQLVESGGGLVQPGGSLRLSCAVTGYSITSDFAWNWIRQAPGKGLEWMGYISYS GNTRYNPSLKSRITISRDTSKNTFYLQMNSLRAEDTAVYYCVTAGRGFPYWGQGTL VTVSS (SEQ ID NO: 5)

[0501] VL DIQMTQSPSSLSASVGDRVTITCHSSQDINSNIGWLQQKPGKAFKGLIYHGTNLDDE VPSRFSGSGSGADYTLTISSLQPEDFATYYCVQYAQFPWTFGQGTKVEIK (SEQ ID NO: 6)Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0502] Alignment of mAb806 scFv and H6T scFv

[0503] mAb806 DVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYISYSGNTRY 60

[0504] +VQL ESG LV+P SL L+C VTGYSITSDFAWNWIRQ PG LEWMGYISYSGNTRY H6T EVQLVESGGGLVQPGGSLRLSCAVTGYSITSDFAWNWIRQAPGKGLEWMGYISYSGNTRY 60

[0505] mAb806 NPSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCVTAGRGFPYWGQGTLVTVSSGGGG 120

[0506] NPSLKSRI+I+RDTSKN F+LQ+NS+ EDTA YYCVTAGRGFPYWGQGTLVTVSSGGGG H6T NPSLKSRITISRDTSKNTFYLQMNSLRAEDTAVYYCVTAGRGFPYWGQGTLVTVSSGGGG 120

[0507] mAb806 SGGGGSGGGGSDILMTQSPSSMSVSLGDTVSITCHSSQDINSNIGWLQQRPGKSFKGLIY 180

[0508] SGGGGSGGGGSDI MTQSPSS+S S+GD V+ITCHSSQDINSNIGWLQQ+PGK+FKGLIY H6T SGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHSSQDINSNIGWLQQKPGKAFKGLIY 180

[0509] mAb806 HGTNLDDEVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPWTFGGGTKLEIK 238

[0510] HGTNLDDEVPSRFSGSGSGADY+LTISSL+ EDFA YYCVQYAQFPWTFG GTK+EIK H6T HGTNLDDEVPSRFSGSGSGADYTLTISSLQPEDFATYYCVQYAQFPWTFGQGTKVEIK 238

[0511] (upper strand = SEQ ID NO:1; lower strand = SEQ ID NO:4)

[0512] H6O scFv QVQLEESGPGLVKPSETLSLTCSVTGYSITSDFAWNWIRQPPGKGLEWMGYISYSGNTRYN PSLKSRITISRDTSKNEFSLRLTSVTAADTAVYYCVTAGRGFPYWGQGTLVTVSSGGGGSG GGGSGGGGSDIVMTQSPSSLSASIGDRVTITCHSSQDINSNIGWLQQKPGKAFKGLIYHGT NLDDEVPSRFSGSGSGAEYTLTIRSLQPEDFATYYCVQYAQFPWTFGQGTKVEIK (SEQ ID NO: 7)

[0513] VH QVQLEESGPGLVKPSETLSLTCSVTGYSITSDFAWNWIRQPPGKGLEWMGYISYSGNTRYN PSLKSRITISRDTSKNEFSLRLTSVTAADTAVYYCVTAGRGFPYWGQGTLVTV (SEQ ID NO: 8)

[0514] VLAttorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0515] DIVMTQSPSSLSASIGDRVTITCHSSQDINSNIGWLQQKPGKAFKGLIYHGTNLDDEVPSRF SGSGSGAEYTLTIRSLQPEDFATYYCVQYAQFPWTFGQGTKVEIK (SEQ ID NO: 9)

[0516] Alignment of mAb 806 scFv and H6O scFv

[0517] mAb806 DVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYISYSGNTRYN 61

[0518] +VQL+ESGP LVKPS++LSLTC+VTGYSITSDFAWNWIRQ PG LEWMGYISYSGNTRYN H6O QVQLEESGPGLVKPSETLSLTCSVTGYSITSDFAWNWIRQPPGKGLEWMGYISYSGNTRYN 61

[0519] mAb806 PSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCVTAGRGFPYWGQGTLVTVSSGGGGS 122

[0520] PSLKSRI+I+RDTSKN+F L+L SVT DTA YYCVTAGRGFPYWGQGTLVTVSSGGGGS H6O PSLKSRITISRDTSKNEFSLRLTSVTAADTAVYYCVTAGRGFPYWGQGTLVTVSSGGGGS 122

[0521] mAb806 GGGGSGGGGSDILMTQSPSSMSVSLGDTVSITCHSSQDINSNIGWLQQRPGKSFKGLIYH 182

[0522] GGGGSGGGGSDI+MTQSPSS+S S+GD V+ITCHSSQDINSNIGWLQQ+PGK+FKGLIYH H6O GGGGSGGGGSDIVMTQSPSSLSASIGDRVTITCHSSQDINSNIGWLQQKPGKAFKGLIYH 181

[0523] mAb806 GTNLDDEVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPWTFGGGTKLEIK 238

[0524] GTNLDDEVPSRFSGSGSGA+Y+LTI SL+ EDFA YYCVQYAQFPWTFG GTK+EIK H6O GTNLDDEVPSRFSGSGSGAEYTLTIRSLQPEDFATYYCVQYAQFPWTFGQGTKVEIK 238

[0525] (upper strand = SEQ ID NO: 1; lower strand = SEQ ID NO: 7)

[0526] H3T scFv QVQLQESGPGLVKPSQTLSLTCTVSGYSITSDFAWNWIRQPPGKGLEWIGYISYSGNTRYNPSLKSRV SISVDTSKNQFSLKLNSVTAADTAVYYCVTAGRGFPYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQ MTQSPSSLSASVGDRVTITCHSSQDINSNIGWYQQRPGKAPKLLIYHGTNLDDGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCVQYAQFPWTFGQGTKVEIK (SEQ ID NO: 10)

[0527] VH QVQLQESGPGLVKPSQTLSLTCTVSGYSITSDFAWNWIRQPPGKGLEWIGYISYSGNTRYNPSLKSR VSISVDTSKNQFSLKLNSVTAADTAVYYCVTAGRGFPYWGQGTLVTV (SEQ ID NO: 11)

[0528] VL DIQMTQSPSSLSASVGDRVTITCHSSQDINSNIGWYQQRPGKAPKLLIYHGTNLDDGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCVQYAQFPWTFGQGTKVEIK (SEQ ID NO: 12)Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0529] Alignment of mAb 806 scFv and H3T

[0530] mAb806 DVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYISYSGNTRYN 61

[0531] +VQLQESGP LVKPSQ+LSLTCTV+GYSITSDFAWNWIRQ PG LEW+GYISYSGNTRYN H3T EVQLQESGPGLVKPSQTLSLTCTVSGYSITSDFAWNWIRQPPGKGLEWIGYISYSGNTRYN 61

[0532] mAb806 PSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCVTAGRGFPYWGQGTLVTVSSGGGGS 121

[0533] PSLKSR+SI+ DTSKNQF L+LNSVT DTA YYCVTAGRGFPYWGQGTLVTVSSGGGGS H3T PSLKSRVSISVDTSKNQFSLKLNSVTAADTAVYYCVTAGRGFPYWGQGTLVTVSSGGGGS 121

[0534] mAb806 GGGGSGGGGSDILMTQSPSSMSVSLGDTVSITCHSSQDINSNIGWLQQRPGKSFKGLIYH 181

[0535] GGGGSGGGGSDI MTQSPSS+S S+GD V+ITCHSSQDINSNIGW QQRPGK+ K LIYH H3T GGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHSSQDINSNIGWYQQRPGKAPKLLIYH 181

[0536] mAb806 GTNLDDEVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPWTFGGGTKLEIK 238

[0537] GTNLDD VPSRFSGSGSG D++LTISSL+ EDFA YYCVQYAQFPWTFG GTK+EIK H3T GTNLDDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCVQYAQFPWTFGQGTKVEIK 238

[0538] (upper strand = SEQ ID NO: 10; lower strand = SEQ ID NO: 3)

[0539] EXAMPLE 2: Construction and Characterization of EGFR CAR

[0540] 806scFv, 6T scFv, 60 svFv or 3H scFv were used to create CAR that include a lgG4(H(S228P)L-CH3) spacer (SEQ ID NO: 31), a CD4 transmembrane domain (SEQ ID NO: 18), a 41 BB co-stimulatory domain (SEQ ID NO: 38) and a CD3Zeta signaling domain (SEQ ID NO: 35). The mature CAR sequence is preceded by a human GM-CSF receptor alpha signal sequence (SEQ ID NO: 41). The CAR sequence is followed by a T2A skip sequence (SEQ ID NO: 41) and a truncated, signaling incompetent CD19 receptor (SEQ ID NO: 43; CD19t), permitting co-expression of the CAR and cell surface CD19t, which can be used to monitor expression of the CAR. The CAR were expressed using epHIV7 lentiviral vector as described previously (Wang et al. 2012 J Immunother 35:689-701).

[0541] The amino acid sequences of the EGFR CAR used in this Example and FIGS. 1A-5D are: EGFRscFv(h6T)-lgG4(HL-CH3)-CD4tm-41BB-CD3ZetaAttorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0542] EVQLVESGGGLVQPGGSLRLSCAVTGYSITSDFAWNWIRQAPGKGLEWMGYISYSGN TRYNPSLKSRITISRDTSKNTFYLQMNSLRAEDTAVYYCVTAGRGFPYWGQGTLVTVSS GGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHSSQDINSNIGWLQQKPGK AFKGLIYHGTNLDDEVPSRFSGSGSGADYTLTISSLQPEDFATYYCVQYAQFPWTFGQ GTKVEIKESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSC SVMHEALHNHYTQKSLSLSLGKMALIVLGGVAGLLLFIGLGIFFKRGRKKLLYIFKQPFMR PVQTTQEEDGCSCRFPEEEEGGCELGGGRVKFSRSADAPAYQQGQNQLYNELNLGR REEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 65)

[0543] EGFRscFv(h6O)-lgG4(HL-CH3)-CD4tm-41BB-CD3Zeta QVQLEESGPGLVKPSETLSLTCSVTGYSITSDFAWNWIRQPPGKGLEWMGYISYSGNT RYNPSLKSRITISRDTSKNEFSLRLTSVTAADTAVYYCVTAGRGFPYWGQGTLVTVSSGG GGSGGGGSGGGGSDIVMTQSPSSLSASIGDRVTITCHSSQDINSNIGWLQQKPGKAFK GLIYHGTNLDDEVPSRFSGSGSGAEYTLTIRSLQPEDFATYYCVQYAQFPWTFGQGTKV EIKESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGKMALIVLGGVAGLLLFIGLGIFFKRGRKKLLYIFKQPFMRPVQTT QEEDGCSCRFPEEEEGGCELGGGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYD VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL YQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 80)

[0544] EGFRscFv(h3T)-lgG4(HL-CH3)-CD4tm-41BB-CD3Zeta QVQLQESGPGLVKPSQTLSLTCTVSGYSITSDFAWNWIRQPPGKGLEWIGYISYSGNT RYNPSLKSRVSISVDTSKNQFSLKLNSVTAADTAVYYCVTAGRGFPYWGQGTLVTVSS GGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHSSQDINSNIGWYQQRPGK APKLLIYHGTNLDDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCVQYAQFPWTFGQG TKVEIKESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS VMHEALHNHYTQKSLSLSLGKMALIVLGGVAGLLLFIGLGIFFKRGRKKLLYIFKQPFMR PVQTTQEEDGCSCRFPEEEEGGCELGGGRVKFSRSADAPAYQQGQNQLYNELNLGR REEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 96)

[0545] EGFRscFv(806)-lgG4(HL-CH3)-CD4tm-41BB-CD3Zeta

[0546] DVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYISYSGNT RYNPSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCVTAGRGFPYWGQGTLVTVSSGG GGSGGGGSGGGGSDILMTQSPSSMSVSLGDTVSITCHSSQDINSNIGWLQQRPGKSF KGLIYHGTNLDDEVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPWTFGGGTK LEIKESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVM HEALHNHYTQKSLSLSLGKMALIVLGGVAGLLLFIGLGIFFKRGRKKLLYIFKQPFMRPVAttorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0547] QTTQEEDGCSCRFPEEEEGGCELGGGRVKFSRSADAPAYQQGQNQLYNELNLGRRE EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 130)

[0548] Blood products were obtained from healthy donors under protocols approved by the City of Hope (COH) Internal Review Board. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation over Ficoll-Paque (GE Healthcare, Little Chalfont, UK) and then underwent sequential rounds of CliniMACS / AutoMACS depletion (to remove CD45RA+ naive T cells, CD25+ regulatory T cells, and CD14+ monocytes) and selection to enrich for the CD45RO+ CD62L+ Tern population. In brief, PBMCs were incubated with clinical-grade anti-CD25, anti-CD14, and anti-CD45RA microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) for 30 min at room temperature (RT) in X Vivo15 media (BioWhittaker, Walkersville, MD) containing 10% fetal calf serum (FCS) (HyClone, GE Healthcare). CD25+, CD14+, and CD45RA+ cells were then immediately depleted using the CliniMACS depletion mode according to the manufacturer’s instructions (Miltenyi Biotec). After centrifugation, the unlabeled negative fraction of cells was resuspended in CliniMACS PBS / EDTA buffer (Miltenyi Biotec) containing 0.5% human serum albumin (HSA) (CSL Behring, King of Prussia, PA) and then labeled with clinical grade biotinylated-DREG56 monoclonal antibody (mAb) (City of Hope Center for Biomedicine and Genetics) at 0.1 pg / 106cells for 30 min at RT. The cells were then washed and resuspended in a final volume of 100 mL CliniMACS PBS / EDTA containing 0.5% HSA. After 30 min incubation with 1.25 mL anti-biotin microbeads (Miltenyi Biotec), the CD62L+ fraction (Tern) was purified with positive selection on CliniMACS according to the manufacturer’s instructions and resuspended in X Vivo15 media containing 10% FCS.

[0549] Tern were stimulated with Dynabeads Human T expander CD3 / CD28 (Invitrogen, Carlsbad, CA) at a 1:3 ratio (T cell:bead) and transduced with at an MOI of 0.3 or greater in X Vivo15 containing 10% FCS with 5 pg / mL protamine sulfate (APP Pharmaceuticals, Schaumburg, IL), 50 U / mL rhlL-2, and 0.5 ng / mL rhlL-15. Cultures were then maintained at 37°C, 5% CO2, with addition of X-Vivo15, 10% FCS as required to keep cell density between 3 x 105and 2 x 106viable cells / mL, with cytokine supplementation (final concentration of 50 U / mL rhlL-2 and 0.5 ng / mL rhlL-15) every Monday, Wednesday, and Friday of culture. On day 7+ of culture, the CD3 / CD28 Dynabeads were removed from cultures using the DynaMag-50 magnet (Invitrogen).Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0550] Total T cell counts were measured on 7 and 14 days after CD3 / CD28 beads activation. Mock T cells without lentivirus transduction were included as a control. The results of this analysis are presented in FIG 1A, which shows good expansion for each CAR construct.

[0551] Cells expressing the various CAR constructs were stained with either anti-CD19-PECy7 or anti-G4S-PE to detect the percentage of cells that express CD19t or CAR transgenes. The results of this analysis are presented in FIG 1B and FIG 1C. The percentage of CD4+ cells and the percentage of CD8+ cells relative to total CAR T cells was assessed by antibody staining. The results of this analysis are presented in FIG 1D.

[0552] EXAMPLE 3: EGFR CAR T Cells Mediate EGFR Selective Cytotoxicity In Vitro

[0553] Peripheral blood mononuclear cells (PBMC) were collected from discard apheresis kits of 5 different human donors as approved by City of Hope (COH) Internal Review Board (IRB) oversight. Central memory T cells (Tern) were then selected from the PBMC by magnetically depleting CD14+and CD25+cells followed by magnetic selection of the CD62L+population, transduced with lentivirus to express either of the four CAR variants, and expanded with IL2 and IL15, all carried out as described. The CD3 / 28 stimulation beads (Thermo Fisher Scientific, catalog no. 11141D) were removed on day 7, successfully transduced cells were enriched on the basis of CD19t expression on days 11-15 using EasySep Human CD19 Positive Selection Kit II (Stemcell Technologies, Inc., catalog no. 17854), and the resulting CAR+ T cells were cryopreserved at day 15 or 17. Unless otherwise indicated, thawed cells were then rested overnight in media with IL2 / IL15 prior to analysis or use in the assays.

[0554] The CAR T cells were assessed for their cytotoxicity towards patient derived glioblastoma GBM) cells. FIG 2A shows the results of flow cytometric analysis of EGFR expression on various patient-derived GBM cells used in the study. The percentage of immunoreactive cells (red line, to the right) compared to isotype antibody staining Ctrl (grey line) are depicted in each histogram. Cell lytic activity was compared by impedance-based assay. Mock T cells or anti-EGFR CAR T cells (effector cells) and the tumor cells (target cells) were seeded at an E: T ratio of 1:5 or 1:20. The detachment and death of adherent tumor cells was monitored as a decrease in normalized cell index upon the addition of effector cells. The results of this analysis are shown in FIG 2B. PBT206 cells were engineered to express EGFRvlll at a low, medium or high level or were knocked out for EGFRvlll. The expression of EGFR and EGFRvlll on engineered GBM cells was measure by flow cytometry. The results are shown in FIG 2C and the geometric mean of EGFR expression in each engineered cell line isAttorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0555] shown in FIG 2D. The cell lytic activity of the various EGFR CAR T cells towards the engineered cell lines was assessed and the results are presented in FIG 2E.

[0556] EXAMPLE 4: Humanized anti-EGFR CAR T Cells induce comparable cytotoxicity against EGFRvlll-expressing tumor cells as 806 CAR T Cells

[0557] Mock T cells or anti-EGFR CAR T cells and tumor cells were cocultured at an E: T ratio of 1:20 for 72 hrs, then number of CART cells and tumor cells were counted by flow cytometry. Percentage of cytotoxicity (% Cytotoxicity) was calculated by dividing the tumor cell count in each treatment group by the average count from the tumor-alone control. The results of this analysis are shown in FIG 3A. Mock T cells or anti-EGFR BB T cells were stimulated with the various engineered tumor cells lines described in Example 3 at an E: T ratio of 2:1 and both IFN-y and IL2 levels in supernatant were collected and measured. The results of this analysis are presented in FIG 3B. It can be seen that CAR T cells targeted with scFv 6T, scFv 60 or scFv 3H were comparable to those targeted with mAb806.

[0558] EXAMPLE 5: Humanized anti-EGFR CAR T cells retain cytotoxicity through multiple rounds of tumor challenge

[0559] A repetitive rechallenge study used to assess anti-EGFR CAR T cells (FIG 4A). Briefly, anti-EGFR CAR T cells were cocultured with PBT030-EGFRvlllmed(2000 CAR T cells and 0.01 M tumor cells), repetitively challenged with either 0.01 M or 0.02 M tumor cells every one or two days. CART cells and tumor cells were counted by flow cytometry at the indicated time points at day 3 and day 10. The results of this study are presented in FIG 4B, where it can be seen that humanized anti-EGFR CAR T cells retain cytotoxicity across multiple rounds of tumor challenge.

[0560] EXAMPLE 6: Humanized anti-EGFR(6T) CAR T cells exhibit comparable antitumor activity as 806-CART in vivo.

[0561] An in vivo study was used to assess the activity of various anti-EGFR CAR T cells. FIG 5A is a schematic depiction of the study. Briefly, orthotopic xenograft GBM tumor model. NSG mice were inoculated intracranial (i.c.) with 0.1M ffLuc+ PBT030-EGFRvlllmedand then treated with 0.1M anti-EGFR CAR T cells by intraventricular injection 3 weeks after tumor implantation. Tumor growth and survival were monitored by bioluminescence imaging in mice, and the results are presented in FIG 5B and FIG 5C. Kaplan-Meier survival analysis demonstrate comparable survival for mice treated with humanized anti-EGFR(6T) BB TAttorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0562] cells as 806 BBζ T cells (FIG 5D). Survival analysis following tumor rechallenge is presented in FIG. 5E, where cured mice after treatment with either 806 BB T cells or 6T BB T cells or 60 BB T cells were rechallenged with 0.05M ffLuc+ PBT030-EGFRvlllmedi.c. Naive mice without prior treatment were included as control.

[0563] EXAMPLE 7: Humanized anti-EGFR(6T) BBC T cells exhibit comparable antitumor activity with various spacer and transmembrane domain

[0564] The anti-EGFR CAR having scFv 6T, HL CH3 spacer, CD4 TM, 4-1 BB co-stim and CD3 Zeta (6T-HLCH3-CD4TM) was modified by replacing the HL CH3 spacer with a CD8 hinge and replacing the CD4 TM with the CD8TM3. (6T-CD8H-CD8TM). An anti-EGFR CAR having cetuximab scFv, HL CH3 spacer, CD4 T, 41-BB co-stim and CD3 Zeta (Cetux-HLCH3-CD4TM) was generated. Vectors encoding each of these CAR and a vector encoding the 806-HLCH3-CD4TM CAR were used to transduce T cells derived from healthy donor. The transduced cells were stained with anti-G4S-PE to detect the percentage of cells that express CAR transgenes. The results of this analysis are shown in FIG 6A. Mock T cells or anti-EGFR CAR T cells (effector cells) and PBT206neg, PBT206-EGFRvllllowor PBT206-EGFRvllhitumor cells (target cells) were seeded at an E: T ratio of 1:40 and cell lytic activity was measured. The results of this study are shown in FIG 6B where it can be seen that CAR T cells 6T-HLCH3-CD4TM, 6T-CD8H-CD8TM, and 806-HLCH3-CD4TM exhibit superior tumor cell killing activity compared to Cetux-HLCH3-CD4TM CAR T cells.

[0565] EXAMPLE 8

[0566] One desirable EGFR targeted CAR includes a 6T scFv, an lgG4(H(S228P)L-CH3) spacer, CD4 transmembrane domain, a 41 BB co-stimulatory domain and a CD3zeta signaling domain. A vector (based on epHIV7) that can be used to produce this CAR in T cells includes the following sequence elements (the coding sequences are underlined and noncoding sequences are not underlined). The sequence of this vector is presented in FIGS 31A-31C as a single sequence, starting with the EFlalpha promoter. The amino acid sequence of the mature CAR encoded by this vector in presented in FIG. 32A.

[0567] EF1 alpha promoter GGATCTGCGATCGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCC CCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCG GGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGG AGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCG CCAGAACACAGCTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCC CTACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGAttorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0568] GTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGG CCTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGC CTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGTTACAGA TCCAAGCTGTGACCGGCGCCTAC

[0569] 5’ Multiple Cloning Site

[0570] GGCTAGC

[0571] Kozak Sequence

[0572] GCCGCCACC GMCSF Signal Peptide ATGCTGCTGCTCGTGACATCTCTGCTGCTGTGCGAGCTGCCCCACCCCGCCTTTCTGC TGATCCCT

[0573] 6T-VH GAGGTGCAGCTGGTGGAGAGCGGTGGTGGGTTGGTGCAGCCAGGAGGCTCCCTCCG CCTGTCATGCGCCGTCACCGGCTACTCCATCACTAGTGACTTTGCTTGGAATTGGATCC GCCAGGCCCCGGGGAAAGGTCTGGAGTGGATGGGCTATATTAGCTACTCTGGCAACAC ACGCTACAACCCTTCGCTAAAGTCCCGCATCACCATCTCTCGGGATACCTCCAAGAACA CGTTCTACCTGCAGATGAACAGCCTTAGGGCGGAAGACACCGCCGTGTACTACTGCGT GACTGCAGGACGTGGCTTCCCCTATTGGGGCCAGGGCACCCTGGTTACCGTGTCGTCT

[0574] (G4S)3

[0575] GGAGGCGGAGGATCTGGCGGCGGAGGAAGTGGCGGAGGGGGATCC

[0576] Humanized 6T-VL GACATCCAGATGACCCAGAGCCCTAGCTCTCTGTCCGCTTCCGTTGGTGATCGTGTCAC CATCACCTGCCATTCGTCCCAGGACATTAATAGCAACATCGGTTGGCTCCAACAGAAGC CCGGCAAGGCCTTCAAAGGCCTTATCTATCACGGGACCAACCTGGATGACGAGGTGCC CTCTCGCTTTTCAGGCTCCGGCTCGGGGGCGGACTACACCCTGACTATTTCTAGTTTGC AGCCAGAGGACTTCGCCACTTACTACTGCGTGCAGTACGCACAGTTCCCGTGGACCTT CGGACAGGGCACAAAGGTGGAGATCAAG

[0577] Hinge

[0578] GAGTCTAAGTACGGCCCTCCCTGCCCCCCTTGTCCA

[0579] Linker

[0580] GGGGGAGGATCTTCCGGAGGAGGAAGCGGA

[0581] lgG4(CH3) GGCCAGCCCAGAGAACCTCAGGTGTACACACTGCCCCCTAGCCAGGAAGAGATGACCA AGAATCAGGTGTCCCTGACATGCCTCGTGAAGGGCTTCTACCCCTCCGATATCGCCGTG GAATGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGG ACAGCGACGGCTCATTCTTCCTGTACAGCAGGCTGACCGTGGACAAGAGCCGGTGGCA GGAAGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACC CAGAAGTCCCTGAGCCTGTCCCTGGGCAAG CD4tm ATGGCCCTGATCGTGCTGGGCGGAGTGGCCGGACTGCTGCTGTTTATCGGCCTGGGCA TCTTCTTC

[0582] 41 BB cytoAttorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0583] AAGCGGGGCAGAAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGGCCCGTGC AGACCACCCAGGAAGAGGACGGCTGCAGCTGCCGGTTCCCCGAGGAAGAGGAAGGC GGCTGCGAGCTG

[0584] G3 linker

[0585] GGAGGCGGC CD3 Zeta AGAGTGAAGTTCAGCCGGTCCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAACCAG CTGTACAACGAGCTGAACCTGGGCAGGCGGGAGGAATACGACGTGCTGGACAAGCGG AGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAACCCCCAGGAAGG CCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATG AAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGCCTGTATCAGGGCCTGTCCAC CGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCAAGG

[0586] T2A CTCGAGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAG AATCCCGGCCCTAGG CD19 truncated ATGCCACCTCCTCGCCTCCTCTTCTTCCTCCTCTTCCTCACCCCCATGGAAGTCAGGCC CGAGGAACCTCTAGTGGTGAAGGTGGAAGAGGGAGATAACGCTGTGCTGCAGTGCCTC AAGGGGACCTCAGATGGCCCCACTCAGCAGCTGACCTGGTCTCGGGAGTCCCCGCTTA AACCCTTCTTAAAACTCAGCCTGGGGCTGCCAGGCCTGGGAATCCACATGAGGCCCCT GGCCATCTGGCTTTTCATCTTCAACGTCTCTCAACAGATGGGGGGCTTCTACCTGTGCC AGCCGGGGCCCCCCTCTGAGAAGGCCTGGCAGCCTGGCTGGACAGTCAATGTGGAGG GCAGCGGGGAGCTGTTCCGGTGGAATGTTTCGGACCTAGGTGGCCTGGGCTGTGGCC TGAAGAACAGGTCCTCAGAGGGCCCCAGCTCCCCTTCCGGGAAGCTCATGAGCCCCAA GCTGTATGTGTGGGCCAAAGACCGCCCTGAGATCTGGGAGGGAGAGCCTCCGTGTGTC CCACCGAGGGACAGCCTGAACCAGAGCCTCAGCCAGGACCTCACCATGGCCCCTGGC TCCACACTCTGGCTGTCCTGTGGGGTACCCCCTGACTCTGTGTCCAGGGGCCCCCTCT CCTGGACCCATGTGCACCCCAAGGGGCCTAAGTCATTGCTGAGCCTAGAGCTGAAGGA CGATCGCCCTGCCAGAGATATGTGGGTAATGGAGACGGGTCTGTTGTTGCCCCGGGCC ACAGCTCAAGACGCTGGAAAGTATTATTGTCACCGTGGCAACCTGACCATGTCATTCCA CCTGGAGATCACTGCTCGGCCAGTACTATGGCACTGGCTGCTGAGGACTGGTGGCTGG AAGGTCTCAGCTGTGACTTTGGCTTATCTGATCTTCTGCCTGTGTTCCCTTGTGGGCATT CTTCATCTTCAAAGAGCCCTGGTCCTGAGGAGGAAAAGA

[0587] 3’ Multiple Cloning Site

[0588] TAATCTAGACCCGGGCTGCAGGAATTCGATATCAAGCTT WPRE ATCGATAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGT TGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCC CGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGT TGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCC CACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCC CTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGG CTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCT TGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCC CTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGC CTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTC CCCGCATCGATAttorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0589] Spacer

[0590] ACCGTCGACTAGCC

[0591] Del U3 GTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAA AGGGGGGACTGGAAGGGCTAATTCACTCCCAAAGAAGACAAGAT

[0592] R region CTGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTC TGGCTAACTAGGGAACCCACTG

[0593] U5 CTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGT GACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCA

[0594] Spacer GAATTCGATATCAAGCTTATCGATACCGTCGACCTCGAGGGGGGGCCCGGTACCCAATT CGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGG AAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGG CGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATG GCGAATGGAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCA GCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGAC CGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGG ACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACC ATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAA AGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGA AGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCT GCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCAGGTGG CACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATA TGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAG T

[0595] Ampicillin Resistance Cassette ATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTG TTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCA CGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCC CGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATC CCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACT TGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAA TTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACG ATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCG CCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACC ACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACT CTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACT TCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGC GTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTA GTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGA GATAGGTGCCTCACTGATTAAGCATTGG

[0596] Spacer TAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTT

[0597] CoE1 oriAttorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0598] AAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGT TTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTT TTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTT TGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGC GCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTC TGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTG GCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAG CGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTAC ACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGA GAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGG GAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTG ACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCC AGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTT CCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACC GCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGA G

[0599] Spacer CGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCA CGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAG CTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGA ATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTC GAAATTAACCCTCACTAAAGGGAACAAAAGCTGGAGCTCCACCGCGGTGGCGGCCTCG A SV40 ori GGTCGAGATCCGGTCGACCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCC CCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTAT GCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTT TTGGAGGCCTAGGCTTTTGCAAA

[0600] Spacer

[0601] AAGCTTCGACGGT CMV promoter ATCGATTGGCTCATGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAAT AGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACT TACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATA ATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAG TATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCC CCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTA TGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGC GGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGT CTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCC AAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGAATT C

[0602] Spacer

[0603] GGAGTGGCGAGCCCTCAGATCCTGCATATAAGCAG R Region CTGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTC TGGCTAACTAGGGAACCCACTGAttorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0604] U5 CTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGT GACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCA

[0605] Spacer GTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACG CAGGA HIV-1 Psi Ψ CTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACG CCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGT

[0606] Spacer ATTAAGCGGGGGAGAATTAGATCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGA AAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAA TCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCAT CCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTG TGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAG AGCAAAACAAAAGTAAGAAAAAAGCACAGCAAGCAGCAGCTGACACAGGACACAGCAA TCAGGTCAGCCAAAATTACCCTATAGTGCAGAACATCCAGGGGCAAATGGTACATCAGG CCATATCACCTAGAACTTTAAATGCATGGGTAAAAGTAGTAGAAGAGAAGGCTTTCAGCC CAGAAGTGATACCCATGTTTTCAGCATTATCAGAAGGAGCCACCCCACAAGATTTAAACA CCATGCTAAACACAGTGGGGGGACATCAAGCAGCCATGCAAATGTTAAAAGAGACCATC AATGAGGAAGCTGCAG

[0607] Rev Response Element (RRE) GCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCC TTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGT ACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTAT TGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCA AGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTG CTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGATC

[0608] Flap TACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTG CAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACA AATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAGATCCAGTTT

[0609] Spacer GGGGATCAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGA EXAMPLE 9

[0610] One desirable EGFR target CAR includes a 6T scFv, an CD8H spacer, CD8-3 transmembrane domain, a 41 BB co-stimulatory domain and a CD3zeta signaling domain. A vector (based on epHIV7) that can be used to produce this CAR in T cells includes the same sequence elements described in Example 7, except that the coding sequences in Example 7 are replaced by the following coding sequences. The sequence of this vector is presented inAttorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0611] FIGS 33A-33C as a single sequence, starting with the EF1 alpha promoter. The amino acid sequence of the mature CAR encoded by this vector in presented in FIG. 32B.

[0612] GMCSF Signal Peptide ATGCTGCTGCTCGTGACATCTCTGCTGCTGTGCGAGCTGCCCCACCCCGCCTTTCTGC TGATCCCT

[0613] 6T-VH GAGGTGCAGCTGGTGGAGAGCGGTGGTGGGTTGGTGCAGCCAGGAGGCTCCCTCCG CCTGTCATGCGCCGTCACCGGCTACTCCATCACTAGTGACTTTGCTTGGAATTGGATCC GCCAGGCCCCGGGGAAAGGTCTGGAGTGGATGGGCTATATTAGCTACTCTGGCAACAC ACGCTACAACCCTTCGCTAAAGTCCCGCATCACCATCTCTCGGGATACCTCCAAGAACA CGTTCTACCTGCAGATGAACAGCCTTAGGGCGGAAGACACCGCCGTGTACTACTGCGT GACTGCAGGACGTGGCTTCCCCTATTGGGGCCAGGGCACCCTGGTTACCGTGTCGTCT

[0614] (G4S)3

[0615] GGAGGCGGAGGATCTGGCGGCGGAGGAAGTGGCGGAGGGGGATCC

[0616] 6T-VL GACATCCAGATGACCCAGAGCCCTAGCTCTCTGTCCGCTTCCGTTGGTGATCGTGTCACCATCACCTGC CATTCGTCCCAGGACATTAATAGCAACATCGGTTGGCTCCAACAGAAGCCCGGCAAGGCCTTCAAAGG CCTTATCTATCACGGGACCAACCTGGATGACGAGGTGCCCTCTCGCTTTTCAGGCTCCGGCTCGGGGG CGGACTACACCCTGACTATTTCTAGTTTGCAGCCAGAGGACTTCGCCACTTACTACTGCGTGCAGTACG CACAGTTCCCGTGGACCTTCGGACAGGGCACAAAGGTGGAGATCAAG

[0617] CD8 hinge ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGC GCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGT GAT

[0618] CD8tm3 ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACT GC

[0619] 41BB AAGCGGGGCAGAAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGGCCCGTGCAGACCACCC AGGAAGAGGACGGCTGCAGCTGCCGGTTCCCCGAGGAAGAGGAAGGCGGCTGCGAGCTG

[0620] G3 linker

[0621] GGAGGCGGCAttorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0622] Zeta AGAGTGAAGTTCAGCCGGTCCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAACCAGCTGTACAACG AGCTGAACCTGGGCAGGCGGGAGGAATACGACGTGCTGGACAAGCGGAGAGGCCGGGACCCTGAG ATGGGCGGCAAGCCTCGGCGGAAGAACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAG ATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGG CCTGTATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGCCCC CAAGG

[0623] T2A CTCGAGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGG CCCTAGG

[0624] CD19 truncated

[0625] ATGCCACCTCCTCGCCTCCTCTTCTTCCTCCTCTTCCTCACCCCCATG G AAGTCAG G CCCG AG G AACCTC TAGTGGTGAAGGTGGAAGAGGGAGATAACGCTGTGCTGCAGTGCCTCAAGGGGACCTCAGATGGCC CCACTCAGCAGCTGACCTGGTCTCGGGAGTCCCCGCTTAAACCCTTCTTAAAACTCAGCCTGGGGCTG CCAGGCCTGGGAATCCACATGAGGCCCCTGGCCATCTGGCTTTTCATCTTCAACGTCTCTCAACAGATG GGGGGCTTCTACCTGTGCCAGCCGGGGCCCCCCTCTGAGAAGGCCTGGCAGCCTGGCTGGACAGTC AATGTGGAGGGCAGCGGGGAGCTGTTCCGGTGGAATGTTTCGGACCTAGGTGGCCTGGGCTGTGGC CTGAAGAACAGGTCCTCAGAGGGCCCCAGCTCCCCTTCCGGGAAGCTCATGAGCCCCAAGCTGTATG TGTGGGCCAAAGACCGCCCTGAGATCTGGGAGGGAGAGCCTCCGTGTGTCCCACCGAGGGACAGCC TGAACCAGAGCCTCAGCCAGGACCTCACCATGGCCCCTGGCTCCACACTCTGGCTGTCCTGTGGGGTA CCCCCTGACTCTGTGTCCAGGGGCCCCCTCTCCTGGACCCATGTGCACCCCAAGGGGCCTAAGTCATT GCTGAGCCTAGAGCTGAAGGACGATCGCCCTGCCAGAGATATGTGGGTAATGGAGACGGGTCTGTTG TTGCCCCGGGCCACAGCTCAAGACGCTGGAAAGTATTATTGTCACCGTGGCAACCTGACCATGTCATTC CACCTGGAGATCACTGCTCGGCCAGTACTATGGCACTGGCTGCTGAGGACTGGTGGCTGGAAGGTCT CAGCTGTGACTTTGGCTTATCTGATCTTCTGCCTGTGTTCCCTTGTGGGCATTCTTCATCTTCAAAGAGC CCTGGTCCTGAGGAGGAAAAGA

[0626] EXAMPLE 18: Humanized 88ti-EGFR(6T)

[0627]

[0628] 7 eteis i n weed tew cytotoxicity against WT-EGFR expressing normal astrocytes arte showed selective recognition of lEGERteil over” WT EGER.

[0629] A study was performed to test cytotoxicity of the anti-EGFR CAR T ceils in human astrocytes (FIGS.

[0630] 34A-34C. Fiow cytometric analysis of EGFR expression (red) on normal human astrocytes, negative control almS ceils, or positive control almS-EGFRvlli ceils as compared to control staining (grey) In FIG. 3 A. T cells that were either mock-transduced (Mock), or transduced to express the parental EGFR-CAR (806), the humanized 6T EGFR-CAR (h806-6T) or the Cetuximab-based EGFR-CAR (Cetux) were used as effectors in a flow cytometry based 43-hour astrocyte killing assay at the indicated F: T ratios as shown In FIG. 24B. % Astrocyte kill Ing was calculated by comparing the viable numbers of the indicated target line co-ctetored with CAM- I ceils to that of astrocytes only. Mock T ceils or theAttorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034

[0631] anti-EGFR BBζ T cells were co-cultured with each cell line depicted in FIG. 34A at an E:T ratio of 2:1 for 19 hours. Harvested supernatants were evaluated for IFNγ levels by ELISA (FIG. 34C).

[0632] Humanized anti-EGFR(6T) CAR also showed selective recognition of EGFRvIII over WT EGFR (FIG. 35). Flow cytometric analysis of WT EGFR (FIG. 35, left panel) and EGFRvIII (FIG. 35, right panel) binding to Jurkat cells that did not express a CAR (None), or Jurkat cells transduced to express either 6T-CAR (h806-6T) or the Cetuximab-based EGFR-CAR (Cetux).

[0633] OTHER EMBODIMENTS

[0634] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034CLAIMS:

1. A nucleic acid molecule comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) comprising:(i) an scFv selected from:a scFv comprising a VH comprising amino acids 21-105 of SEQ ID NO: 5 and a VL comprising amino acids 11-104 of SEQ ID NO: 6;a scFv comprising a VH comprising amino acids 5-101 of SEQ ID NO: 8 and a VL comprising amino acids 11-104 of SEQ ID NO: 9;a scFv comprising a VH comprising amino acids 17-105 of SEQ ID NO: 11 and a VL comprising amino acids 11-97 of SEQ ID NO: 12;(ii) a spacer domain;(iii) a transmembrane domain;(iv) a costimulatory domain; and(v) a CD3ζ signaling domain.

2. The nucleic acid molecule of claim 1, wherein the scFv comprises: a VH comprising or consisting of the amino acid sequence of SEQ ID NO:5 and a VL comprising or consisting of the amino acid sequence of SEQ ID NO:6.

3. The nucleic acid molecule of claim 1, wherein the scFv comprises: a VH comprising or consisting of the amino acid sequence of SEQ ID NO:8 and a VL comprising or consisting of the amino acid sequence of SEQ ID NO:9.

4. The nucleic acid molecule of claim 1, wherein the scFv comprises: a VH comprising or consisting of the amino acid sequence of SEQ ID NO:11 and a VL comprising or consisting of the amino acid sequence of SEQ ID NO: 12.

5. The nucleic acid molecule of claim 1, wherein the scFv comprises or consists of the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 131.

6. The nucleic acid molecule of claim 1, wherein the scFv comprises or consists of the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 132.

7. The nucleic acid molecule of claim 1, wherein the scFv comprises or consists of the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO:133.Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-0348. The nucleic acid molecule of claim 1, wherein the scFv comprises.

9. The nucleic acid molecule of claim 1, wherein the chimeric antigen receptor comprises the amino acid sequence of any of SEQ ID NOs: 54-100 and 127-129.

10. A nucleic acid molecule comprising a nucleotide sequence encoding a chimeric antigen receptor comprising:(i) a single chain Fab comprising an amino acid sequence selected from: SEQ ID NOs: 105, 106, 109, 110, 119, 120, 123 and 124, a single chain Fab comprising SEQ ID NOs: 135 and 136, a single chain Fab comprising SEQ ID NOs: 107 and 108, a single chain Fab comprising SEQ ID NOs: 137 and 138, and a single chain Fab comprising SEQ ID NOs: 121 and 122,(ii) a spacer domain;(iii) a transmembrane domain;(iv) a costimulatory domain; and(v) a CD3ζ signaling domain.

11. The nucleic acid molecule of any of the forgoing claims, wherein: the spacer domain comprises a sequence selected from the group consisting of: SEQ ID NOs: 24-34 and 134; the transmembrane domain comprises a sequence selected from the group consisting of SEQ ID NOs: 15-23; the costimulatory domain comprises a sequence selected from the group consisting of SEQ ID NOs: 36-40, and the CD3ζ signaling domain comprises a sequence selected from the group consisting of SEQ ID NOs: 35 and 47-53.

12. The nucleic acid molecule of any of the forgoing claims, wherein the transmembrane domain selected from the group consisting of:a CD4 transmembrane domain,a CD8 transmembrane domain,a CD28 transmembrane domain, anda CD3ζ transmembrane domain.

13. The nucleic acid molecule of any of the forgoing claims, wherein the costimulatory domain selected from the group consisting ofa CD28 costimulatory domain,a 41-BB costimulatory domain,an 0X40 costimulatory domain, andAttorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034a 2B4 costimulatory domain.

14. The nucleic acid molecule of any of the forgoing claims, further comprising a linker consisting of 1 - 5 amino acids between one or more of: the scFV and the spacer domain, the spacer domain and the transmembrane domain, the transmembrane domain and the co-stimulatory domain, and the costimulatory domain and the CD3ζ signaling domain.

15. The nucleic acid molecule of any of the forgoing claims, wherein the transmembrane domain comprises or consists of the amino acid sequence of any of SEQ ID NO: 15-23.

16. The nucleic acid molecule of any of the forgoing claims, wherein the costimulatory domain comprises or consists of the amino acid sequence of any of SEQ ID NO: 36-40.

17. The nucleic acid molecule of any of the forgoing claims, wherein the CD3ζ signaling domain comprises or consists of the amino acid sequence of any of SEQ ID NO: 35 and 47-53.

18. A human cell harboring the nucleic acid molecule of any of the forgoing claims.

19. A polypeptide comprising:a VH comprising amino acids 21-105 of SEQ ID NO: 5 and a VL comprising amino acids 11-104 of SEQ ID NO: 6;a VH comprising amino acids 5-101 of SEQ ID NO: 8 and a VL comprising amino acids 11-104 of SEQ ID NO: 9;a VH comprising amino acids 17-105 of SEQ ID NO: 11 and a VL comprising amino acids 11-97 of SEQ ID NO: 12;a VH comprising SEQ ID NO: 5 and a VL comprising SEQ ID NO: 6;a VH comprising SEQ ID NO: 8 and a VL comprising SEQ ID NO: 9;a VH comprising SEQ ID NO: 11 and a VL comprising SEQ ID NO: 12;SEQ ID NO: 4;SEQ ID NO: 7;SEQ ID NO: 10;Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034SEQ ID NO: 131;SEQ ID NO: 132; orSEQ ID NO: 133.

20. A polypeptide comprising:a scFv comprising a VH comprising amino acids 21-105 of SEQ ID NO: 5 and a VL comprising amino acids 11-104 of SEQ ID NO: 6;a scFv comprising a VH comprising amino acids 5-101 of SEQ ID NO: 8 and a VL comprising amino acids 11-104 of SEQ ID NO: 9;a scFv comprising a VH comprising amino acids 17-105 of SEQ ID NO: 11 and a VL comprising amino acids 11-97 of SEQ ID NO: 12;a scFv comprising a VH comprising SEQ ID NO: 5 and a VL comprising SEQ ID NO: 6;a scFv comprising a VH comprising SEQ ID NO: 8 and a VL comprising SEQ ID NO: 9;a scFv comprising a VH comprising SEQ ID NO: 11 and a VL comprising SEQ ID NO: 12;a scFv comprising SEQ ID NO: 4;a scFv comprising SEQ ID NO: 7;a scFv comprising SEQ ID NO: 10;a scFv comprising SEQ ID NO: 131;a scFv comprising SEQ ID NO: 132;a scFv comprising SEQ ID NO: 133;21. A single chain Fab comprising an amino acid sequence selected from: SEQ ID NOs: 105, 106, 109, 110, 119, 120, 123, and 124.

22. An antibody having:a) a heavy chain comprising SEQ ID NO: 13 and a light chain comprising SEQ ID NO: 14;b) a heavy chain comprising SEQ ID NO: 101 and a light chain comprising SEQ ID NO: 102;c) a heavy chain comprising SEQ ID NO: 103 and a light chain comprising SEQ ID NO: 104;d) a heavy chain comprising SEQ ID NO: 111 and a light chain comprising SEQ ID NO: 112;Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034e) a heavy chain comprising SEQ ID NO: 113 and a light chain comprising SEQ ID NO: 114;f) a heavy chain comprising SEQ ID NO: 115 and a light chain comprising SEQ ID NO: 116;g) a heavy chain comprising SEQ ID NO: 117 and a light chain comprising SEQ ID NO: 118; orh) a heavy chain comprising SEQ ID NO: 125 and a light chain comprising SEQ ID NO: 126.

23. An antibody comprising:(a) a heavy chain VH-CH1 comprising SEQ ID NO: 107 and a light chain VL-CL comprising SEQ ID NO: 108; or(b) a heavy chain VH-CH1 comprising SEQ ID NO: 121 and a light chain VL-CL comprising SEQ ID NO: 122.

24. An antibody comprising:a VH comprising amino acids 21-105 of SEQ ID NO: 5 and a VL comprising amino acids 11-104 of SEQ ID NO: 6;a VH comprising amino acids 5-101 of SEQ ID NO: 8 and a VL comprising amino acids 11-104 of SEQ ID NO: 9;a VH comprising amino acids 17-105 of SEQ ID NO: 11 and a VL comprising amino acids 11-97 of SEQ ID NO: 12;a VH comprising SEQ ID NO: 5 and a VL comprising SEQ ID NO: 6;a VH comprising SEQ ID NO: 8 and a VL comprising SEQ ID NO: 9; ora VH comprising SEQ ID NO: 11 and a VL comprising SEQ ID NO: 12.

25. A single chain Fab comprisingSEQ ID NOs: 107 and 108,SEQ ID NOs: 137 and 138, orSEQ ID NOs: 121 and 122.

26. A method of treating a cancer expressing a constitutively active EGFR and / or EGFRVIII, the method comprising administer the patient a T cell harboring the nucleic acid molecule of any of claims 1-17.Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-03427. A method of treating glioblastoma comprising administer the patient a T cell harboring the nucleic acid molecule of any of claims 1-17.

28. The method of claim 25, wherein the cancer is any of a Glioma, Glioblastoma, Lung Cancer, Colorectal Cancer, Genitourinary Cancers, Breast Cancer, Non-Small Cell Lung Cancer (NSCLC), Head and Neck Squamous Cell Carcinoma (HNSCC), Glioblastoma Multiforme (GBM), Triple-Negative Breast Cancer (TNBC), Esophageal Cancer, Gastric Cancer, Pancreatic Cancer, Cervical Cancer, Bladder Cancer, Ovarian Cancer, Prostate Cancer, Hepatocellular Carcinoma (HCC), Cholangiocarcinoma (Bile Duct Cancer), a Soft Tissue Sarcoma, Thyroid Cancer, Mesothelioma, Renal Cell Carcinoma, a Skin Cancer.

29. A method of treating a subject suffering from a cancer comprising cells expressing EGFR and / or EGFRVIII on the surface of a cancer cell, the method comprising administering to the subject an effective amount of a composition comprising:(a) human T cells expressing a encoding a chimeric antigen receptor (CAR) comprising:(i) an scFv selected from:a scFv comprising a VH comprising amino acids 21-105 of SEQ ID NO: 5 and a VL comprising amino acids 11-104 of SEQ ID NO: 6;a scFv comprising a VH comprising amino acids 5-101 of SEQ ID NO: 8 and a VL comprising amino acids 11-104 of SEQ ID NO: 9;a scFv comprising a VH comprising amino acids 17-105 of SEQ ID NO: 11 and a VL comprising amino acids 11-97 of SEQ ID NO: 12;(ii) a spacer domain;(iii) a transmembrane domain;(iv) a costimulatory domain; and(v) a CD3ζ signaling domain; or(b) an antibody or polypeptide comprising:a VH comprising amino acids 21-105 of SEQ ID NO: 5 and a VL comprising amino acids 11-104 of SEQ ID NO: 6;a VH comprising amino acids 5-101 of SEQ ID NO: 8 and a VL comprising amino acids 11-104 of SEQ ID NO: 9;a VH comprising amino acids 17-105 of SEQ ID NO: 11 and a VL comprising amino acids 11-97 of SEQ ID NO: 12;a VH comprising SEQ ID NO: 5 and a VL comprising SEQ ID NO: 6;a VH comprising SEQ ID NO: 8 and a VL comprising SEQ ID NO: 9;Attorney Docket No.: 40056-0094W01 / COH Ref.: TEC 24-034a VH comprising SEQ ID NO: 11 and a VL comprising SEQ ID NO: 12;SEQ ID NO: 4;SEQ ID NO: 7;SEQ ID NO: 10;SEQ ID NO: 131;SEQ ID NO: 132;SEQ ID NO: 133;SEQ ID NO: 105;SEQ ID NO: 106;SEQ ID NO: 109;SEQ ID NO: 110;SEQ ID NO: 119;SEQ ID NO: 120;SEQ ID NO: 123;SEQ ID NO: 124;SEQ ID NOs: 107 and 108;SEQ ID NOs: 137 and 138 andSEQ ID NOs: 121 and 122.

30. The method of claim 29, wherein the cancer is a glioma or glioblastoma.

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