EA044329B1 - CHIMERIC ANTIGENE RECEPTORS TARGETED BY EPIDERMAL GROWTH FACTOR RECEPTOR VARIANT III - Google Patents

Description

Field of technology to which the invention relates

The invention relates to chimeric antigen receptors (CARs). CARs are capable of retargeting immune cell specificity and reactivity to a selected target, developing the properties of a ligand-binding domain. In particular, the invention relates to CARs that specifically bind to epidermal growth factor receptor variant III (EGFRvIII specific CARs). The invention further relates to polynucleotides encoding EGFRvIII specific CARs, and isolated cells expressing EGFRvIII specific CARs on their surface. The invention further relates to methods for constructing immune cells expressing EGFRvIII specific CARs on their surface. The invention is particularly useful for the treatment of solid tumors such as glioblastoma multiforme (GBM), non-small cell lung cancer, head and neck cancer, breast cancer, ovarian cancer and prostate cancer. The invention further relates to immune cells containing EGFRvIII specific CARs (EGFRvIII specific CAR-T cells), compositions containing EGFRvIII specific CAR-T cells, and methods of using EGFRvIII specific CAR-T cells for the treatment of EGFRvIII-mediated pathologies.

Prerequisites for creating the invention

EGFR variant III (EGFRvIII), a tumor-specific EGFR mutant, is the product of a genomic rearrangement that is often associated with amplification of the wild-type EGFR gene. EGFRvIII is formed by an in-frame deletion of exons 2–7, resulting in a 267 amino acid deletion with a glycine substitution at the junction. The truncated receptor loses its ability to bind ligands but gains constitutive kinase activity. Of interest is the fact that EGFRvIII is always co-expressed with full-length wild-type EGFR in the same tumor cells.

In addition, cells expressing EGFRvIII exhibit increased proliferation, invasion, angiogenesis, and resistance to apoptosis.

EGFRvIII is most commonly found in glioblastoma multiforme (GBM). It is estimated that 25-35% of GBM carries its truncated receptors. In addition, its expression reflects a more aggressive phenotype and poor prognosis. Besides GBM, EGFRvIII expression has also been described in other solid tumors such as non-small cell lung cancer, head and neck cancer, breast cancer, ovarian cancer and prostate cancer. Conversely, EGFRvIII is not expressed in healthy tissues. The lack of expression in normal tissues makes EGFRvIII an ideal target for the development of tumor-specific targeted therapies.

Adoptive transfer of T cells genetically modified to recognize antigens associated with cancer has shown promise as a new approach for the treatment of cancer (see, for example, Brenner et al., Current Opinion in Immunology, 22(2): 251- 257 (2010); Rosenberg et al., Nature Reviews Cancer, 8(4): 299-308 (2008)). T cells can be genetically modified to express chimeric antigen receptors (CARs), which are fusion proteins consisting of an antigen recognition component and T cell activating domains (see, for example, Eshhar et al., Proc. Natl. Acad. Sci. USA, 90(2): 720-724 (1993), and Sadelain et al., Curr. Opin. Immunol, 21(2): 215-223 (2009)). Thus, treating solid tumors such as glioblastoma multiforme using an anti-EGFRvIII antagonist, including EGFRvIII specific CARs and EGFRvIII specific CAR-T cells, will be a promising therapeutic agent. Summary of the invention

Chimeric antigen receptors (CARs) are provided that bind to EGFRvIII. Certain EGFRvIII specific CARs have been shown to be effective when expressed in T cells to activate T cells upon contact with EGFRvIII. Advantageously, the EGFRvIII specific CARs provided in the present invention bind human EGFRvIII. Also advantageously, the EGFRvIII specific CAR-T cells provided by the present invention exhibit degranulation activity, increased interferon gamma production, and/or cytotoxic activity when contacted with cells expressing EGFRvIII.

In one aspect, the invention provides an EGFRvIII specific CAR comprising an extracellular ligand binding domain, a first transmembrane domain, and an intracellular signaling domain, wherein the extracellular ligand binding domain comprises (a) a heavy chain variable region (VH) containing (i) a VH hypervariable region one (CDR1) containing the sequence shown in SEQ ID NO: 62, 63, 64, 74, 75, 76, 80, 81, 82, 88, 89, 90, 109, 110, 111, 115, 116, 117 , 121, 122, 123, 137, 138, or 139; (II) VH CDR2 containing the sequence shown in SEQ ID NO: 70, 71, 77, 78, 83, 84, 86, 87, 91, 92, 112, 113, 118, 119, 124, 125, 127, 128 , 140, or 141; and III) VH CDR3 containing the sequence shown in SEQ ID NO: 73, 79, 85, 114, 120, 126, 129, or 142, and/or (b) a light chain variable region (VL) containing (I) VL CDR1 containing the sequence shown in SEQ ID NO: 149, 156, 159, 162, 165, 182, 185, 187, or 195; (II) VL CDR2 containing the sequence shown in SEQ ID NO: 152, 157, 160, 163, 183, 186, 188, or 196; and (III) VL CDR3 containing the sequence shown in SEQ ID NO: 153, 158, 161, 164, 184, 189, or 197.

In another aspect, the invention provides an EGFRvIII specific CAR containing extracellular

- 1 044329 a precise ligand binding domain, a first transmembrane domain, and an intracellular signaling domain, wherein the extracellular ligand binding domain comprises a single chain Fv fragment (scFv) containing a heavy chain variable region (VH) containing three CDRs from the VH region containing the sequence represented by SEQ ID NO: 5, 9, 11, 13, 15, 37, 39, 41, 43, or 48; and/or a light chain variable region (VL) comprising three CDRs from the VL region containing the sequence set forth in SEQ ID NO: 6, 10, 12, 14, 16, 38, 40, 42, or 49. In some embodiments, The VH region may contain the sequence set forth in SEQ ID NO: 5, 9, 11, 13, 15, 37, 39, 41, 43, or 48, or a variant thereof with one or more conservative amino acid substitutions in regions that are not within the CDR, and/or VL region may contain the amino acid sequence presented in SEQ ID NO: 6, 10, 12, 14, 16, 38, 40, 42, or 49, or a variant thereof with one or more amino acid substitutions in the amino acids , which are not within the CDR. For example, in some embodiments, the VH or VL region of a scFv may comprise the amino acid sequence described above, or a variant thereof with no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 conserved substitutions in areas that are not within the CDR.

In some embodiments, the invention provides an EGFRvIII specific CAR comprising an extracellular ligand binding domain, a first transmembrane domain, and an intracellular signaling domain, wherein the extracellular ligand binding domain comprises a single chain Fv fragment (scFv) containing a heavy chain variable region (VH), containing the sequence shown in SEQ ID NO: 11, 15, 30, 37, or 41; and/or a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 12, 16, 31, 38, or 42. In some embodiments, the VH contains the sequence shown in SEQ ID NO: 11, and VL contains the sequence presented in SEQ ID NO: 12. In some embodiments, VH contains the sequence presented in SEQ ID NO: 15, and VL contains the sequence presented in SEQ ID NO: 16. In some embodiments, VH contains the sequence presented in SEQ ID NO: 30, and VL contains the sequence presented in SEQ ID NO: 31. In some embodiments, VH contains the sequence presented in SEQ ID NO: 37 and VL contains the sequence presented in SEQ ID NO: 38 In some embodiments, VH contains the sequence presented in SEQ ID NO: 41, and VL contains the sequence presented in SEQ ID NO: 42.

In some embodiments, the intracellular signaling domain comprises a CD3zeta signaling domain. In some embodiments, the intracellular signaling domain comprises a 4-1BB signaling domain. In some embodiments, the CAR may further comprise a second intracellular signaling domain. In some embodiments, the second intracellular signaling domain may comprise a 4-1BB signaling domain. In some embodiments, the first intracellular signaling domain comprises a CD3zeta signaling domain and the second intracellular signaling domain comprises a 4-1BB signaling domain.

In some embodiments, the CAR may comprise a stem domain between the extracellular ligand binding domain and the first transmembrane domain. In some embodiments, the stem domain may be selected from the group consisting of: human CD8a loop, human CD28 loop, IgG1 loop, and FcyRIIIa loop.

In some embodiments, the first transmembrane domain may comprise a CD8a chain transmembrane domain.

In some embodiments, the CAR may contain another extracellular ligand binding domain that is not specific for EGFRvIII.

In some embodiments, the CAR, extracellular ligand binding domain(s), first transmembrane domain, and intracellular signaling domain(s) are on the same polypeptide.

In some embodiments, the CAR may comprise a second transmembrane domain, wherein the first transmembrane domain and extracellular ligand-binding domain(s) are on the first polypeptide, and wherein the second transmembrane domain and intracellular signaling domain(s) are present on the first polypeptide. the domain(s) are located on the second polypeptide, where the first transmembrane domain contains a transmembrane domain from the α chain of the high affinity IgE receptor (FcεRI) and the second transmembrane domain contains a transmembrane domain from the γ or β chain of FcsRI. In some embodiments, the CAR may comprise a third polypeptide comprising a third transmembrane domain fused to an intracellular signaling domain from a co-stimulatory molecule, wherein the third transmembrane domain comprises a transmembrane domain from the γ or β chain of FcεRI.

In another aspect, the invention provides an isolated polynucleotide containing a nucleotide sequence encoding an EGFRvIII specific CAR, as described in the present invention.

In another aspect, the invention provides an expression vector containing a nucleotide

- 2044329 sequence encoding an EGFRvIII specific CAR antibody as described in the present invention.

In another aspect, the invention provides an engineered immune cell expressing EGFRvIII specific CARs on its outer cell membrane, as described in the present invention. In some embodiments, the engineered immune cell may contain another CAR that is not specific for EGFRvIII.

In some embodiments, the engineered immune cell may contain a polynucleotide encoding a suicide polypeptide. In some embodiments, the suicide polypeptide is RQR8. In some embodiments, the polynucleotide encoding the suicide polypeptide is on a different nucleic acid molecule than the polynucleotide containing the nucleotide sequence encoding the EGFRvIII specific CAR. In some embodiments, the polynucleotide encoding the suicide polypeptide is part of the same nucleic acid molecule as the polynucleotide containing the nucleotide sequence encoding the EGFRvIII specific CAR.

In some embodiments, the engineered immune cell containing the EGFRvIII-specific CAR may contain a suicide polypeptide on a separate polypeptide chain from the polypeptide chain of the EGFRvIII-specific CAR.

In some embodiments, the EGFRvIII specific CAR, as described in the present invention, also contains a suicide polypeptide on the same polypeptide chain as the CAR. For example, the suicide polypeptide may be located between the scFv and the CAR loop sequence. In some embodiments, the suicide polypeptide in the CAR may be in R2 format, as provided in the present invention. In some embodiments, the suicide polypeptide contains an epitope that is recognized by rituximab.

Also provided by the present invention is a polynucleotide encoding an EGFRvIII specific CAR, which also encodes a suicide polypeptide in the CAR.

In some embodiments, the immune cell may be derived from an inflammatory T lymphocyte, a cytotoxic T lymphocyte, a regulatory T lymphocyte, a memory T lymphocyte, a helper T lymphocyte, a natural killer T lymphocyte, or a natural killer cell.

In some embodiments, the engineered immune cell may comprise a disruption in one or more endogenous genes, wherein the endogenous gene encodes TCRa, TCRe, CD52, glucocorticoid receptor (GR), deoxycytidine kinase (dCK), or an immune checkpoint protein, such as, for example , programmed death-1 (PD-1).

In some embodiments, the immune cell is obtained from a healthy donor. In some embodiments, the immune cell is obtained from a patient.

In another aspect, the invention provides an engineered immune cell expressing EGFRvIII specific CARs on its outer cell membrane as described herein for use as a drug. In some embodiments, the medicament is for use in the treatment of an EGFRvIII-associated malignancy (e.g., any malignancy with EGFRvIII expression) selected from the group consisting of glioblastoma multiforme, anaplastic astrocytoma, giant cell glioblastoma, gliosarcoma, anaplastic oligodendroglioma, anaplastic ependymoma , anaplastic oligoastrocytoma, choroid plexus carcinoma, anaplastic ganglioglioma, pineoblastoma, pineocytoma, meningioma, medulloepithelioma, ependymoblastoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, atypical teratoid-rhabdoid tumor, mixed glioma, head and neck cancer, not small cell lung cancer, breast cancer, ovarian cancer, prostate cancer, medullobastomy, colorectal cancer, anal cancer, cervical cancer, kidney cancer, skin cancer, pancreatic cancer, liver cancer, bladder cancer, stomach cancer, thyroid cancer, mesothelioma, cancer uterus, lymphoma and leukemia.

In another aspect, the invention provides a method for constructing an immune cell, comprising: providing an immune cell; and expressing on the cell surface at least one EGFRvIII specific CAR, as described in the present invention.

In some embodiments, the method includes: providing an immune cell; introducing into the cell at least one polynucleotide encoding said EGFRvIII specific CAR; and expressing said polynucleotide in the cell.

In some embodiments, the method includes providing an immune cell; introducing into the cell at least one polynucleotide encoding said EGFRvIII specific CAR; and introducing at least one other CAR that is not specific for EGFRvIII.

In another aspect, the invention provides a method of treating a subject suffering from a condition associated with malignant cells, where the method includes: providing an immune cell expressing on the surface of an EGFRvIII specific CAR, as described in the present invention; And

- 3 044329 introduction of the specified immune cells to the specified patient.

In another aspect, the invention provides a pharmaceutical composition containing an engineered immune cell as described in the present invention.

In another aspect, the invention provides a method of treating a condition associated with EGFRvIII-expressing malignant cells in a subject, comprising administering to the subject in need thereof an effective amount of a pharmaceutical composition comprising an engineered immune cell as described herein. In some embodiments, the condition is a malignancy. In some embodiments, the cancer is an EGFRvIII-associated malignancy selected from the group consisting of glioblastoma multiforme, anaplastic astrocytoma, giant cell glioblastoma, gliosarcoma, anaplastic oligodendroglioma, anaplastic ependymoma, anaplastic oligoastrocytoma, choroid plexus carcinoma, anaplastic ic ganglioglioma, pineoblastoma, pineocytoma, meningioma, medulloepithelioma, ependymoblastoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, atypical teratoid-rhabdoid tumor, mixed glioma, head and neck cancer, non-small cell lung cancer, breast cancer, ovarian cancer, prostate cancer, medullobastomy, colon and rectal cancer colon, anal cancer, kidney cancer, cervical cancer, liver cancer, pancreatic cancer, stomach cancer, thyroid cancer, mesothelioma, uterine cancer and bladder cancer.

In another aspect, the invention provides a method of inhibiting the growth or progression of a tumor in a subject who has cancer cells expressing EGFRvIII, comprising administering to the subject in need thereof an effective amount of a pharmaceutical composition comprising an engineered immune cell as described herein.

In another aspect, the invention provides a method of inhibiting metastasis of malignant cells expressing EGFRvIII in a subject, comprising administering to the subject in need thereof an effective amount of a pharmaceutical composition comprising an engineered immune cell as described herein.

In another aspect, the invention provides a method of inducing tumor regression in a subject who has cancer cells expressing EGFRvIII, comprising administering to the subject in need thereof an effective amount of a pharmaceutical composition containing an engineered immune cell as described herein.

Brief description of figures/drawings

In fig. 1A, fig. 1B, and FIG. Figure 1C shows examples of FACS binding histograms of three EGFRvIII antibodies: mAbs 42G9 (Fig. 1A), 32A10 (Fig. 1B), and 32G8 (Fig. 1C), with three F98 cell lines: F98 (EGFR negative), F98-EGFRwt, and F98 -EGFRvIII. The x-axis represents the fluorescence intensity; the Y-axis represents the percentage of the maximum/normalized to the modal value.

In fig. 2A is a bar graph that summarizes EGFRvIII CAR-specific expression for CARs containing different EGFRvIII specific clones.

In fig. 2B is a bar graph summarizing the percentage of EGFRvIII specific CAR T cells that are associated with recombinant EGFR-mFc and EGFRvIII-mFc.

In fig. 2C is a bar graph that summarizes the expression of CARs containing 10 different EGFRvIII specific clones in CAR T cells.

In fig. 3 is a bar graph summarizing the EGFRvIII degranulation activities of specific CAR T cells expressing different EGFRvIII specific clones, alone or when cocultured with different cell lines: cells that do not express any EGFR protein (NCI-H522 and U87-KO ), cells express high levels of wild-type EGFR (U87-KO-EGFRwt), or cells that express low (NCI-H522-EGFRvIII) and high (U87-KO-EGFRvIII) levels of EGFRvIII.

In fig. 4 is a bar graph summarizing IFNy secretion by EGFRvIII specific CAR T cells expressing different EGFRvIII specific clones, alone or when co-cultured with different cell lines: cells that do not express any EGFR protein (NCI-H522 and U87 -KO), cells express high levels of wild-type EGFR (U87-KO-EGFRwt), or cells that express low (NCI-H522-EGFRvIII) and high (U87-KO-EGFRvIII) levels of EGFRvIII.

In fig. 5 is a bar graph comparing the cytotoxicity of EGFRvIII specific CAR T cells expressing different EGFRvIII specific clones relative to a cell line expressing wild type EGFR (U87-KO-EGFRwt) relatively high (U87-KOEGFRvIII) and low EGFRvIII (NCI-H522- EGFRvIII) expressing cell lines.

In fig. 6 is a graph summarizing the antitumor activities of EGFRvIII specific CAR T cells expressing various EGFRvIII specific clones against GBM cells in a subcutaneous GBM xenograft model.

In fig. 7 is a bar graph that summarizes CAR expression by CAR T cells expressing four different EGFRvIII specific clones in CAR and carrying intra- 4 044329

CAR suicide sequence R2 in CAR on Day 4, Day 9/10, and Day 14/15 after T cell transduction.

In fig. 8 is a bar graph summarizing CAR expression by CAR T cells expressing four different EGFRvIII specific clones in the CAR and carrying the intraCAR R2 suicide sequence at Day 4 and Day 14/15 after T cell transduction.

In fig. 9 is a bar graph summarizing the cytotoxicity of CAR T cells expressing four different EGFRvIII specific clones and carrying the intra-CAR suicide sequence R2 relative to a cell line expressing high levels (U87-KOEGFRvIII) and low levels of EGFRvIII (NCI-H522- EGFRvIII).

Detailed description

The invention disclosed herein provides chimeric antigen receptors (CARs) and CAR-containing immune cells (eg CAR-T cells) that specifically bind to EGFRvIII (eg humEGFRvIII). The invention also provides polynucleotides encoding these CARs, compositions containing these CAR-T cells, and methods for producing and using these CARs and CAR-T cells. The invention also provides methods for treating a condition associated with EGFRvIII-mediated pathologies in a subject, such as a cancer.

General techniques

In carrying out the invention, unless specifically stated otherwise, generally accepted techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, immunology, virology, and monoclonal antibody generation and construction will be used, which are known to one skilled in the art. Such techniques are described in detail in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., and., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995).

Definitions

The term extracellular ligand-binding domain as used in the present invention refers to an oligo- or polypeptide that is capable of binding to a ligand. Preferably, the domain is capable of interacting with a molecule on a cell surface. For example, the extracellular ligand binding domain may be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state.

The term stem domain or loop domain, as used interchangeably in the present invention, refers to any oligo or polypeptide that functions to link a transmembrane domain to an extracellular ligand binding domain. In particular, stem domains are used to provide greater flexibility and accessibility to the extracellular ligand binding domain.

The term intracellular signaling domain refers to the portion of a protein that transmits an effector functional signal and directs cells to perform a specialized function.

A costimulatory molecule, as used in the present invention, refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and receptor Toll ligand. Examples of co-stimulatory molecules include CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C , B7-H3 and a ligand that specifically binds to CD83 and others.

A costimulatory ligand refers to a molecule on an antigen presenting cell that specifically binds a cognate costimulatory signaling molecule on a T cell, thereby providing a signal that, in addition to the primary signal provided by, for example, TCR/CD3 complex binding, to the MHC the molecule loaded on the peptide mediates the T-cell response, including, but not limited to, activation of proliferation, differentiation, cytokine production and others. The co-stimulatory ligand may include, but is not limited to

- 5 044329 im, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin receptor β, 3/TR6, ILT3, ILT4, antagonist or antibody that binds Toll ligand receptor and ligand that specifically binds B7-H3 A costimulatory ligand also includes, but is not limited to, an antibody that specifically binds to a costimulatory molecule presented on a T cell, such as, but not limited to, CD27, CD28, 41BB, OX40, CD30, CD40, PD-1 , ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-I3, a ligand that specifically binds to CD83.

An antibody is an immunoglobulin molecule capable of specifically binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., using at least one antigen recognition site located on the variable region of the immunoglobulin molecule. As used herein, the term covers not only intact polyclonal or monoclonal antibodies, but also their antigen-binding fragments (such as Fab, Fab', F(ab') ₂ , Fv), single chain (scFv) and antibody domains (including , for example, shark and camel antibodies), and fusion proteins containing the antibody, and any other modified configuration of the immunoglobulin molecule, which contain an antigen recognition site. An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or a subclass thereof), and the antibody need not be of any preferred class. Depending on the amino acid sequence of the constant region of its antibody heavy chains, immunoglobulins can be divided into different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of them can be divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant regions that correspond to different classes of immunoglobulins are designated alpha, delta, epsilon, gamma and mu, respectively. The subunit structures and three-dimensional configurations of the various classes of immunoglobulins are known.

The term antigen-binding fragment or antigen-binding moiety of an antibody, as used herein, refers to one or more intact antibody fragments that retain the ability to specifically bind a given antigen (eg, EGFRvIII). The antigen-binding functions of an antibody can be carried out by fragments of an intact antibody. Examples of binding fragments included within the term antigen-binding fragment of an antibody include Fab; Fab';F(ab')₂; Fd fragment consisting of VH and CH1 domains; Fv fragment consisting of the VL and VH domains of a single-arm antibody; a single domain antibody (dAb) fragment (Ward et al., Nature 341:544-546, 1989), and a dedicated hypervariable region (CDR).

An antibody, antibody conjugate, or polypeptide that preferentially binds or specifically binds (used interchangeably in the present invention) a target (e.g., EGFRvIII protein) is a term well known in the art, and methods for determining such specific or preferential binding are also well known in the art. this field of technology. A molecule is considered to exhibit specific binding or preferential binding if it interacts or associates more frequently, more rapidly, for a longer duration and/or with greater affinity with a particular cell or substance than it exhibits with alternative cells or substances. An antibody specifically binds or binds preferentially to a target if it binds with greater affinity, avidity, more readily, and/or for a longer duration than it binds to other substances. For example, an antibody that specifically or preferentially binds to an EGFRvIII epitope is an antibody that binds that epitope with greater affinity, avidity, more readily, and/or longer duration than it binds other EGFRvIII epitopes or non-EGFRvIII epitopes. It is also understood that upon reading this definition, for example, an antibody (or part or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. As such, specific binding or preferential binding does not necessarily require (although it may include) exclusive binding. Typically, but not necessarily, a binding reference denotes a preferred binding.

An antibody variable region refers to an antibody light chain variable region or an antibody heavy chain variable region, either alone or in combination. As is known in the art, the heavy and light chain variable regions each consist of four framework regions (FRs) connected by three hypervariable regions (CDRs), also known as complementarity determining regions. The CDRs on each strand are held together in close proximity by the FR and, with the CDRs from the other strand, facilitating the formation of the antigen binding site of the antibodies. There are at least two techniques for determining CDRs: (1) an approach based on interspecies sequence variability (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda MD)) ; and (2) an approach based on crystallographic studies of antigen-antibody complexes (Alazikani et al., 1997, J. Molec. Biol. 273:927-948). As used in the present invention, CDR may refer to

- 6 044329

CDRs determined using either approach or a combination of both approaches.

A variable domain CDR is an amino acid residue within a variable region that is identified according to Kabat, Chothia, accumulation of both Kabat and Chothia, AbM, contact, and/or conformational definitions, or any CDR determination method well known in the art. Antibody CDRs can be identified as hypervariable regions originally defined by Kabat et al. See, for example, Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington D.C. CDR positions can also be identified using the structural loop structures originally defined by Chothia and others. See, for example, Chothia et al., Nature 342:877-883, 1989. Other approaches to identifying CDRs include the AbM definition, which is a compromise between Kabat and Chothia and is obtained using Oxford Molecular's AbM antibody modeling software (now Accelrys®), or contact determination of CDR based on observed antigen contacts, as described in MacCallum et al., J. Mol. Biol., 262:732-745, 1996. In another approach, referred to herein as CDR conformational determination, CDR positions can be identified as residues that are enthalpically significant for antigen binding. See, for example, Makabe et al., Journal of Biological Chemistry, 283:1156-1166, 2008. Also, still other definitions of CDR boundaries may not strictly adhere to one of the above approaches, but will nevertheless overlap with at least part of the Kabat CDR, although they may be shorter or longer in light of predictions or experimental evidence that specific residues or groups of residues, or even entire CDRs, will not have a significant effect on antigen binding. As used in the present invention, a CDR may refer to a CDR determined using any approach known in the art, including a combination of approaches. The methods used in the present invention may employ CDRs determined in accordance with any of these approaches. For any given embodiment containing more than one CDR, the CDR may be defined in accordance with any of the Kabat, Chothia, common, AbM, contact, and/or conformational definitions.

As used in the present invention, a monoclonal antibody refers to an antibody obtained from a population of essentially homogeneous antibodies, that is, the individual antibodies contained in the population are identical except for possible naturally occurring mutations that may be present in small quantities.

Monoclonal antibodies are highly specific, targeting a single antigenic site. Additionally, unlike polyclonal antibody preparations, which typically include different antibodies targeting different determinants (epitopes), each monolonal antibody targets a single determinant on an antigen. The modifier monoclonal indicates the characteristic of the antibody that it was obtained from an essentially homogeneous population of antibodies, and should not be construed as requiring that the antibody be obtained by any preferred method. For example, monoclonal antibodies used in accordance with the invention can be produced using the hybridoma method first described by Kohler and Milstein, Nature 256:495, 1975, or can be produced using recombinant DNA methods such as described in US Pat. No. 4,816,567 . Monoclonal antibodies can also be isolated from phage libraries using techniques described in McCafferty et al., Nature 348:552-554, 1990, for example.

As used herein, a humanized antibody refers to forms of non-human (eg, murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab') ₂ or other antigen-binding antibody subsequences) that contain minimal sequence originating from non-human immunoglobulin. Preferably, the humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the hypervariable region (CDR) of the recipient are replaced by residues from the CDR of a non-human species (donor antibody), such as mouse, rat or rabbit, having the desired specificity, affinity and potency. In some cases, the Fv framework region (FR) residues of a human immunoglobulin are replaced by corresponding non-human residues. In addition, the humanized antibody may contain residues that were not found in either the recipient antibody or the imported CDR or framework sequences, but are included for further purification and performance of the antibody. In general, a humanized antibody will contain substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions correspond to those of the consensus human immunoglobulin sequences. The humanized antibody will optimally also contain at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Preferred are antibodies having Fc regions modified as described in WO 99/58572. Other forms of humanized antibodies have one or more CDRs (CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, or CDR H3) that are altered relative to the parent antibody, which also

- 7 044329 are designated as one or more CDRs derived from one or more CDRs from the parent antibody.

As used herein, a human antibody means an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and/or that has been produced using any of the techniques for preparing human antibodies known to those skilled in the art or disclosed in the present invention. This definition of human antibody includes antibodies containing at least one human heavy chain polypeptide or at least one human light chain polypeptide. One such example is an antibody containing murine light chain and human heavy chain polypeptides. Antibodies can be produced using various techniques known in the art. In one embodiment, the human antibody is selected from a phage library, wherein the phage library expresses human antibodies (Vaughan et al., Nature Biotechnology, 14:309-314, 1996; Sheets et al., Proc. Natl. Acad. Sci. (USA ) 95:6157-6162, 1998; Hoogenboom and Winter, J. Mol. Biol., 227:381, 1991; Marks et al., J. Mol. Biol., 222:581, 1991). Human antibodies can also be obtained by immunizing animals in which human immunoglobulin loci have been transgenically introduced in place of endogenous loci, for example, mice in which endogenous immunoglobulin genes have been partially or completely inactivated. This approach is described in US Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016. Alternatively, a human antibody can be prepared by immortalizing human B lymphocytes that produce antibody directed to the target antigen (such B lymphocytes can be recovered from the individual or from single cell cDNA cloning, or can be immunized in vitro). See, for example, Cole et al. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77, 1985; Boerner et al., J. Immunol., 147(l):86-95, 1991; and U.S. Pat. No. 5,750,373.

The term chimeric antibody refers to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, for example, an antibody in which the variable region sequences are derived from a murine antibody and the constant region sequences are derived from a human antibody. .

The terms polypeptide, oligopeptide, peptide and protein are used interchangeably in the present invention to refer to amino acid chains of any length. For example, the chain may be relatively short (eg, 10-100 amino acids), or longer. The chain may be linear or branched, it may contain modified amino acids, and/or may be interrupted by non-amino acids. The terms also cover an amino acid chain that has been modified naturally or by intervention; for example, formation of disulfide bonds, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification such as conjugation to a labeled moiety. Also included in the definition are, for example, polypeptides containing one or more amino acid analogues (including, for example, non-naturally occurring amino acids, etc.), as well as other modifications known in the art. It is understood that the polypeptides may occur as single chains or associated chains.

A monovalent antibody contains one antigen-binding site per molecule (eg, IgG or Fab). In some cases, a monovalent antibody may have more than one antigen-binding site, but the binding sites are from different antigens.

A bivalent antibody contains two antigen-binding sites per molecule (eg, IgG). In some cases, the two binding sites have similar antigenic specificities. However, bivalent antibodies can be bispecific.

Bispecific or bispecific is a hybrid antibody having two different antigen-binding sites. The two antigen-binding sites of a bispecific antibody bind to two different epitopes, which may be located on the same or different protein targets.

A bifunctional is an antibody that has identical antigen-binding sites (that is, identical amino acid sequences) in two arms, but each binding site can recognize two different antigens.

Antibodies of the invention can be produced using techniques well known in the art, for example, recombinant technologies, phage display technologies, synthetic technologies or combinations of such technologies, or other technologies well known in the art (see, for example, Jayasena, S.D. , Clin. Chem., 45: 1628-50, 1999 and Fellowse, F. A., et al., J. Mol. Biol., 373(4):924-40, 2007).

As is known in the art, polynucleotide, or nucleic acid, which is used interchangeably in the present invention, refers to chains of nucleotides of any length, and includes DNA and RNA. The nucleotides may be decosyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or analogs thereof, or any substrate that can be incorporated into the chain by DNA or RNA polymerase. The polynucleotide may contain modified nucleotides, such as methylated nucleotides and analogs thereof. If present, modification of the nucleotide structure can occur before or after chain assembly. The nucleotide sequence can be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, for example, by conjugation with a labeled moiety. Other types of modifications include, for example, caps, replacement of one or more naturally occurring nucleotides with an analog, intranucleotide modifications such as, for example, modifications with uncharged bonds (for example, methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged bonds (for example, phosphorothioates, phosphorodithioates, etc.) that contain side components, such as, for example, proteins (for example, nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), with intercalators (for example, acridine , psoralen, etc.), which contain chelators (for example, metals, radioactive metals, boron, oxidizing metals, etc.), containing alkylators, with modified bonds (for example, alpha anomeric nucleic acids, etc.), as well as unmodified form of polynucleotide (s). Additionally, any hydroxyl groups originally present on the sugars can be replaced, for example, by phosphonate groups, phosphate groups protected by standard protecting groups, or activated to provide additional linkages to additional nucleotides, or can be conjugated to solid supports. The 5' and 3' terminal OH may be phosphorylated or replaced by amines or organic components of cap groups containing 1-20 carbon atoms. Other hydroxyls can also be derivatized to standard protecting groups. The polynucleotides may also contain similar forms of ribose or deoxyribose sugars that are commonly known in the art, including, for example, 2'-O-methyl-, 2'-Oallyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, alpha or beta anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and nucleoside analogs with the nitrogenous base removed, for example methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which the phosphate is replaced by P(O)S (thioate), P(S)S (dithioate), (O)NR ₂ (amidate), P(O )R, P(O)OR', CO or CH ₂ (formacetal), and each R or R' independently represents H or substituted or unsubstituted alkyl (1-20 C), optionally containing ether (-O-) bond, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. It is not necessary that all bonds in a polynucleotide be identical. The previous description applies to all polynucleotides provided in the present invention, including RNA and DNA.

As is known in the art, an antibody constant region refers to an antibody light chain constant region or an antibody heavy chain constant region, either alone or in combination.

As used herein, substantially pure refers to a material that is at least 50% pure (i.e., free of contaminants), more preferably at least 90% pure, more preferably at least 95% pure , even more preferably at least 98% pure, and most preferably at least 99% pure.

A host cell includes an individual cell or cell culture that may be or has been the recipient for the vector(s) for incorporation of polynucleotide inserts. Host cells include the progeny of a single host cell, and the progeny need not be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or intentional mutation. The host cell includes cells transfected in vivo with the polynucleotide(s) of the present invention.

As used in the present invention, an immune cell refers to a cell of hematopoietic origin involved in the initiation and/or implementation of the innate and/or adaptive immune response.

As is known in the art, the term Fc region is used to define the C-terminal region of an immunoglobulin heavy chain. The Fc region may be a native sequence Fc region or a variant Fc region. Although the boundaries of the immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region typically extends from the amino acid residue at position Cys226, or Pro230, to its carboxyl terminus. The numbering of residues in the Fc region corresponds to the EU index, as in Kabat. Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991. The immunoglobulin F region typically contains two constant regions, CH2 and CH3.

As used in the art, Fc receptor and FcR describe a receptor that binds to the Fc region of an antibody. A preferred FcR is a native human FcR sequence. In addition, a preferred FcR is one that binds to an IgG antibody (receptor gamma) and includes receptors of the FcyRI, FcyRII, and

- 9 044329

FcyRIII, including allelic variants and alternatively spliced forms of these receptors. FcyRII receptors include FcyRIIA (activating receptor) and FcyRIIB (inhibitory receptor), which have similar amino acid sequences, differing in most cases in their cytoplasmic domains. FcR is reviewed in Ravetch and Kinet, Ann. Rev. Immunol., 9:457-92, 1991; Capel et al., Immunomethods, 4:25-34, 1994; and de Haas et al., J. Lab. Clin. Med., 126:330-41, 1995. FcR also includes a neonal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol., 117:587, 1976; and Kim et al., J Immunol., 24:249, 1994).

The term compete, as used herein with respect to an antibody, means that the first antibody, or antigen-binding portion (or portion) thereof, binds to an epitope in a manner sufficiently similar to the binding of the second antibody, or antigen-binding portion thereof, such that such that the binding effect of the first antibody to its recognized epitope is clearly reduced in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody. Alternatively, where the binding of the second antibody to its epitope is also clearly reduced in the presence of the first antibody, this may, but need not, occur. In other words, the first antibody may inhibit the binding of the second antibody to its epitope without the second antibody inhibiting the binding of the first antibody to its corresponding epitope. However, if each antibody clearly inhibits the binding of another antibody to its recognized epitope or ligand, whether to a similar, greater or lesser extent, then the antibodies are cross-competing with each other for binding of their respective epitope(s) ). Both competitive and cross-competitive antibodies are covered by the invention. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or portion thereof), one skilled in the art will understand based on the knowledge provided in the present invention that such competitive and/or cross-competitive antibodies are covered and may be useful in the methods disclosed in the present invention.

As used in the present invention, autologous means that the cells, cell line, or population of cells used to treat patients are derived from the specified patient or from a human leukocyte antigen (HLA)-matched donor.

As used in the present invention, allogeneic means that the cells or population of cells used to treat patients do not originate from the specified patient, but from a donor.

As used in the present invention, treatment is an approach to obtain favorable or desirable clinical results. For the purposes of the present invention, beneficial or desirable clinical results include, but are not limited to, one or more of the following: reduction in proliferation (or destruction) of neoplastic or malignant cells, inhibition of metastasis of neoplastic cells, shrinkage or reduction in tumor size expressing EGFRvIII, remission of EGFRvIII associated disease (eg, cancer), reducing symptoms that develop due to an EGFRvIII associated disease (eg, cancer), improving the quality of life of those suffering from an EGFRvIII associated disease (eg, cancer), reducing the dose of other medications needed for treating EGFRvIII associated disease (eg, cancer), slowing the progression of EGFRvIII associated disease (eg, cancer), curing EGFRvIII associated disease (eg, cancer), and/or prolonging the survival of patients who have EGFRvIII associated disease (eg, cancer) ).

Relief refers to the reduction or improvement of one or more symptoms compared to the absence of administration of EGFRvIII specific CARs.

Relief also includes shortening or decreasing the duration of symptoms.

As used in the present invention, an effective dose or effective amount of a drug, compound or pharmaceutical composition is an amount sufficient to obtain any one or more beneficial or desired results. For prophylactic use, beneficial or desirable results include eliminating or reducing the risk, reducing the severity, or delaying the onset of disease, including biochemical, histological and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presented during the development of the disease. For therapeutic use, beneficial or desirable results include clinical results such as reduction in the prevalence or attenuation of one or more symptoms of various EGFRvIII associated diseases or conditions (such as, for example, glioblastoma multiforme), reduction in the dose of other drugs needed to treat the disease, enhancement effect of another drug, and/or slowing of EGFRvIII progression is associated

- 10 044329 disease in patients. An effective dose may be administered in one or more administrations. For purposes of the present invention, an effective dose of a drug, compound or pharmaceutical composition is an amount sufficient to effect prophylactic or therapeutic treatment, either directly or indirectly. As understood in the clinical context, an effective dose of a drug, compound or pharmaceutical composition can be achieved with or without another drug, compound or pharmaceutical composition. Thus, an effective dose may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered for administration in such an effective amount if, in combination with one or more other agents, the desired result can be achieved or achieved.

The individual or subject is a mammal, more preferably a human. Mammals also include, but are not limited to, primates, horses, dogs, cats, mice and rats.

As used in the present invention, a vector refers to a construct that is capable of delivering, and preferably expressing, one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

As used in the present invention, an expression control sequence refers to a nucleotide sequence that directs the transcription of a nucleic acid. The expression control sequence may be a promoter, such as a constitutive or inducible promoter, or an enhancer. The expression control sequence is operably linked to the nucleotide sequence to be transcribed.

As used herein, a pharmaceutically acceptable carrier or pharmaceutically acceptable excipient includes any material that, when combined with an active ingredient, enables the ingredient to retain biological activity and does not react with the host's immune system. Examples include, but are not limited to, any standard pharmaceutical carriers such as phosphate-buffered saline, water, emulsions such as oil-in-water emulsion, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate-buffered saline (PBS) or physiological (0.9%) saline. Compositions containing such carriers are formulated using well known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy 21st Ed. Mack Publishing, 2005).

The term _kon or k _a as used in the present invention refers to the rate constant of association of an antibody with an antigen. Specifically, rate constants (kon/k _a and k _off /kd) and equilibrium dissociation constants can be determined using, for example, full-length antibodies and/or Fab fragments of antibodies and the corresponding antigen.

The term k _off or kd as used in the present invention refers to the rate constant for the dissociation of an antibody from the antibody/antigen complex.

The term Kd, as used in the present invention, refers to the equilibrium dissociation constant of the antibody-antigen interaction.

By determining the rate constants of association and dissociation, k _on and k _off respectively, a surface plasmon resonance based biosensor can be used to characterize analyte/ligand interactions under conditions where the analyte is monovalent to the binding of the ligand, which is immobilized at low capacity onto the sensor surface by capture reagent. The analysis is carried out using kinetic titration methodology as described in Karlsson et al., Anal. Biochem 349, 136-147, 2006. The sensor chip, capture reagent, and assay buffer used for this assay are selected to ensure stable ligand capture to the sensor surface, minimize nonspecific analyte binding to surfaces, and produce analyte-binding responses that are suitable for kinetic analysis, as recommended in Myszka, J. Mol. Recognit 12, 279-284, 1999. Analyte-binding responses for analyte/ligand interactions are dual-linked and fit to a 1:1 Langmuir limited mass transfer model with k _a , k d and R _max as global parameters, as described in Myszka & Morton et al., Biophys. Chem 64, 127-137 (1997). The equilibrium dissociation constant, K _D , is derived from the ratio of the kinetic rate constants, K _D =k _off /k _on . Such determinations are preferably carried out at 25°C or 37°C.

Reference to approximately a value or parameter in the present invention includes (and describes) embodiments that are directed to that value or parameter per se. For example, a description with reference to approximately X includes a description of X. Numerical ranges x

- 11 044329 are numbers determined by the range. In general, the term approximately refers to a specified value of a variable and to all values of the variable that are within the experimental error of the specified value (for example, within a 95 percent confidence interval for the mean) or within 10 percent of the specified value, whichever , which one is greater. When a term is approximately used in the context of a period of time (years, months, weeks, days, etc.), the term approximately denotes a period of time plus or minus one value of the next subordinate time period (for example, approximately 1 year indicates 11-13 months; approximately 6 months means 6 months plus or minus 1 week; approximately 1 week means 6-8 days, etc.), or within 10 percent of the specified value, whichever is greater.

It is intended that, in all applicable cases where embodiments are described in the present invention with the word including, similar embodiments described by the terms consisting of and/or essentially consisting of are also provided.

If aspects or embodiments of the invention are described using a Markush group or other grouping of alternatives, then the invention covers not only the entire group described as a whole, but also each member of the group individually and all possible subgroups of the main group, as well as the main group in which one is missing or more group members. The invention also provides for the precise exclusion of one or more of any group members in the claimed invention.

Unless specifically stated otherwise, all technical and scientific terms used in the present invention have the same meanings as commonly understood by one skilled in the art to which the claimed invention relates. In the event of a conflict, the present description, including definitions, will control. For the entire description and claims, the word includes, or variations such as include or including will be intended to include the specified integer or group of integers, but not to exclude any other integers or groups of integers. Unless specifically required by context, terms in the singular will include plural forms and terms in the plural will include the singular.

The methods and materials described herein are exemplary, although methods and materials similar or equivalent to those described herein may also be used in practicing or testing the invention. The materials, methods, and examples are illustrative only and are not intended to be limiting in any way.

EGFRvIII specific CARs and methods for their preparation

The invention provides CARs that bind to EGFRvIII (eg, humEGFRvIII (eg, SEQ ID NO: 201, accession number: P00533 feature identifier VAR_066493, or GenBank accession number AJN69267)). EGFRvIII specific CARs provided by the present invention include single chain CARS and multi chain CARs. CARs have the ability to redirect the specificity and reactivity of T cells to EGFRvIII in a manner that is not limited by the MHC, developing the antigen-binding properties of monoclonal antibodies. Non-MHC-restricted antigen recognition provides CAR-expressing T cells with the ability to recognize antigen independent of antigen processing, thereby bypassing the underlying tumor escape mechanism.

In some embodiments, the CARs provided by the present invention comprise an extracellular ligand binding domain (eg, single chain fragment variable (scFv)), a transmembrane domain, and an intracellular signaling domain. In some embodiments, the extracellular ligand binding domain, the transmembrane domain, and the intracellular signaling domain are in a single polypeptide, that is, single chain. Multi-chain CARs and polypeptides are also provided in the present invention. In some embodiments, the multichain CARs comprise: a first polypeptide comprising a transmembrane domain and at least one extracellular ligand binding domain, and a second polypeptide comprising a transmembrane domain and at least one intracellular signaling domain, wherein the polypeptides assemble together to form the multichain CARs .

In some embodiments, EGFRvIII specific multichain CARs are based on high affinity receptor for IgE (FcεRI). FcεRI expressed on mast cells and basophils trigger allergic reactions. FcεRI is a tetrameric complex consisting of one α subunit, one β subunit, and two disulfide-linked γ subunits, the α subunit containing the IgE-binding domain. The β and γ subunits contain ITAMs, which mediate signal transduction. In some embodiments, the extracellular domain of the FcRa chain is removed and replaced with an EGFRvIII specific extracellular ligand binding domain. In some embodiments, the multichain EGFRvIII specific CARs comprise a scFv that binds specifically to the EGFRvIII CD8a loop, and the ITAM FcRP chain. In some embodiments, the CAR may or may not contain an FcRy chain.

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In some embodiments, the extracellular ligand binding domain comprises a scFv containing a light chain variable region (VL) and a heavy chain variable region (VH) of a target antigen-specific monoclonal antibody linked by a flexible linker. Single chain variable region fragments are prepared by linking light and/or heavy chain variable regions using a short peptide linker (Bird et al., Science 242:423-426, 1988). An example of a linker peptide is a GS linker having the amino acid sequence (GGGGS) ₄ (SEQ ID NO: 202), forms a bridge of approximately 3.5 nm between the carboxyl terminus of one variable region and the amino terminus of another variable region. Linkers of other sequences have been created and are used (Bird et al., 1988, supra). In general, linkers can be short, flexible polypeptides and preferably contain approximately 20 amino acid residues or fewer. Linkers, in turn, can be modified to provide additional functions, such as attaching drugs or attaching solid supports. Single-chain variants can be produced either recombinantly or synthetically. To synthetically obtain scFv, you can use an automatic synthesizer. For recombinant production of scFv, a suitable plasmid containing a polynucleotide that encodes the scFv can be introduced into a suitable host cell, either eukaryotic, such as yeast, plant, insect or mammalian cells, or prokaryotic, such as E. coli. Polynucleotides encoding the scFv presenter can be obtained using conventional manipulations such as polynucleotide ligation. The resulting scFv can be isolated using standard protein purification techniques known in the art.

In some embodiments, the extracellular ligand binding domain comprises (a) a VH region comprising (i) VH hypervariable region one (CDR1) comprising the sequence set forth in SEQ ID NO: 62, 63, 64, 74, 75, 76, 80, 81, 82, 88, 89, 90, 93, 94, 95, 99, 100, 101, 109, 110, 111, 115, 116, 117, 121, 122, 123, 132, 133,134, 137, 138, 139, 143, 144, or 145; (II) VH CDR2 containing the sequence shown in SEQ ID NO: 65, 66, 68, 69, 70, 71, 77, 78, 83, 84, 86, 87, 91, 92, 96, 97, 98, 102 , 103, 105, 106, 112, 113, 118, 119, 124, 125, 127, 128, 130, 131, 135, 136, 140, 141, 146, or 147; and III) VH CDR3 containing the sequence shown in SEQ ID NO: 67, 72, 73, 79, 85, 104, 107, 108, 114, 120, 126, 129, 142, or 148; and/or a VL region containing (I) VL CDR1 containing the sequence presented in SEQ ID NO: 149, 154, 156, 159, 162, 165, 166, 168, 169, 170, 171, 173, 174, 176, 178, 181, 182, 185, 187, 190, 192, 195, or 198; (II) VL CDR2 containing the sequence shown in SEQ ID NO: 150, 152, 155, 157, 160, 163, 172, 175, 179, 183, 186, 188, 191, 193, 196, or 199; and (III) VL CDR3 comprising the sequence set forth in SEQ ID NO: 151, 153, 158, 161, 164, 167, 177, 180, 184, 189, 194, 197, or 200. In some embodiments, VH and The VLs are linked together using a flexible linker. In some embodiments, the flexible linker comprises the amino acid sequence set forth in SEQ ID NO: 202.

In some embodiments, the extracellular ligand binding domain comprises (a) a VH region comprising (i) VH hypervariable region one (CDR1) comprising the sequence set forth in SEQ ID nO: 62, 63, 64, 74, 75, 76, 80, 81, 82, 88, 89, 90, 109, 110, 111, 115, 116, 117, 121, 122, 123, 137, 138, or 139; (II) VH CDR2 containing the sequence shown in SEQ ID NO: 70, 71, 77, 78, 83, 84, 86, 87, 91, 92, 112, 113, 118, 119, 124, 125, 127, 128 , 140, or 141; and iii) a VH CDR3 containing the sequence shown in SEQ ID NO: 73, 79, 85, 114, 120, 126, 129, or 142, and/or (b) a VL region containing (i) a VL CDR1 containing the sequence represented by SEQ ID NO: 149, 156, 159, 162, 165, 182, 185, 187, or 195; (II) VL CDR2 containing the sequence shown in SEQ ID NO: 152, 157, 160, 163, 183, 186, 188, or 196; and (III) VL CDR3 containing the sequence shown in SEQ ID NO: 153, 158, 161, 164, 184, 189, or 197.

In another aspect, a CAR is provided that specifically binds to EGFRvIII, wherein the CAR comprises an extracellular ligand binding domain comprising: a VH region containing VH CDR1, VH CdR2, and VH CDR3 from the VH sequence set forth in SEQ ID NO: 1, 3 , 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 30, 32, 34, 35, 37, 39, 41, 43, 44, 46, 48, or 50; and/or a VL region containing VL CDR1, VL CDR2, and VL CDR3 from the VL sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 29, 31, 33, 36, 38, 40, 42, 45, 47, 49, or 51. In some embodiments, VH and VL are linked together via a flexible linker. In some embodiments, the flexible linker comprises the amino acid sequence set forth in SEQ ID NO: 202.

In some embodiments, the CAR comprises an extracellular ligand binding domain comprising: a VH region comprising VH CDR1, VH CDR2, and VH CDR3 from the VH sequence set forth at SEQ ID NO: 5, 9, 11, 13, 15, 37, 39, 41, 43, or 48; and/or a VL region comprising VL CDR1, VL CDR2, and VL CDR3 from the VL sequence set forth in SEQ ID NO: 6, 10, 12, 14, 16, 38, 40, 42, or 49. In some embodiments, VH and VL are linked together using a flexible linker. In some embodiments, the flexible linker comprises the amino acid sequence set forth in SEQ ID NO: 202.

In some embodiments, the CAR of the invention comprises an extracellular ligand binding domain having any one of the partial light chain sequences such as

- 13 044329 is presented in table. 1, and/or any one of the particular heavy chain sequences as presented in table. 1. In table. 1, the underlined sequences are CDR sequences according to Kabat and those in bold according to Chothia. Various mAbs from table. 1 may also be referred to in the present invention as various anti-EGFRvIII antibody clones.

Table 1

mAb Light chain Heavy chain m62G7 DVVMTQTPLTLSVTIGQPASISCKSSQS LLYSNGKTYLNW LLQ R Р GQ S Р KRLIYLV SKLDSGVPDRFTGSGSGTDFTLKISRVE AEDLGFYYCVQDTHFPLTFGAGTKLELK (SEQ ID NO: 2) EVQLQQSGPELVKPGASVKISCKTSGYTF TDYTLHWVKQSHVKSLEWIGGIDPINGGT TYNQKFKGKAT LTVDKSSS TAYM E L R S L T SEDSAVYYCARGEAMDSWGQGTSVTVSS (SEQ ID NO: 1) H62G7 DVVMTQSPLSLPVTLGQPASISCKSSQS LLYSNGKTYLNWFQQRPGQSPRRLIYLV QVQLVQSGAEVKKPGASVKVSCKASGYTF TDYTLHWVRQAPGQGLEWMGGINPINGGT SKLDSGVPDRFSGSGSGTDFTLKISRVE AEDVGVYYCVQDTHFPLTFGGGTKVEIК (SEQ ID NO: 4) TYNQKFKGRVTMTRDTSTSTVYMELSSLR S E D TAVY Y CARGEAMDSWGQGTLVTVSS (SEQ ID NO: 3) H62G7L6/EQ DVVMTQSPLSLPVTLGQPASISCKSSQS LLYSNGKTYLNWFQQRPGQSPRRLIYQV SKLDSGVPDRFSGSGSGTDFTLKISRVE AEDVGVYYCGQDTHFPLTFGGGTKVEIK (SEQ ID NO: 6) QVQLVQSGAEVKKPGASVKVSCKASGYTF TDYTLHWVRQAPGQGLEWMGGIWPITGGT TYNQKFKGRVTMTRDTSTSTVYMELSSSLR S E D TAVY Y CARGEAQGSWGQGTLVTVSS (SEQ ID NO: 5) H62G7H14/L1-DV DVVM TQSPLSLPVTLGQPASISCKSSQS LLYSNDKTYTNWFQQRPGQSPRRLIYEV SKLDVGVPDRFSGSGSGTDFTLKISRVE AEDVGVYYCGQDTHFPLTFGGGTKVEIK (SEQ ID NO: 8) QVQLVQSGAEVKKPGASVKVSCKASGYTF TDYTLHWVRQAPGQGLEWMGGIWPITGGT TYNQKFKGRVTMTRDTSTSTVYMELSSLR S E D TAVY Y CARGEAEGSWGQGTLVTVSS (SEQ ID NO: 7) 42G9 EVVLTQSPATLSVSPGERATLLSCRASQS VRSNLAWYQQKSGQAPRLLIYGSTIRAT GVPARFSGSGSGTEFTLTISSLQSEDFA VYYCQQYSDWPFTFGPGTKVDIK (SEQ ID NO: 10) QVTLKESGPVLLKPTETLTLTCTVSGFSL SNPRMGVSWIRQ P PGKALEW FAHIFSTDE KSLKLSLRSRLTLSKDTSKSQVVLTMTNM APVDSATYY CARDSSNYEGYFDFWGQ GT L VTVSS (SEQ ID NO: 9) 32A10 EVVMTQSPATLSVSPGERVTLSCRASQS VSSNFAWYQQR P GQAP RLLLYGATTRAT GLPGRFSGSGSGTENILTISSLQSEDFA IYFCQQYKDWPFTFGPGSKVDIK (SEQ ID NO: 12) QVTLKESGPVLVKPTETLTLTCTVSGFSL SNARMGVSWIRQPPGKALEWLAHIFSTDE KSIRRSLRSRLTLSKDTSKSQVVLTMTNM D PVD TATY F CARDSSNYEGYFDYWGQ GT L VTVSS (SEQ ID NO: 11) 20В9 EIVMTQSPATLSVSPGERATLLSCRVSQS IGANLAWYQQKFGQAPRLLIYGASTRAT GIPVRFSGGGSGTEFTLTISSLQSEDFA IY S CQQYIYWPFFTGPGTTVDIK (SEQ ID NO: 14) QVTLKESGPVLVKPTETLTLTCTVSGFSL SNARMGVSWIRQPPGKALEWLGHIFSTDE KSYSTSLRGRITISKDTSRGLVVLTLTNM DPVDTATYY CARDSSNYEGYFDFWGP GF L VTVSS (SEQ ID NO: 13) 14С11 EIVMTQSPATLSVSPGERATLLSCRASQS VSNNLAWYQQKPGQAPRLLIYGASTRAT GVPARFSGSDSGTEFSLTISSLQSEDFA VYFCQQYKDWPFTFGPGTKVEIК (SEQ ID NO: 16) QVTLKESGPVLVKPTETLTLTCTVSGFSL NNARMGVSWIRQ P PGKALEW FAHIFSTDE KSFRTSLRSRLTLSKDTSKSQVVLTMTNM DPVDTATYYCARDSSNYEGYFDYWGQGIL VTVSS (SEQ ID NO: 15) 21E11 DMWTQSPATLSVSPGERATLLSCRASQS VGSDLAWYQQPPGQSPRLLIYGASTRAT GVPARFSGSGSGTDFTLTITSLESEDFA VYYCQQYNDWPFTFGPGTKVDIK (SEQ ID NO: 18) QVTLKESGPVLVKPTETLTLTCTVSGFSL SNVRMGVSWIRQ P PGKALEW FAHIFSSDE KSIRRSLRSRLTLSKDTSKSQVVLTMTNM D PVDTATYYCARDSSNYEGYFDFWGQ GT L VTVSSN (SEQ ID NO: 17) 49В11 EMEVTQSPATLSVSPGERATLLSCRASQN IGSDLAWYQQQSGQAPRLLISGASTRAT GVPTRFSGSGSGTDFTLTITSLQSEDFA VYYCQQYNDWPFTFGPGTKVDIK (SEQ ID NO: 20) QVTLKESGPVLVKPTETLTLTCTVSGFSL SNVRMGVSWIRQ P PGKALEW FAHIFSSDE KSIRRSLRSRLTLSKDTSKSQVVLTMTNM D PVDTATYYCARDSSNYEGYFDYWGQ GT L VTVSS (SEQ ID NO: 19)

- 14 044329

46E10 EVVMTQSPPNLSVSPGERATLLSCRASQS VTSNFAWYQQRPGQSPRLLLYGASTRAT GVPGRFSGSGSGTENILTISSLQSEDFA VYFCQQYKDWPFTFGGSKVDIK (SEQ ID NO: 22) QVTLKESGPVLVKPTETLTLTCTVSGFSL SNARMGVSWIRQPPGKALEWLAHIFSTDE KSIRRSLRSRLTLSKDTSKSQVVLIMTNM D PVDTATYYCARDSSNYEGYFDYWGQ GT L VTVSS (SEQ ID NO: 21) 12H6 EVVMTQSPATLSVSPGERATLLSCRASQG VSSNFAWYQQRPGQSPRLLLYGASTRAT GVPGRFSGSGSGTENILTISSLQSEDFA IYFCQQYKDWPFTFGGSKVDIK (SEQ ID NO: 24) QVTLKESGPVLVKPTETLTLTCTVSGFSL SNARMGVSWIRQPPGKALEWLAHIFSTDE KSIRRSLRSRLTLSKDTSKSQVVLTMTNM D PVDTATYYCARDSSNYEGYFDYWGQ GT L VTVSS (SEQ ID NO: 23) 19A9 EVVMTQSPATLSVSPGERATLLSCRASQS VNRNLAWYQQKPGQAPRLLIFGTSTRAT GIPARFSGSGSGTEFTLTIDSLQSEHSG LYYCQQYNDWPFTFGPGTKVDIK (SEQ ID NO: 26) QVTLEESGPVLVKPTETLTLTCTVSGFSL SNARMGVSWIRQPPGKAPEWFAHIFSTDE KSLRLSLRSRLTLSKDTSKSQVVLTMTNM D PVDTATYYCARDSSNYEGYFDYWGQ GT L VTVSS (SEQ ID NO: 25) 11B11 EVLMTQSPATLSVSPGERATLLSCRASQS VSTNFAWYQQRPGQAPRLLL FGASTRAT GIPGRFSGSGSGTENILTISSLQSEDFA IYFCQQYKDWPFTFGGSKVEIK (SEQ ID NO: 28) QVTLKESGPVLVKPTETLTLTCTVSGFSL SNAKMGVSWIRQPPGKALEWLAHIFSTDE KSIRRSLRSRLTMS KD T S KS QVVL TM TNM D PVD TAT Y Y CVRDSSNYEGYFDYWGQ GT L VTVSS (SEQ ID NO: 27) 21E7 DVVLTQSPATLSVSPGERATLLSCRASQS VNSNLAWYQQNPGQAPRLLIFGSSTRAT GIPASFSGSGSGTEFTLTINSLQSEHSA VYYCQQYNDWPFTFGPGTKVDIK (SEQ ID NO: 29) QVTLEESGPVLVKPTETLTLTCTVSGFSL SNARMGVSWIRQPPGKAPEWFAHIFSTDE KSLRLSLRSRLTLSKDTSKSQVVLTMTNM D PVDTATYYCARDSSNYEGYFDYWGQ GT L VTVSS (SEQ ID NO: 25) 12B2 EVVMTQSPATLSVSPGERATLSCRASQS VINNLAWYQQKPGQAPRLLIYGTSTRAT QVTLKESGPVLVKPTETLTLTCTVSGFSL SNPRMGVSWIRQPPGKALEWLGHIFSSDE DIPARFSGSGSGTEFTLTISSLQSEDFA VYYCQDYNNWPFTFGPGTKVDIK (SEQ ID NO: 31) KSYRLSLRSRLSISKDTSKSQVVLTM TNM D PVD TAT Y Y CVRDSSNYGGYFDYWGQ GT L VTVSS (SEQ ID NO: 30) HER EIVMTQSPATLSVSPGERTTLSCRASQS VGSNLAWYQQKPGQAPRLLIYGASTRAS QVTLKESGPVLVKPIETLTLTCTVCGFSL SNPRMGVSWIRQPPGKALEWLGHIFSSDE GVPARFSGSGSGTEFTLTISSLQSEDFA VYSCQEYNNWPFTFGQGTKVEIK (SEQ ID NO: 33) KSYRLFLRSRLSISKDTSKSQVVLTMTNM D PVDTATYYCARDSSDYEGYFDYWGQ GT L VTVSS (SEQ ID NO: 32) 17G11 EVVMTQSPATLSVSPGERATLLSCRASQS VINNLAWYQQKPGQAPRLLIYGTSTRAT DIPARFSGSGSGTEFTLTISSLQSEDFA VYYCQDYNNWPFTFGPGTKVDIK (SEQ ID NO: 31) QVTLKESGPVLVKPTETLTLTCTVFGFSL SNPRMGVSWIRQPPGKAPEWLGHIFSSDE KSYRLSLRSRLSISKDTSKSQVVFXMTNM DPGDPATYY CVRDSSNYEEYFDYWGQ GT L VTVSS (SEQ ID NO: 34) 29D5 KIVMTQSPATLSVSPGERATLSCRANQI VSSNLAWYQQКP GQAP RLLVFGTSTRAT GIPIRFSGSGSGTEFTLTVSSLQSEDFA VYVCQQYNDWPFTFGPGTKVDIК (SEQ ID NO: 36) QVTLKESGPVLVKPTETLTLTCTVSGFSL SNPRMGVSWLRQP PGKALEW FAHIFSTDE KSYSPSLRGRLTVSKDTSKSQVVLTLTNM D PVDTATYYCARDSSNYEGYFDYWGQ GT L VTVSS (SEQ ID NO: 35) 30D8 DIVMTQSPLSLPVTPGEPASISCRSSQS LLHNKRNNYLDWFLQKPGQSPQLLIYLA EVQLVESGGGLVKPGGSLRLSCEASGFTF SDAWMSWVRQAPGKGLEWVGRIKSKTDGG SNRASGVPDRFSGGGSGTDFTLKISRVE AE DV GVY Y CMQAQQTPITFGQGTRLEIK (SEQ ID NO: 38) TTDYVVPLNGRF11S RDD S RNT LY LQ LNN LKTEDTAVYYCTTVPGSYGYWGQGTLVTV SS (SEQ ID NO: 37) 20E12 DIVLTQSPLLSSVTPGEPASISCRSSQS LLYSNGKNYLDWFLHKPGQSPQLLIYLG SNRASGVPDRFSGSGSGIDFILKISRVE AE DV GVY Y CMQAQQTPITFGQGTRLEIK (SEQ ID NO: 40) EVNLVESGGGLVKPGGSLRLSCEASGFTF SYAWMSWVRQAPGKGLEWVGRIKSIADGG ATDYAAPVRNRFTISRDDSRNTLYLEMHS L KT E D TAVY Y S T TIPGNDAFDMWGQ GTMV TVSS (SEQ ID NO: 39) 26B9 DIVLTQSPLSLPVTPGEPASISCRSSQS LLHRDGFNYLDWFLQKPGQSPQLLIYLA SSRASGVPDRFSGSDSGTDFTLKISRVE AE DV GVY Y CMQALQTPITFGQGTRLEIK (SEQ ID NO: 42) EVQLVESWGVLVKPGGSLRLSCAASGFIF NNAWMSWVRQAPGKGLEWIGRIKSKSDGG TTDYAAPVKDRFTISRDDSKDTLYLQMNG LKTEDTAVYFCTTAPGGPFDYWGQGTLVT VSS (SEQ ID NO: 41)

- 15 044329

32G8 DIVLTQSPLLSSVTPGEPASISCRSSQS LLYSNGKNYLDWFLHKPGQSPQLLIYLG SNRASGVPDRFSGSGSGIDFILKISRVE AEDVGVYYCMQAQQTPITFGQGTRLEIK (SEQ ID NO: 40) EVNLVESGGGLVKPGGSLRLSCEASGFTF SYAWMSWVRQAPGKGLEWVGRIKSITDGG VIDYAAPVRNRСTISRDDSRNTLYLEΜHS L KT E D TAVY Y S T TIPGNDDFDMWGQ GRMV TVSS (SEQ ID NO: 43) 34E7 DIVLTQSPLLSSVTPGEPASISCRSTQS LLYSNGKNYLDWFLHKPGQSPQLLIFLG SIRASGVPDRFSGSGSGIDFILKISRVE AEDVGVYYCMQAQQTPITFGQGTRLEIK (SEQ ID NO: 45) EVNLVESGGGLVKPGGSLRLSCEASGFTF SYAWMSWVRQAPGKGLEWVGRIKSINDGG ATDYASPVRNRFTISRDDSRNMLYLEΜHS L KT E D TAVY Y S T TIPGNDAFDMWGQ GTLV TVSS (SEQ ID NO: 44) 20G5 DI VLTQSPLSLPVTPGEPASISCRSSQS LLYSDRRNYLDWFLQKPGQSPHLLIYLG EVQLVESGGDLVKPGGSLRLSCAASGFTF TNAWMSWVRQAPGKGLEWVGRIKSKIDGG SYRASGVPDRFSGSGSGTDFTLKISRVE AE DV GVY Y CMQALQIPITFGQGTRLEIK (SEQ ID NO: 47) TTDYAAPVKGRFIISRDDSKNTLSLQΜNS LКTEDTAMYYСTTAPGGPFDYWGQGSLVT VSS (SEQ ID NO: 46) C6 ELQSVLTQPPSASGTPGQRVTISCSGSS SNIGSNYVYWYQ Q LP GTAP КILIYRNNQ RPSGVPDRFSGSKSGTSASLAISGLRSE DEADYYCAAWDDNLSGWVFGTGTKLTVL QVQLVQSGAEVKKPGSSVKVSCKASGDTF SSNAISWVRQAPGQGLEWMGVIIPIFGTA DYAQKFQGRVTITADE STS TAYME LS S LR SEDTAVYYCARHTYHEYAGGYYGGAMDPW (SEQ ID NO: 49) GQGTLVTVSS (SEQ ID NO: 48) AT 5 DIQMTQSPSSLSSASVGDRVTITCRASQS ISSYLNWYQQKPGKAPKLLIYAASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQSYSTPLTFGQGTKVEIK (SEQ ID NO: 51) EVQLLESGGGLVQPGGSLRLSCAASGFTF SNYAMSWVRQAPGKGLEWVSDISGGGGRT YYADSVKGRFTISRDNSKNTLYLQMNSLR AE D TAVY Y CARAGLLYGGGVYPMDIWGQ G TLVTVSS (SEQ ID NO: 50)

Also provided in the present invention are the CDR portions of the extracellular ligand binding domains of the EGFRvIII CARs (including the Chothia, Kabat CDR, and CDR contact regions). The determination of CDR regions is within the skill of one skilled in the art. It is understood that in some embodiments, the CDRs may be a combination of Kabat and Chothia CDRs (also referred to as combined CRs or extended CDRs). In some embodiments, the CDRs are Kabat CDRs. In other embodiments, the CDRs are Chothia CDRs. In other words, in embodiments with more than one CDR, the CDRs may be any of Kabat, Chothia, a combination of CDRs, or combinations thereof. Table 2 provides examples of CDR sequences provided in the present invention.

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table 2

Heavy chain mAb CDRH1 CDRH2 CDRH3 m62G7 TDYTLH (SEQ ID NO: 62) (Rabat); GYTFTD (SEQ ID NO: 63) (Chothia); GYTFTDYTLH (SEQ ID NO: 64) (EXTENDED) GIDPINGGTTYNQRFR G (SEQ ID NO: 65) (Rabat) GIDPINGGTTY (SEQ ID NO: 66) (Chothia) GEAMDS (SEQ ID NO: 67) h62G7 TDYTLH (SEQ ID NO: 62) (Rabat); GYTFTD (SEQ ID NO: 63) (Chothia); GYTFTDYTLH (SEQ ID NO: 64) (EXTENDED) GINPINGGTTYNQRFR G (SEQ ID NO: 68) (Rabat) GINPINGGTTY (SEQ ID NO: 69) (Chothia) GEAMDS (SEQ ID NO: 67) h62G7-H14 TDYTLH (SEQ ID NO: 62) (Rabat); GYTFTD (SEQ ID NO: 63) (Chothia); GYTFTDYTLH (SEQ ID NO: 64) (EXTENDED) GIWPITGGTTYNQRFR G (SEQ ID NO: 7 0) (Rabat) GIWPITGGTTY (SEQ ID NO: 71) (Chothia) GEAEGS (SEQ ID NO: 72) h62G7-EQ TDYTLH (SEQ ID NO: 62) (Rabat); GYTFTD (SEQ ID NO: 63) (Chothia); GYTFTDYTLH (SEQ ID NO: 64) (EXTENDED) GIWPITGGTTYNQRFR G (SEQ ID NO: 7 0) (Rabat) GIWPITGGTTY (SEQ ID NO: 71) (Chothia) GEAQGS (SEQ ID NO: 73) 42G9 SNPRMGVS (SEQ ID NO: 74) (Rabat); GFSLSNPR (SEQ ID NO: 75) (Chothia); GFSLSNPRMGVS (SEQ ID NO: 76) (EXTENDED) HIFSTDERSLRLSLRS (SEQ ID NO: 77) (Rabat) HIFSTDERSL (SEQ ID NO: 78) (Chothia) DSSNYEGYFDF (SEQ ID NO: 79) 32 AYU SNARMGVS (SEQ ID NO: 8 0) (Rabat); GFSLSNAR (SEQ ID NO: 81) (Chothi a); GFSLSNARMGVS (SEQ ID NO: 82) (EXTENDED) HIFSTDERSIRRSLRS (SEQ ID NO: 83) (Rabat) HIFSTDERSI (SEQ ID NO: 84) (Chothia) DSSNYEGYFDY (SEQ ID NO: 85) 20В9 SNARMGVS (SEQ ID NO: 8 0) (Rabat); GFSLSNAR (SEQ ID NO: 81) (Chothia); GFSLSNARMGVS (SEQ ID NO: 82) (EXTENDED) HIFSTDERSYSTSLRG (SEQ ID NO: 86) (Rabat) HIFSTDERSY (SEQ ID NO: 87) (Chothia) DSSNYEGYFDF (SEQ ID NO: 79) 14S11 NNARMGVS (SEQ ID NO: 8 8) (Rabat); GFSLNNAR (SEQ ID NO: 89) (Chothia); GFSLNNARMGVS (SEQ ID NO: 90) (EXTENDED) HIFSTDERSFRTSLRS (SEQ ID NO: 91) (Rabat) HIFSTDERSF (SEQ ID NO: 92) (Chothia) DSSNYEGYFDY (SEQ ID NO: 85) 21E11 SNVRMGVS (SEQ ID NO: 93) (Rabat); GFSLSNVR (SEQ ID NO: 94) (Chothia); GFSLSNVRMGVS (SEQ ID HIFSSDERSIRRSLRS (SEQ ID NO: 96) (Rabat) HIFSSDERSI (SEQ ID NO: 97) DSSNYEGYFDF (SEQ ID NO: 79)

- 17 044329

NO: 95) (EXTENDED) (Chothia) 49B11 SNVRMGVS (SEQ ID NO: 93) (Rabat); GFSLSNVR (SEQ ID NO: 94) (Chothia); GFSLSNVRMGVS (SEQ ID NO: 95) (EXTENDED) HIFSSDERSIRRSLRS (SEQ ID NO: 96) (Rabat) HIFSSDERSI (SEQ ID NO: 97) (Chothia) DSSNYEGYFDY (SEQ ID NO: 85) 46E10 12H6 SNARMGVS (SEQ ID NO: 8 0) (Rabat); GFSLSNAR (SEQ ID NO: 81) (Chothia); GFSLSNARMGVS (SEQ ID NO: 82) (EXTENDED) HIFSTDERSIRRSLRS (SEQ ID NO: 83) (Rabat) HIFSTDERSI (SEQ ID NO: 84) (Chothia) DSSNYEGYFDY (SEQ ID NO: 85) 19A9 21E7 SNARMGVS (SEQ ID NO: 8 0) (Rabat); GFSLSNAR (SEQ ID NO: 81) (Chothia); GFSLSNARMGVS (SEQ ID NO: 82) (EXTENDED) HI FSTDERSLRLSLRS (SEQ ID NO: 98) (Rabat) HIFSTDERSL (SEQ ID NO: 78) (Chothia) DSSNYEGYFDY (SEQ ID NO: 85) 11B11 SNARMGVS (SEQ ID NO: 99) (Rabat); GFSLSNAR (SEQ ID NO: 100) (Chothia); GFSLSNARMGVS (SEQ ID NO: 101) (EXTENDED) HIFSTDERSIRRSLRS (SEQ ID NO: 83) (Rabat) HIFSTDERSI (SEQ ID NO: 84) (Chothia) DSSNYEGYFDY (SEQ ID NO: 85) 12B2 SNPRMGVS (SEQ ID NO: 74) (Rabat); GFSLSNPR (SEQ ID NO: 75) (Chothia); GFSLSNPRMGVS (SEQ ID NO: 76) (EXTENDED) HIFSSDERSYRLSLRS (SEQ ID NO: 102) (Rabat) HIFSSDERSY (SEQ ID NO: 103) (Chothia) DSSNYGGYFDY (SEQ ID NO: 104) HER SNPRMGVS (SEQ ID NO: 74) (Rabat); GFSLSNPR (SEQ ID NO: 75) (Chothia); GFSLSNPRMGVS (SEQ ID NO: 76) (EXTENDED) HI FSSDERSYRLFLRS (SEQ ID NO: 105) (Rabat) HIFSSDERSY (SEQ ID NO: 103) (Chothia) DSSDYEGYFDY (SEQ ID NO: 107) 17G11 SNPRMGVS (SEQ ID NO: 74) (Rabat); GFSLSNPR (SEQ ID NO: 7 5) (Chothi a); GFSLSNPRMGVS (SEQ ID NO: 76) (EXTENDED) HIFSSDERSYRLSLRS (SEQ ID NO: 102) (Rabat) HIFSSDERSY (SEQ ID NO: 103) (Chothia) DSSNYEEYFDY (SEQ ID NO: 108) 29D5 SNPRMGVS (SEQ ID NO: 74) (Rabat); GFSLSNPR (SEQ ID NO: 75) (Chothia); GFSLSNPRMGVS (SEQ ID NO: 76) (EXTENDED) HI FSTDERSYSPSLRG (SEQ ID NO: 106) (Rabat) HIFSTDERSY (SEQ ID NO: 87) (Chothia) DSSNYEGYFDY (SEQ ID NO: 85) 30D8 SDAWMS (SEQ ID NO: 109) (Rabat); GFTFSD (SEQ ID NO: 110) (Chothia); GFTFSDAWMS (SEQ ID NO: 111) (EXTENDED) RIRSRTDGGTTDYVVP LNG (SEQ ID NO: 112) (Rabat) RIRSRTDGGTTDY (SEQ ID NO: 113) (Chothia) VPGSYGY (SEQ ID NO: 114) 20E12 SYAWMS (SEQ ID NO: 115) (Rabat); GFTFSY (SEQ ID NO: 116) (Chothia); GFTFSYAWMS (SEQ ID RIRSIADGGATDYAAP VRN (SEQ ID NO: 118) (Rabat) RIRSIADGGATDY (SEQ ID NO: 119) IPGNDAFDM (SEQ ID NO: 120)

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NO: 117) (EXTENDED) (Chothia) 2 6B9 NNAWMS (SEQ ID NO: 121) (Rabat); GFIFNN (SEQ ID NO: 122) (Chothia); GFIFNNAWMS (SEQ ID NO: 123) (EXTENDED) RIRSRSDGGTTDYAAP VRD (SEQ ID NO: 124) (Rabat) RIRSRSDGGTTDY (SEQ ID NO: 125) (Chothia) APGGPFDY (SEQ ID NO: 126) 32G8 SYAWMS (SEQ ID NO: 115) (Rabat); GFTFSY (SEQ ID NO: 116) (Chothia); GFTFSYAWMS (SEQ ID NO: 117) (EXTENDED) RIRSITDGGVIDYAAP VRN (SEQ ID NO: 127) (Rabat) RIRSITDGGVIDY (SEQ ID NO: 128) (Chothia) IPGNDDFDM (SEQ ID NO: 129) 34E7 SYAWMS (SEQ ID NO: 115) (Rabat); GFTFSY (SEQ ID NO: 116) (Chothia); GFTFSYAWMS (SEQ ID NO: 117) (EXTENDED) RIRSINDGGATDYASP VRN (SEQ ID NO: 130) (Rabat) RIRSINDGGATDY (SEQ ID NO: 131 (Chothia) IPGNDAFDM (SEQ ID NO: 120) 20G5 TNAWMS (SEQ ID NO: 132) (Rabat); GFTFTN (SEQ ID NO: 133) (Chothia); GFTFTNAWMS (SEQ ID NO: 134) (EXTENDED) RIRSRIDGGTTDYAAP VRG (SEQ ID NO: 135) (Rabat) RIRSRIDGGTTDY (SEQ ID NO: 136) (Chothia) APGGPFDY (SEQ ID NO: 126) C6 SSNAIS (SEQ ID NO: 137) (Rabat); GDTFSS (SEQ ID NO: 138) (Chothia); GDTFSSNAIS (SEQ ID NO: 139) (EXTENDED) VII PIFGTADYAQRFQ G (SEQ ID NO: 140) (Rabat) VIIPIFGTADY (SEQ ID NO: 141) (Chothia) HTYHEYAGGYYGG AMDP (SEQ ID NO: 142) B5 SNYAMS (SEQ ID NO: 143) (Rabat); GFTFSN (SEQ ID NO: 144) (Chothia); GFTFSNYAMS (SEQ ID NO: 145) (EXTENDED) DISGGGGRTYYADSVR G (SEQ ID NO: 146) (Rabat) DISGGGGRTYY (SEQ ID NO: 14 7) (Chothia) AGLLYGGGVYPMD I (SEQ ID NO: 148) Light chain mAb CDRL1 CDRL2 CDRL3 m62G7 h62G7 RSSQSLLYSNGRTYLN (SEQ ID NO: 149) LVSRLDS (SEQ ID NO: 150) VQDTHFPLT (SEQ ID NO: 151) h62G7—L6 RSSQSLLYSNGRTYLN (SEQ ID NO: 149) QVSRLDS (SEQ ID NO: 152) GQDTHFPLT (SEQ ID NO: 153) h62G7-Ll-DV RSSQSLLYSNDRTYTN (SEQ ID NO: 154) EVSRLDV (SEQ ID NO: 155) GQDTHFPLT (SEQ ID NO: 153) 42G9 RASQSVRSNLA (SEQ ID NO: 156) GSTIRAT (SEQ ID NO: 157) QQYSDWPFT (SEQ ID NO: 158) 32A10 RASQSVSSNFA (SEQ ID NO: 159) GATTRAT (SEQ ID NO: 160) QQYRDWPFT (SEQ ID NO: 161) 20B9 RVSQSIGANLA (SEQ ID NO: 162) GASTRAT (SEQ ID NO: 163) QQYIYWPFT (SEQ ID NO: 164) 14C11 RASQSVSNNLA (SEQ ID NO: 165) GASTRAT (SEQ ID NO: 163) QQYRDWPFT (SEQ ID NO: 161)

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21E11 RASQSVGSDLA (SEQ ID NO: 166) GASTRAT (SEQ ID NO: 163) QQYNDWPFT (SEQ ID NO: 167) 49В11 RASQNIGSDLA (SEQ ID NO: 168) GASTRAT (SEQ ID NO: 163) QQYNDWPFT (SEQ ID NO: 167) 46E10 RASQSVTSNFA (SEQ ID NO: 169) GASTRAT (SEQ ID NO: 163) QQYNDWPFT (SEQ ID NO: 161) 12N6 RASQGVSSNFA (SEQ ID NO: 170) GASTRAT (SEQ ID NO: 163) QQYNDWPFT (SEQ ID NO: 161) 19A9 RASQSVNRNLA (SEQ ID NO: 171) GTSTRAT (SEQ ID NO: 172) QQYNDWPFT (SEQ ID NO: 167) 11В11 RASQSVSTNFA (SEQ ID NO: 173) GASTRAT (SEQ ID NO: 163) QQYNDWPFT (SEQ ID NO: 161) 21E7 RASQSVNSNLA (SEQ ID NO: 174) GSSTRAT (SEQ ID NO: 175) QQYNDWPFT (SEQ ID NO: 167) 12V2 17G11 RASQSVINNLA (SEQ ID NO: 176) GTSTRAT (SEQ ID NO: 172) QDYNNWPFT (SEQ ID NO: 177) HER RASQSVGSNLA (SEQ ID NO: 178) GASTRASG (SEQ ID NO: 179) QEYNNWPFT (SEQ ID NO: 180) 29D5 RANQIVSSNLA (SEQ ID NO: 181) GTSTRAT (SEQ ID NO: 172) QQYNDWPFT (SEQ ID NO: 167) 30D8 RSSQSLLHNKRNNYLD (SEQ ID NO: 182) LASNRAS (SEQ ID NO: 183) MQAQQTPIT (SEQ ID NO: 184) 20E12 32G8 RSSQSLLYSNGKNYLD (SEQ ID NO: 185) LGSNRAS (SEQ ID NO: 186) MQAQQTPIT (SEQ ID NO: 184) 26V9 RSSQSLLHRDGFNYLD (SEQ ID NO: 187) LASSRAS (SEQ ID NO: 188) MQALQTPIT (SEQ ID NO: 189) 34E7 RSTQSLLYSNGNNYLD (SEQ ID NO: 190) LGSIRAS (SEQ ID NO: 191) MQAQQTPIT (SEQ ID NO: 184) 20G5 RSSQSLLYSDRRNYLD (SEQ ID NO: 192) LGSYRAS (SEQ ID NO: 193) MQALQIPIT(SEQ ID NO: 194 ) C6 SGSSSNIGSNYVY (SEQ ID NO: 195) RNNQRPS (SEQ ID NO: 196) AAWDDNLSGWV (SEQ ID NO: 197) AT 5 RASQSISSYLN (SEQ ID NO: 198) AASSLQS (SEQ ID NO: 199) QQSYSTPLT(SEQ ID NO: 200 )

The invention covers modifications of CARs and polypeptides according to the embodiments of the invention presented in table. 1, including functional equivalents of CARs having modifications that do not significantly affect their properties, and variants that increase or decrease activity and/or affinity. For example, the amino acid sequence can be mutated to produce an antibody with the desired binding affinity for EGFRvIII. Modifications of polypeptides are common practice in the art and need not be described in more detail in the present invention. Examples of modified polypeptides include polypeptides with conservative substitutions of amino acid residues, one or more deletions or additions of amino acids that do not significantly adversely change functional activity, or that mature (enhance) the affinity of the polypeptide for its ligand, or use chemical analogues.

Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from a single residue to polypeptides containing hundreds or more residues, as well as intrasequence insertions of one or more amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue or an antibody fused to an epitope tag. Other insertional variants of the antibody molecule include fusion of the N or C terminus of the antibody to an enzyme or polypeptide, which increases the half-life of the antibody in the circulation.

Substitution variants have at least one amino acid residue in the antibody molecule removed and a different residue inserted in its place. Sites of greatest interest for replacement metagenesis include hypervariable regions, but FR changes are also covered. Conservative substitutions are presented in table. 3 under the heading Conservative substitutions. If such substitutions lead to changes in biological activity, then in the future there may be

- 20 044329 more significant changes were introduced, designated as typical replacements in the table. 3, or as further described below with reference to amino acid classes, and the products are screened. In some embodiments, the antibody substitution variants provided by the present invention have no more than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 conservative substitution in the VH or VL region compared to the reference parent antibody. In some embodiments, replacements are not made within the CDR of the VH or VL region.

Table 3

Amino acid substitutions

Original residue (naturally occurring amino acid) Conservative Substitutions Typical substitutions Ala (A) Val Val; Leu; Ile Arg(R) Lys Lys; Gln; Asn Asn(N) Gln Gln; His; Asp, Lys; Arg Asp (D) Glu Glu; Asn Cys(C) Ser Ser; Ala Gln(Q) Asn Asn; Glu Glu (E) Asp Asp; Gln Gly (G) Ala Ala His(H) Arg Asn; Gln; Lys; Arg He(I) Leu Leu; Val; Met; Ala; Ph; Norleucine Leu (L) lie Norleucine; lie; Val; Met; Ala; Phe Lys (K) Arg Arg; Gln; Asn Met(M) Leu Leu; Ph; lie Phe(F) Tyr Leu; Val; lie; Ala; Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp(W) Tyr Tyr; Phe Tyr (Y) Phe Trp; Ph; Thr; Ser Val(V) Leu lie; Leu; Met; Ph; Ala; Norleucine

In some embodiments, the invention provides a CAR containing an extracellular ligand binding domain that binds to EGFRvIII and competes for binding to EGFRvIII with antibodies described in the present invention, or CARs described in the present invention (for example, Table 5A), including m62G7, h62G7, h62G7-H14/Ll-DV, h62G7-L6/EQ, 42G9, 32A10, 20B9, 14C11, 21E11, 49B11, 46E10, 12H6, 19A9, 21E7, 11B11, 12B2, 11F1 0, 17G11, 29D5, 30D8, 20E12, 26B9, 32G8, 34E7, 20G5, C6, and B5.

In some embodiments, the invention provides CARs comprising CDR portions of anti-EGFRvIII antibodies based on CDR contact regions. CDR contact regions are regions of an antibody that confer specificity to the antibody for an antigen. In general, CDR contact regions include portions of residues in the CDR and vernier regions that set limits to maintain the correct loop structure for the antibody to bind a specific antigen. See, for example, Makabe et al., J. Biol. Chem., 283:1156-1166, 2007. Determination of CDR contact sites is well known to one skilled in the art.

The binding affinity ( _KD ) of EGFRvIII specific CARs as described in the present invention with EGFRvIII (such as humEGFRvIII (e.g., (SEQ ID NO: 201)) can be from about 0.001 nM to about 5000 nM. In some embodiments, the binding affinity is about any of 5000 nM, 4500 nM, 4000 nM, 3500 nM, 3000 nM, 2500 nM, 2000 nM, 1789 nM, 1583 nM, 1540 nM, 1500 nM, 1490 nM, 1064 nM, 1000 nM, 933 nM, 894 nM, 750 nM, 705 nM, 678 nM, 532 nM, 500 nM, 494 nM, 400 nM, 349 nM, 340 nM, 353 nM, 300 nM, 250 nM, 244 nM, 231 nM, 225 nM, 207 nM, 200 nM , 186 nM, 172 nM, 136 nM, 113 nM, 104 nM, 101 nM, 100 nM, 90 nM, 83 nM, 79 nM, 74 nM, 54 nM, 50 nM, 45 nM, 42 nM, 40 nM, 35 nM, 32 nM, 30 nM, 25 nM, 24 nM, 22 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 12 nM, 10 nM, 9 nM, 8 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, 5.5 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.3 nM, 0.1 nM, 0, 01 nM, or 0.001 nM In some embodiments, the binding affinity is less than about any of 5000 nM, 4000 nM, 3000 nM, 2000 nM, 1000 nM, 900 nM, 800 nM, 250 nM, 200 nM, 100 nM , 50 nM, 30 nM, 20 nM, 10 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, 5 nM, 4.5 nM, 4 nM, 3.5 nM, 3 nM, 2, 5 nM, 2 nM, 1.5 nM, 1 nM, or 0.5 nM.

The CAR intracellular signaling domain of the invention is responsible for intracellular signaling followed by binding of the extracellular ligand binding domain to the target, resulting in activation of the immune cell and immune response. The intracellular signaling domain has the ability to activate at least one of the normal effectors

- 21 044329 functions of the immune cell in which CAR is expressed. For example, the effector function of a T cell may be cytolytic activity or helper activity, including cytokine secretion.

In some embodiments, the intracellular signaling domain for use in a CAR may be cytoplasmic sequences, such as, but not limited to, T cell receptor and co-receptors, that act together to initiate signaling upon receptor uptake of an antigen, as well as any derivative or variant of these sequences and any synthetic sequence that has identical functionality. Intracellular signaling domains include two distinct classes of cytoplasmic signal sequences: those that initiate antigen-dependent primary activation, and those that act in an antigen-independent manner to provide a secondary or costimulatory signal. Primary cytoplasmic signal sequences may contain signaling motifs that are known as immunoreceptor tyrosine-based activation motifs ITAM. ITAMs are well-defined signaling motifs found in the intracytoplasmic tail of various receptors that serve as binding sites for syk/zap70 class tyrosine kinases. Examples of ITAMs used in the invention may include, but are not limited to, those derived from the TCRZ FcRy, FcRe, FcRε, CD3y, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b and CD66d. In some embodiments, the intracellular signaling domain of a CAR may comprise a CD3Z (zeta) signaling domain that has an amino acid sequence of at least about 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% , or 99% sequence identical to the amino acid sequence presented in SEQ ID NO: 205. In some embodiments, the intracellular signaling domain of the CAR of the invention comprises a co-stimulatory molecule domain.

In some embodiments, the intracellular signaling domain of the CAR of the invention comprises a portion of a co-stimulatory molecule selected from the group consisting of fragment 41BB (GenBank: AAA53133.) and CD28 (NP_006130.1). In some embodiments, the CAR intracellular signaling domain of the invention comprises an amino acid sequence that contains at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% sequence identity amino acid sequence presented in SEQ. ID NO: 213 (CD28 signaling domain) or SEQ ID NO: 204 (41BB signaling domain).

CARs are expressed on the cell surface membrane. Thus, a CAR may contain a transmembrane domain. Suitable transmembrane domains for the CARs disclosed herein have the ability to (a) be expressed on the surface of a cell, preferably an immune cell, such as, for example, but not limited to, lymphocyte cells or natural killer (NK) cells, and (b ) interact with the ligand binding domain and the intracellular signaling domain to direct the cellular response of the immune cell to a predetermined target cell. The transmembrane domain may be derived from either a natural or synthetic source. The transmembrane domain can be derived from any membrane-bound or transmembrane protein. By way of non-limiting examples, the transmembrane polypeptide may be a T cell receptor subunit such as α, β, γ or δ, a CD3 complex polypeptide, IL-2 receptor p55 (α chain), p75 (β chain) or γ chain , the subunit chain of Fc receptors, especially Fcy receptor III or CD proteins. Alternatively, the transmembrane domain may be synthetic and may contain predominantly hydrophobic residues such as leucine and valine. In some embodiments, said transmembrane domain is derived from the human CD8a chain (eg, NP_001139345.1). The CAR may further comprise a stem domain between an extracellular ligand binding domain and said transmembrane domain. The stem domain may contain up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25-50 amino acids. The stem region may be derived from all or part of a naturally occurring molecule, such as all or part of the extracellular region of CD8, CD4, or CD28, or all or part of the constant region of an antibody. Alternatively, the stem domain may be a synthetic sequence that corresponds to a naturally occurring stem sequence or may be a completely synthetic stem sequence. In some embodiments, said stem domain is part of the human CD8a chain (eg, NP_001139345.1). In another preferred embodiment, said transmembrane and loop domains comprise a portion of the human CD8a chain, preferably comprising at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% sequence identical to the amino acid sequence presented in SEQ ID NO: 210 and SEQ ID NO: 208, respectively. In some embodiments, the CARs disclosed in the present invention may contain an extracellular ligand binding domain that specifically binds EGFRvIII,

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Human CD8a loop and transmembrane domains, CD3Z signaling domain, and 4-1BB signaling domain.

In table Figure 4 shows typical domain sequences that can be used in

CARs disclosed in the present invention.

Table 4

Typical sequences of CAR components

Domain Amino acid sequence SEQ ID NO: CD8cx signal peptide MAL PVTALLPLAL L LHAAR P 206 FcyRIIIa loop GLAVSTISSFFPPGYQ 207 CD8a loop TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD 208 IgGl loop EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK 209 CD8a transmembrane (TM) domain IYIWAPLAGTCGVLLLSLVITLYC 210 41BB intracellular signaling domain (ISD) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 203 0Ό3ζ intracellular signaling domain (ISD) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR KNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR 205 FccRI a-TM-IC (FccRI a chain transmembrane intracellular domain) FFIPLLVVILFAVDTGLFISTQQQVTFLLKIKRTRKGFRLLNPHPKPNPKN N 211 FccRip-ΔΙΤΑΜ (FccRI β chain without ΙΤΑΜ) MDTESNRRANLALPQEPSSVPAFEVLEISPQEVSSGRLLKSASSPPLHTWL TVLKKEQEFLGVTQILTAMICLCFGTVVCSVLDISHI EGDIFSSFKAGYPF WGAIFFSISGMLSIISERRNATYLVRGSLGANTASSIAGGTGITILIINLK KSLAYIHIHSCQKFFETKCFMASFSTEIVVMMLFLTILGLGSAVSLTICGA GEELKGNK VPE 212 41BB-IC (41BB co-signal domain) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 204 CD28-IC (CD28 co-signaling domain) RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS 213 FccRIy-SP (signal peptide) MIPAVVLLLLLLVEQAAA 214 FccRI γ-ΔΙΤΑΜ (FesRI γ chain without ΙΤΑΜ) LGEPQLCYILDAILFLYGIVLTLLYCRLKIQVRKAAITSYEKS 215 GSG-P2A (GSG-P2A ribosomal jump polypeptide) GSGATNFSLLKQAGDVEENPGP 216 GSG-T2A (GSG-T2A ribosomal jump polypeptide) GSGEGRGSLLTCGDVEENPGP 217

In table 5A shows the amino acid sequences of exemplary EGFRvIII specific CARs according to the present invention. In table 5A, the signal/leader peptide sequence is in bold and the GS linker ((GGGGS) ₄ (SEQ ID NO: 202)) is underlined.

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Table 5A

Amino acid sequences of typical EGFRvIII specific CARs

CAR Amino acid sequence of CAR Components (in order, N-terminus to C-terminus) h62G7L6/EQ MALPVTALLLPLALLLHAARPDVVMTQSPLSLPVTLGQPASISCKSSQ SLLYSNGKTYLNWFQQRPGQSPRRLIYQVSKLDSGVPDRFSGSGSGTD FTLKISRVEAEDVGVYYCGQDTHFPLTFGGGTKVEIКGGGGSGGGGSG GGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYTLHW VRQ APGQGLEWMGGIWPITGGTTYNQKFKGRVTMTRDTSTSTVYMELSSSLR SEDTAVYYCARGEAQGSWGQGTLVTVSSTTTPAPRPPTPAPTIASQPL SLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLY CKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFS RSADAPAY QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKN PQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY DALHMQALPPR (SEQ ID NO: 52) CD8a signal peptide; h62G7-L6/EQ VL (Table 1 SEQ ID NO: 6) ; GS linker [(GGGGS) ₄ (SEQ ID NO: 202)]; h62G7—L6/EQ VH (Table 1 SEQ ID NO: 5) ; CD8a loop; CD8aTM; 4-IBB signaling domain; CD3zeta signaling domain 42G9 MALPVTALLLPLALLLHAARPQVTLKESGPVLLKPTETLTLTCTVSGF SLSNPRMGVSWIRQPPGKALEWFAHIFSTDEKSLKLSLRSRLTLSKDT SKSQVVLMTTNMAPVDSATYYCARDSSNYEGYFDFWGQGTLVTVSSGG GGSGGGGSGGGGSGGGGSEVVLTQSPATLSVSPGERATLSCRASQSVR SNLAWYQQ KSGQAPRLLIYGSTIRATGVPARFSGSGSGTEFTLTISSL QSEDFAVYYCQQYSDWPFTFGPGTKVDIKTTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITL YCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKF SRSADAPAYQQGQN QLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR (SEQ ID NO: 53) CD8a signal peptide; 42G9 VH (Table 1 SEQ ID NO: 9); GS linker; 42G9 VL (Table 1 SEQ ID NO: 10); CD8a loop; CD8aTM; 4-IBB signaling domain; CD3zeta signaling domain 32A10 MALPVTALLLPLALLLHAARPQVTLKESGPVLVKPTETLTLTCTVSGF SLSNARMGVSWIRQPPGKALEWLAHIFSTDEKSIRRSLRSRLTLSKDT SKSQVVLMTTNMDPVDTATYFCARDSSNYEGYFDYWGQGTLVTVSSGG GGSGGGGSGGGGSGGGGSEVVMTQSPATLSVSPGERVTLSCRASQSVS SNFAWYQQRPG QAPRLLLYGATTRATGLPGRFSGSGSGTENILTISSL QSEDFAIYFCQQYKDWPFTFGPGSKVDIKTTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITL YCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKF SRSADAPAYQQGQNQLYN ELNLGRREEYDVLDKRRGRDPEMGGKPRRK NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR(SEQ ID NO: 54) CD8a signal peptide; 32A10 VH (Table 1 SEQ ID NO: 11); GS linker; 32A10 VL (Table 1 SEQ ID NO: 12); CD8a loop; CD8aTM; 4-IBB signaling domain; CD3zeta signaling domain 20В9 MALPVTALLLPLALLLHAARPQVTLKESGPVLVKPTETLTLTCTVSGF SLSNARMGVSWIRQPPGKALEWLGHIFSTDEKSYSTSLRGRITISKDT SRGLVVLTLTNMDPVDTATYYCARDSSNYEGYFDFWGPGFLVTVSSGG GGSGGGGSGGGGSGGGGSEIVM T Q S PAT L SVS P G E RAT L S C RV S Q SIG ANL AWYQQKFGQAPRLLIYGASTRATGIPVRFSGGGSGTEFTLTISSL CD8a signal peptide; 20B9 VH (Table 1, SEQ ID NO: 13); GS linker;

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QSEDFAIYSCQQYIYWPFTFGPGTTVDIKTTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITL YCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKF SRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK NP QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR (SEQ ID NO: 55) 20B9 VL (Table 1, SEQ ID NO: 14) ; CD8a loop; CD8aTM; 4-IBB signal domain; CD3zeta signal domain 14C11 MALPVTALLLPLALLLHAARPQVTLKESGPVLVKPTETLTLTCTVSGF SLNNARMGVSWIRQPPGKALEWFAHIFSTDEKSFRTSLRSRLTLSKDT SKSQVVLTMTNMDPVDTATYYCARDSSNYEGYFDYWGQGILVTVSSGG GGSGGGGSGGGGSGGGGSEIVM T Q S PAT L SVS P G E RAT L S C RAS Q SV S N NLAWYQQKPGQAPRLLIYGASTRATGVPARFSGSDSGTEFSLTISSL QSEDFAVYFCQQYKDWPFTFGPGTKVEIKTTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITL YCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKF SRSADAPAY QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR (SEQ ID NO: 56) CD8a signal peptide; 14С11 VH (Table 1, SEQ ID NO: 15); GS linker; 14C11 VL (Table 1, SEQ ID NO: 16); CD8a loop; CD8aTM; 4-IBB signaling domain; CD3zeta signaling domain 20E12 MALPVTALLLPLALLLHAARPEVNLVESGGGLVKPGGSLRLSCEASGF TFSYAWMSWVRQAPGKGLEWVGRIKSIADGGATDYAAPVRNRFTI3RD DSRNTLYLEMHSLKTEDTAVYYCTTIPGNDAFDMWGQGTMVTVSSGGG GSGGGGSGGGGSGGGGSDIVLTQSPLLSSVTPGEPASISCRSSQSLLY SNGKNYLDWFLHKPGQSP QLLIYLGSNRASGVPDRFSGSGSGIDFILK ISRVEAEDVGVYYCMQAQQTPITFGQGTRLEIК TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYI WAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDG CSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYD VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERR RGK GHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 57) CD8c< signal peptide; 20E12 VH (Table 1, SEQ ID NO: 39); GS linker; 20E12 VL (Table 1, SEQ ID NO: 40); CD8a loop; CD8aTM; 4-IBB signaling domain; CD3zeta signaling domain 32G8 MALPVTALLLPLALLLHAARPEVNLVESGGGLVKPGGSLRLSCEASGF TFSYAWMSWVRQAPGKGLEWVGRIKSITDGGVIDYAAPVRNRCTI3RD DSRNTLYLEMHSLKTEDTAVYYCTTIPGNDDFDMWGQGRMVTVSSGGG GSGGGGSGGGGSGGGGSDIVLTQSPLLSSVTPGEPASISCRSSQSLLY SNGKNYLDWFLHKPGQSPQ LLIYLGSNRASGVPDRFSGSGSGIDFILK ISRVEAEDVGVYYCMQAQQTPITFGQGTRLEIК TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYI WAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDG CSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYD VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERR RGK GHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 58) CD8a signal peptide; 32G8 VH (Table 1, SEQ ID NO: 43); GS linker; 32G8 VL (Table 1, SEQ ID NO: 40) ; CD8a loop; CD8aTM; 4—IBB signaling domain; CD3zeta signaling domain 26B9 MALPVTALLLPLALLLHAARPEVQLVESWGVLVKPGGSLRLSCAASGF IFNNAWMSWVRQAPGKGLEWIGRIKSKSDGGTTDYAAPVKDRFTI3RD DSKDTLYLQMNGLKTEDTAVYFCTTAPGGPFDYWGQGTLVTVSSGGGG SGGGGSGGGGSGGGGSDIVLTQSPLSLPVTPGEPASISCRSSQSLLHR DGFNYLDWFLQK PGQSPQLLIYLASSRASGVPDRFSGSDSGTDFTLKI SRVEAEDVGVYYCMQALQTPITFGQGTRLEIК TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYI WAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDG CSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYD VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERR RGK GHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 59) CD8a signal peptide; 26B9 VH (Table 1, SEQ ID NO: 41); GS linker; 26B9 VL (Table 1, SEQ ID NO: 42) ; CD8a loop; CD8aTM; 4-IBB signaling domain; CD3zeta signaling domain 30D8 MALPVTALLLPLALLLHAARPEVQLVESGGGLVKPGGSLRLSCEASGF TFSDAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYVVPLNGRFII 3RD DSRNTLYLQLNNLKTEDTAVYYCTTVPGSYGYWGQGTLVTVSSGGGGS GGGGSGGGGSGGGGSDIVMTQ3PL3LPVTPGEPA31 3CR33Q3LLΗ NK RNNYLDWFLQKPGQSPQLLIYLASNRASGVPDRFSGGGSGTDFTLKI3 CD8a signal peptide; 30D8 VH (Table 1, SEQ ID NO: 37); GS linker;

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RVEAEDVGVYYCMQAQQTPITFGQGTRLEIKTTTTPAPRPPTPAPTIAS QPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVI TLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRV KFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR RK NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR (SEQ ID NO: 60) 30D8 VL (Table 1, SEQ ID NO: 38); CD8a loop; CD8aTM; 4-IBB signaling domain; CD3zeta signal domain C6 MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGSSVKVSCKASGD TFSSNAISWVRQAPGQGLEWMGVII PIFGTADYAQKFQGRVTITADES TSTAYMELSSLRSEDTAVYYCARHTYHEYAGGYYGGAMDPWGQGTLVT VSSGGGGSGGGGSGGGGSGGGGSELQSVLTQPPSASGTPGQRVTISCS GSSS NIGSNYVYWYQQLPGTAPKILIYRNNQRPSGVPDRFSGSKSGTS ASLAISGLRSEDEADYYCAAWDDNLSGWVFGTGTKLTVLTTTPAPRPP TPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCG VLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEE EGGCELRVKFSRS ADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRD PEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPR (SEQ ID NO: 61) CD8a signal peptide; C6 VH (Table 1, SEQ ID NO: 48); GS linker; C6 VL (Table 1, SEQ ID NO: 49); CD8a loop; CD8aTM; 4-1BB signaling domain; CD3zeta signaling domain

In table 5B shows the nucleotide sequences of representative scFvs for EGFRvIII specific CARs according to the present invention. In table 5B, the CD8a signal/leader peptide coding sequence is underlined.

Table 5 B

Nucleotide sequences of typical EGFRvIII specific scFvs

CAR scFv Nucleotide sequence h62G7L6/EQ s cFv ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCCCTGGCTCTGCTGCTGCACGCTGCTCGCCCTGATG TGGTCATGACTCAGTCTCCCCTGTCTCTGCCCGTCACCCTGGGACAGCCCGCCAGCATCTCCTGCAA GAGCTCCCAGAGCCTGCTGTACTCCAACGGCAAGACCTATCTGAATTGGTTCCAGCAGAGACCCGGC CAGAGCCCTCGGAGACTGATCTACCAGGTGTCTAAGCT GGACAGCGGCGTGCCTGATCGCTTCTCTG GAAGCGGATCCGGAACCGACTTTACACTGAAGATCAGCCGGGTGGAGGCAGAGGACGTGGGCGTGTA CTATTGCGGCCAGGATACCCACTTCCCACTGACATTTGGCGGCGGCACCAAGGTGGAGATCAAGGGA GGAGGAGGAAGCGGAGGAGGAGGAAGCGGCGGCGGCGGCTCTGGCGGCGGCGGCAGCCAGGTGCAGC TGGTGCAGAGCGG AGCAGAGGTGAAGAAGCCTGGCGCCTCCGTGAAGGTGTCTTGTAAGGCCAGCGG CTACACATTCACCGATTATACACTGCACTGGGTGCGGCAGGCCCCTGGCCAGGGACTGGAGTGGATG G GAGGAAT CTGGCCTATCAC C GGAG GAAC CACATACAAC CAGAAG T T TAAG GG CAGAG T GACAAT GA CCAGGGACACATCTACCAGCACAGTGTATATGGAGC TGTCTAGCCTGCGCTCCGAGGATACAGCCGT GTACTATTGCGCCAGAGGCGAGGCACAGGGATCTTGGGGACAGGGCACCCTGGTGACAGTGTCCTCT (SEQ ID NO:228) 42G9s cFv ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCACTGGCCCTGCTGCTGCACGCAGCAAGGCCTCAGG TGACCCTGAAGGAGAGCGGCCCTGTGCTGCTGAAGCCAACAGACCCTGACACTGACCTGCACAGT GTCTGGCTTCAGCCTGTCCAACCCCCGGATGGGCGTGAGCTGGATCAGACAGCCCCCTGGCAAGGCC CTGGAGTGGTTCGCCCACATCTTTTCTACCGATGAG AAGAGCCTGAAGCTGTCCCTGAGATCTAGGC TGACCCTGAGCAAGGACACATCTAAGAGCCAGGTGGTGCTGACCATGACAAACATGGCCCCTGTGGA CTCCGCCACATACTATTGCGCCAGAGACAGCTCCAATTACGAGGGCTATTTCGACTTTTGGGGCCAG GGCACCCTGGTGACAGTGTCTAGCGGCGGAGGAGGATCCGGAGGAGGAGGATCTGGCGGCGGCGGCT CCGGCGG CGGCGGCTCCGAGGTGGTGCTGACCCAGAGCCCTGCCACACTGTCCGTGTCTCCAGGCGA GAGAGCCACCCTGTCTTGTAGGGCCAGCCAGTCCGTGCGCAGCAATCTGGCCTGGTACCAGCAGAAG TCCGGCCAGGCCCCAAGACTGCTGATCTATGGCTCCACCATCAGGGCCACAGGAGTGCCAGCACGCT TCTCTGGAAGCGGATCCGGCACAGAGTTTACCCTGACAATCTCCTC TCTGCAGTCCGAGGATTTCGC CGTGTACTATTGCCAGCAGTACTCTGACTGGCCCTTCACCTTTGGCCCTGGCACAAAGGTGGATATC AAG (SEQ ID NO: 229) 32A10s cFv ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCACTGGCCCTGCTGCTGCACGCAGCAAGGCCACAGG TGACCCTGAAGGAGTCCGGCCCCGTGCTGGTGAAGCCTACAGACCCTGACACTGACCTGCACAGT GTCCGGCTTCTCTCTGAGCAACGCCCGCATGGGCGTGTCTTGGATCAGGCAGCCCCCTGGCAAGGCC

- 26 044329

CTGGAGTGGCTGGCCCACATCTTTTCCACCGACGAGAAGTCTATCCGGAGAAGCCTGCGCTCCAGGC TGACCCTGAGCAAGGATACATCCAAGTCTCAGGTGGTGCTGACCATGACAAACATGGACCCCGTGGA TACCGCCACATACTTCTGCGCCAGACAGCTCCAATTACGAGGGCTATTTTGATTACTGGGGCCAG GGCACCCTGGTGACAGTGTCTAGCGGAGGAGGAGGAAG CGGAGGAGGAGGATCTGGCGGCGGCGGCT CTGGCGGCGGCGGCAGCGAGGTGGTCATGACCCAGAGCCCAGCCACACTGAGCGTGTCCCCTGGCGA GAGGGTGACCCTGTCCTGTAGGGCATCTCAGAGCGTGTCCTCTAACTTCGCCTGGTATCAGCAGAGA CCAGGCCAGGCACCAAGGCTGCTGCTGTACGGAGCAACCACAAGAGCCACAGGACTGCCCGGCAGGT TTTCCGG ATCTGGAAGCGGCACCGAGAATATCCTGACAATCAGCTCCCTGCAGTCTGAGGACTTCGC CATCTATTTTTGCCAGCAGTACAAGGATTGGCCATTCACCTTTGGCCCCGGCAGCAAGGTGGACATC AAG (SEQ ID NO: 230) 20B9 ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCACTGGCCCTGCTGCTGCACGCAGCAAGACCTCAGG scFv TGACCCTGAAGGAGTCCGGCCCTGTGCTGGTGAAGCCAACAGAGACCCTGACACTGACCTGCACAGT GTCTGGCTTCAGCCTGTCCAACGCAAGGATGGGCGTGAGCTGGATCAGGCAGCCCCCTGGCAAGGCC CTGGAGTGGCTGGGCCACATCTTTAGCACCGACGAGAAGTCTTACAGCACATCCCTGAGAGGCAGGA TCACCATCTCTAAGGATACAAGCAGAGGCCTGGTG GTGCTGACCCTGACAAACATGGACCCCGTGGA TACCGCCACATACTATTGCGCCAGGGACAGCTCCAATTACGAGGGCTATTTCGATTTTTGGGGCCCT GGCTTCCTGGTGACCGTGTCTAGCGGCGGCGGCGGCTCTGGAGGAGGAGGAAGCGGAGGAGGAGGAT CCGGCGGCGGCGGCTCTGAGATCGTGATGACCCAGTCCCCTGCCACACTGTCTGTGAGCCCAGGCGA GAGAG CCACCCTGTCTTGTAGGGTGTCCCAGTCTATCGGCGCCAATCTGGCCTGGTACCAGCAGAAG TTCGGCCAGGCCCCAAGGCTGCTGATCTATGGAGCATCCACCAGAGCCACAGGAATCCCCGTGAGGT TCTCCGGAGGAGGATCTGGAACCGAGTTTACCCTGACAATCTCCTCTCTGCAGAGCGAGGACTTTGC CATCTACTCCTGCCAGCAGTACATCTATTGGCCCTTCACATTTGG CCCTGGCACCACAGTGGATATC AAG (SEQ ID NO: 231) 14C11 ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCACTGGCCCTGCTGCTGCACGCAGCAAGACCACAGG scFv TGACCCTGAAGGAGAGCGGACCCGTGCTGGTGAAGCCTACAGAGACCCTGACACTGACCTGCACAGT GAGCGGCTTCTCCCTGAACAATGCAAGGATGGGCGTGTCCTGGATCAGGCAGCCCCCTGGCAAGGCC CTGGAGTGGTTCGCCCACATCTTTAGCACCGACGAGAAGTCCTTTCGCACATCTCTGAGAAGCAGGC TGACCCTGAGCAAGGATACAAGCAAGTCCCAGGTG GTGCTGACCATGACAAACATGGACCCCGTGGA TACCGCCACATACTATTGCGCCAGAGACAGCTCCAATTACGAGGGCTATTTCGATTACTGGGGCCAG GGCATCCTGGTGACCGTGTCTAGCGGCGGCGGCGGCTCTGGAGGAGGAGGAAGCGGAGGAGGAGGAT CCGGCGGCGGCGGCTCTGAGATCGTGATGACCCAGTCTCCCGCCACACTGTCTGTGAGCCCTGGCGA GAGAGCC ACACTGAGCTGTAGGGCCTCCCAGTCTGTGAGCAACAATCTGGCCTGGTATCAGCAGAAG CCAGGCCAGGCACCAAGGCTGCTGATCTACGGAGCATCCACCAGAGCCACAGGAGTGCCAGCAAGGT TCTCCGGATCTGACAGCGGCACCGAGTTTAGCCTGACAATCTCCTCTCTGCAGTCCGAGGACTTCGC CGTGTATTTTTGCCAGCAGTACAAGGATTGGCCATTCACCTTTGG CCCCGGCACAAAGGTGGAGATC AAG (SEQ ID NO: 232) 20E12 ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCACTGGCCCTGCTGCTGCACGCAGCAAGGCCAGAGG scFv TGAACCTGGTGGAGTCCGGCGGCGGCCTGGTGAAGCCTGGCGGATCCCTGAGGCTGTCTTGCGAGGC AAGCGGCTTCACCTTCAGCTACGCCTGGATGTCCTGGGTGCGCCAGGCCCCCGGCAAGGGACTGGAG TGGGTGGGACGGATCAAGTCCATCGCAGACGGAGGAGCAACCGATTACGCAGCCCCTGTGAGAAACA G GT T CACAAT C T C CAGAGAC GAT T C T AG GAATAC C C T G TAT C T GGAGAT GCAC T C T C T GAAGACAGA GGACACCGCCGTGTACTATTGCACCACAATCCCTGGCAACGACGCCTTTGATATGTGGGGCCAGGGC ACAATGGTGACCGTGAGCTCCGGCGGCGGCGGCTCTGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCG GGGGCGGCGGCTCTGACATCGTGCTGACACAGTCCCCACTGTCCCTG TCTGTGACCCCCGGCGAGCC TGCAAGCATCTCCTGTAGATCTAGCCAGAGCCTGCTGTACTCCAACGGCAAATTATCTGGATTGG TTCCTGCACAAGCCAGGCCAGTCTCCCCAGCTGCTGATCTACCTGGGATCTAATAGGGCAAGCGGAG TGCCAGACCGGTTCTCTGGAAGCGGATCCGGCATCGACTTCATCCTGAAGATCAGCAGGGTGGAGGC CGAGGATGTGGGCGTGTACT ATTGCATGCAGGCCCAGCAGACACCCATCACCTTTCGGCCAGGGCACA AGACTGGAGATCAAG (SEQ ID NO: 233) 32G8 scFv ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCACTGGCCCTGCTGCTGCACGCAGCAAGGCCAGAGG TGAACCTGGTGGAGTCCGGCGGCGGCCTGGTGAAGCCTGGCGGATCCCTGAGGCTGTCTTGCGAGGC AAGCGGCTTCACCTTCAGCTACGCCTGGATGTCCTGGGTGCGCCAGGCCCCCGGCAAGGGACTGGAG TGGGTGGGCCGGATCAAGTCCATCACCGACGGAGGCG TGATCGATTACGCAGCACCTGTGAGAAACA G GT GCACAAT C T C CAGAGAC GAT T C TAG GAATAC CCTGTATCTG GAGAT G CAC T C T C T GAAGACAGA GGACACCGCCGTGTACTATTGTACCACAATCCCTGGCAACGACGATTTCGATATGTGGGGCCAGGGC AGAATGGTGACCGTGAGCTCCGGCGGCGGCGGCTCTGGAGGAGGAGGAAGCGGAGGAGG AGGAAGCG GGGGCGGCGGCTCTGACATCGTGCTGACACAGTCCCCACTGTCCCTGTCTGTGACCCCCGGCGAGCC TGCAAGCATCTCCTGTAGGTCTAGCCAGAGCCTGCTGTACTCCAACGGCAAGAATTATCTGGATTGG TTTCTGCACAAGCCAGGCCAGTCTCCCCAGCTGCTGATCTACCTGGGATCTAATAGGGCAAGCGGAG TGCCAGACCGGTTCTCTGGAAGCGGATCCGG CATCGACTTCATCCTGAAGATCAGCCGCGTGGAGGC AGAGGACGTGGGCGTGTACTATTGCATGCAGGCCCAGCAGACACCCATCACCTTCGGCCAGGGCACA AGACTGGAGATCAAG (SEQ ID NO: 234) 26B9 ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCACTGGCCCTGCTGCTGCACGCAGCAAGGCCAGAGG scFv TGCAGCTGGTGGAGTCTTGGGGCGTGCTGGTGAAGCCTGGCGGATCTCTGAGGCTGAGCTGCGCAGC ATCCGGCTTCATCTTTAACAATGCCTGGATGTCCTGGGTGCGCCAGGCCCCCGGCAAGGGACTGGAG TGGATCGGCCGGATCAAGAGCAAGTCCGACGGAGGAACCACAGATTACGCAGCACCTGTGAAGGACC

- 27 044329

GCTTCACAATCTCTCGGGACGATAGCAAGGATACCCTGTATCTGCAGATGAACGGCCTGAAGACAGA GGACACCGCCGTGTACTTCTGCACCACAGCCCCTGGCGGCCCTTTTGATTATTGGGGCCAGGGCACA CTGGTGACCGTGAGCTCCGGAGGAGGAGGAAGCGGCGGAGGAGGCAGCGGCGGCGGCGGCTCTGGCG GCGGCGGCAGCGACATCGTGCTGACACAGAGCCCTCTG TCCCTGCCAGTGACCCCCGGCGAGCCTGC CTCTATCAGCTGTCGCTCTAGCCAGAGCCTGCTGCACCGGGACGGCTTCAATTACCTGGATTGGTTT CTGCAGAAGCCAGGCCAGTCCCCCCAGCTGCTGATCTATCTGGCCTCCTCTAGAGCCTCTGGCGTGC CAGACAGGTTCTCCGGCTCTGACAGCGGCACAGACTTCACCCTGAAGATCAGCAGAGTGGAGGCCGA GGATGTG GGCGTGTACTATTGCATGCAGGCCCTGCAGACACCCATCACCTTCGGCCAGGGCACAAGA CTGGAGATCAAG (SEQ ID NO: 235) 30D8 scFv ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCACTGGCCCTGCTGCTGCACGCAGCAAGGCCTGAGG TGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGAAGCCTGGCGGATCCCTGAGGCTGTCTTGCGAGGC AAGCGGCTTCACCTTTAGCGACGCATGGATGTCCTGGGTGCGCCAGGCCCCTGGCAAGGGACTGGAG TGGGTGGGACGGATCAAGAGCAAGACAGAGCGG CACCACAGATTACGTGGTGCCACTGAACGGCC GCTTCATCATCTCCCGCGACGATTCTCGGAATACCCTGTATCTGCAGCTGAACAATCTGAAGACAGA GGATACCGCCGTGTACTATTGCACCACAGTGCCAGGCTCCTACGGCTATTGGGGCCAGGGCACACTG GTGACCGTGAGCTCCGGCGGCGGCGGCTCTGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGGGGGCG GCGGCTCT GACATCGTGATGACACAGTCTCCACTGAGCCTGCCAGTGACCCCTGGCGAGCCAGCCTC CATCTCTTGTCGCTCTAGCCAGAGCCTGCTGCACAACAAGCGGAACAATTACCTGGATTGGTTTCTG CAGAAGCCTGGCCAGTCCCCTCAGCTGCTGATCTATCTGGCCAGCAATAGAGCCTCCGGAGTGCCAG ACAGGTTCTCTGGAGGAGGAAGCGGAACAGACTTCACCCTGAAGATCA GCAGAGTGGAGGCCGAGGA CGTGGGCGTGTACTATTGCATGCAGGCCCAGCAGACACCTATCACCTTCGGCCAGGGCACAAGACTG GAGATCAAG (SEQ ID NO: 236) C6 scFv ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCTCTGGCCCTGCTGCTGCACGCAGCAAGGCCACAGG TGCAGCTGGTGCAGTCCGGAGCAGAGGTGAAGAAGCCTGGCAGCTCCGTGAAGGTGAGCTGCAAGGC CTCCGGCGACACATTCTCTAGCAACGCAATCAGCTGGGTGCGCCAGGCCCCTGGCCAGGGACTGGAG TGGATGGGCGTGATCATCCCTATCTTCGGCACCGCCG ACTATGCCCAGAAGTTTCAGGGCCGGGTGA CAATCACCGCCGATGAGTCTACAAGCACCGCCTACATGGAGCTGTCCTCTCTGAGATCCGAGGACAC AGCCGTGTACTATTGTGCCAGGCACACCTATCACGAGTACGCAGGAGGATACTATGGAGGAGCAATG GATCCTTGGGGACAGGGCACACTGGTGACCGTGAGCTCCGGCGGCGGCGGCTCTGGAGGAGGAGGAA GCGGAGG AGGAGGAAGCGGGGGCGGCGGCTCTGAGCTGCAGAGCGTGCTGACCCAGCCACCTTCCGC CTCTGGAACACCAGGCCAGAGGGTGACCATCAGCTGCTCCGGATCTAGCTCCAACATCGGCTCCAAT TACGTGTATTGGTACCAGCAGCTGCCAGGCACAGCCCCCAAGATCCTGATCTACCGCAACAATCAGC GGCCTTCTGGCGTGCCAGATAGATTCTCTGGCAGCAAGTCCGGCACCT CTGCCAGCCTGGCAATCTC CGGCCTGAGGTCTGAGGACGAGGCCGATTACTATTGCGCCGCCTGGGACGATAACCTGAGCGGCTGG GTGTTTGGCACAGGCACCAAGCTGACAGTGCTG (SEQ ID NO: 237)

Table 5C provides representative nucleotide sequences of representative EGFRvIII specific CARs according to the present invention.

Table 5C

Nucleotide sequences of typical EGFRvIII specific CARs

CAR Nucleotide sequence of CAR Components h62G7L6/EQ ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCCCTGGCTCTGCTGCTG CACGCTGCTCGCCCTGATGTGGTCATGACTCAGTCTCCCCTGTCTCTG CCCGTCACCCTGGGACAGCCCGCCAGCATCTCCTGCAAGAGCTCCCAG AGCCTGCTGTACTCCAACGGCAAGACCTATCTGAATTGGTTCCAGCAG AGACCGGCCAGAGCCCTCGGAGACTGATCTACCAGGTGTCTA AGCTG GACAGCGGCGTGCCTGATCGCTTCTCTGGAAGCGGATCCGGAACCGAC TTTACACTGAAGATCAGCCGGGTGGAGGCAGAGGACGTGGGCGTGTAC TATTGCGGCCAGGATACCCACTTCCCACTGACATTTGGCGGCGGCACC AAGGTGGAGATCAAGGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGGC GGCGGCGGCTCTGGCGGCGGCGGCAGCCAGGTGCAGCTGGTG CAGAGC GGAGCAGAGGTGAAGAAGCCTGGCGCCTCCGTGAAGGTGTCTTGTAAG GCCAGCGGCTACACATTCACCGATTATACACTGCACTGGGTGCGGCAG GCCCCTGGCCAGGGACTGGAGTGGATGGGAGGAATCTGGCCTATCACC G GAGGAAC CACATACAAC CAGAAG T T TAAG GG CAGAG T GACAAT GAC C AGGGACACATCTACCAGCACAGTGTATA TGGAGCTGTCTAGCCTGCGC TCCGAGGATACAGCCGTGTACTATTGCGCCAGGCGAGGCACAGGGA TCTTGGGGACAGGGCACCCTGGTGACAGTGTCCTCTACCACAACCCCA GCACCAAGACCACCTACCCCTGCACCAACAATCGCCTCCCAGCCTCTG TCTCTGCGCCCAGAGGCATGTAGGCCAGCAGCAGGAGGAGCAGTGCAC ACCAGGGGCCTGGACTTTGCCTGCGATA TCTACATCTGGGCACCACTG GCAGGAACATGTGGCGTGCTGCTGCTGAGCCTGGTCATCACCCTGTAC CD8a signal peptide; h62G7-L6/EQ VL (Table 1 SEQ ID NO: 6) ; GS linker [(GGGGS) ₄ (SEQ ID NO: 202)]; h62G7—L6/EQ VH (Table 1 SEQ ID NO: 5) ; CD8a loop; CD8aTM; 4-IBB signaling domain; CD3zeta signaling domain

- 28 044329

TGCAAGCGCGGCCGGAAGAAGCTGCTGTATATCTTCAAGCAGCCCTTC ATGAGACCCGTGCAGACAACCCAGGAGGAGGACGGCTGCTCCTGTAGG TTCCCAGAAGAAGAGGAGGGCGGCTGTGAGCTGAGAGTGAAGTTTTCC AGGTCTGCC GAT GCAC CAG CATAC CAGCAG GGACAGAAT CAGC T G TAT AACGAGCTGAATCTGGGCAGGCGCGAGGAGTATG ACGTGCTGGATAAG AGGAGAGGAAGGGACCCTGAGATGGGAGGCAAGCCTAGGCGCAAGAAC CCACAGGAGGGCCTGTACAATGAGCTGCAGAAGGATAAGATGGCCGAG GCCTATTCCGAGATCGGCATGAAGGGCGAGCGGAAGGGGCAAGGGC CAC GACGGGCTGTAC CAGG GAC T G T CAAC C GC TAC CAAG GATAC T TAC GACGCCCTGCATATGCAGGCACT GCCTCCAAGGTGA (SEQ ID NO:238) 42G9 ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCACTGGCCCTGCTGCTG CACGCAGCAAGGCCTCAGGTGACCCTGAAGGAGAGCGGCCCTGTGCTG CTGAAGCCAACAGAGACCCTGACACTGACCTGCACAGTGTCTGGCTTC AGCCTGTCCAACCCCCGGATGGGCGTGAGCTGGATCAGACAGCCCCCT GGCAAGGCCCTGGAGTGGTTCGCCCACATCTTTTCTACCG ATGAGAAG AGCCTGAAGCTGTCCCTGAGATCTAGGCTGACCCTGAGCAAGGACACA TCTAAGAGCCAGGTGGTGCTGACCATGACAAACATGGCCCCTGTGGAC TCCGCCACATACTATTGCGCCAGAGACAGCTCCAATTACGAGGGCTAT TTCGACTTTTGGGGGCCAGGGCACCCTGGTGACAGTGTCTAGCGGCGGA GGAGGATCCGGAGGAGGAGGATCTGGCGGCGGCGGCTC CGGCGGCGGC GGCTCCGAGGTGGTGCTGACCCAGAGCCCTGCCACACTGTCCGTGTCT CCAGGCGAGAGAGCCACCCTGTCTTGTAGGGCCAGCCAGTCCGTGCGC AGCAATCTGGCCTGGTACCAGCAGAAGTCCGGCCAGGCCCAAGACTG CTGATCTATGGCTCCACCATCAGGGCCACAGGAGTGCCAGCACGCTTCTCTGGAAGCGGATCCGGCACAGAGTTTACCCT GACAATCTCCTCTCTG CAGTCCGAGGATTTCGCCGTGTACTATTGCCAGCAGTACTCTGACTGG CCCTTCACCTTTGGCCCTGGCACAAAGGTGGATATCAAGACCACAACC CCTGCACCAAGGCCACCAACCCCAGCACCTACAATCGCAAGCCAGCCA CTGTCCCTGAGACCCGAGGCCTGTAGGCCTGCAGCAGGAGGAGCAGTG CACACCCGCGGCCTGGACTTTGCCTGCGATATCTA TATCTGGGCACCA CTGGCAGGAACCTGTGGCGTGCTGCTGCTGAGCCTGGTCATCACCCTG TACTGCAAGCGCGGCCGGAAGAAGCTGCTGTATATCTTCAAGCAGCCC TTCATGCGGCCCGTGCAGACAACCCAGGAGGAGGATGGCTGCTCCTGT AGATTCCCCTGAGGAGGAGGAGGGAGGATGTGAGCTGAGGGTGAAGTTT TCTCGGAGCGCCGACGCACCAGCATACCAG CAGG GACAGAAC CAG C T G TATAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGAT AAGAGGAGGGGAAGACCCAGAGATGGGAGGCAAGCCACGGAGAAAG AACCCCCAGGAGGGCCTGTACAATGAGCTGCAGAAGGATAAGATGGCC GAGGCCTATTCTGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAG G GACAC GAC G GAC T G TAC CAGGGAC T G T C CAC C G CAACAAAGGACAC C TATGATGCCCTGCATATGCAGGCACTGCCTCCAAGGTGA (SEQ ID NO: 239) CD8a signal peptide; 42G9 VH (Table 1 SEQ ID NO: 9); GS linker; 42G9 VL (Table 1 SEQ ID NO: 10); CD8a loop; CD8aTM; 4-IBB signaling domain; CD3zeta signaling domain 32A10 ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCACTGGCCCTGCTGCTG CACGCAGCAAGGCCACAGGTGACCCTGAAGGAGTCCGGCCCCGTGCTG GTGAAGCCTACAGAGACCCTGACACTGACCTGCACAGTGTCCGGCTTC TCTCTGAGCAACGCCCGCATGGGCGTGTCTTGGATCAGGCAGCCCCCT GGCAAGGCCCTGGAGTGGCTGGCCCACATCTTTTCCACCGAC GAGAAG TCTATCCGGAGAAGCCTGCGCTCCAGGCTGACCCTGAGCAAGGATACA TCCAAGTCTCAGGTGGTGCTGACCATGACAAACATGGACCCCGTGGAT ACCGCCACATACTTCTGCGCCAGACAGCTCCAATTACGAGGGCTAT TTTGATTACTGGGGCCAGGGCACCCTGGTGACAGTGTCTAGCGGAGGA GGAGGAAGCGGAGGAGGAGGATCTGGCGGCGGCGGCTCTGGCGG CGGC GGCAGCGAGGTGGTCATGACCCAGAGCCCAGCCACACTGAGCGTGTCC CCTGGCGAGAGGGTGACCCTGTCCTGTAGGGCATCTCAGAGCGTGTCC TCTAACTTCGCCTGGTATCAGCAGAGACCAGGCCAGGCACCAAGGCTG CTGCTGTACGGAGCAACCACAAGAGCCACAGGACTGCCCGGCAGGTTT TCCGGATCTGGAAGCGGCACCGAGAATATCCTGACAAT CAGCTCCCTG CAGTCTGAGGACTTCGCCATCTATTTTTGCCAGCAGTACAAGGATTGG CCATTCACCTTTGGCCCCGGCAGCAAGGTGGACATCAAGACCACAACC CCTGCACCAAGACCACCAACCCCAGCACCTACAATCGCCTCTCAGCCT CTGAGCCTGCGCCCAGAGGCATGTAGGCCAGCAGCAGGAGGAGCAGTG CACACAAGGGGCCTGGACTTCGCCTGCGATATCTATATCTGG GCACCT CTGGCAGGAACCTGTGGCGTGCTGCTGCTGAGCCTGGTCATCACCCTG TATTGCAAGAGAGGCAGGAAGAAGCTGCTGTACATCTTCAAGCAGCCT TTTATGCGCCCAGTGCAGACAACCCAGGAGGAGGACGGCTGCAGCTGT CGGTTCCCTGAAGAGGAGGAGGGCGGCTGTGAGCTGAGAGTGAAGTTT CD8a signal peptide; 32A10 VH (Table 1 SEQ ID NO: 11) ; GS linker; 32A10 VL (Table 1 SEQ ID NO: 12); CD8a loop; CD8aTM; 4-IBB signaling domain; CD3zeta signaling domain

- 29 044329

TCCAGGTCTGCCGATGCCCCAGCCTATCAGCAGGGCCAGAATCAGCTG TACAACGAGCTGAATCTGGGCAGGCGCGAGGAGTACGACGTGCTGGAT AAGAGGAGAGGAAGGGATCCAGAGATGGGAGGCAAGCCTAGGCGCAAG AACCCACAGGAGGGCCTGTATAATGAGCTGCAGAAGGACAAGATGGCC GAGGCCTACTCCGAGATCGGCATGAAGGGAGAGCGGAGAAGGGGG CAAG GGACACGATGGCCTGTATCAGGGCCTGTCTACCGCCACAAAGGACACC TACGATGCCCTGCATATGCAGGCACTGCCTCCAAGGTGA (SEQ ID NO: 240) 20B9 ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCACTGGCCCTGCTGCTG CACGCAGCAAGACCTCAGGTGACCCTGAAGGAGTCCGGCCCTGTGCTG GTGAAGCCAACAGAGACCCTGACACTGACCTGCACAGTGTCTGGCTTC AGCCTGTCCAACGCAAGGATGGGCGTGAGCTGGATCAGGCAGCCCCCT GGCAAGGCCCTGGAGTGGCTGGGCCACATCTTTAGCACCGA CGAGAAG TCTTACAGCACATCCCTGAGAGGCAGGATCACCATCTCTAAGGATACA AGCAGAGGCCTGGTGGTGCTGACCCTGACAAACATGGACCCCGTGGAT ACCGCCACATACTATTGCGCCAGGGACAGCTCCAATTACGAGGGCTAT TTCGATTTTTGGGGCCCTGGCTTCCTGGTGACCGTGTCTAGCGGCGGC GGCGGCTCTGGAGGAGGAGGAAGCGGAGGAGGAGGATCCGGC GGCGGC GGCTCTGAGATCGTGATGACCCAGTCCCCTGCCACACTGTCTGTGAGC CCAGGCGAGAGAGCCACCCTGTCTTGTAGGGTGTCCCAGTCTATCGGC GCCAATCTGGCCTGGTACCAGCAGAAGTTCGGCCAGGCCCCAAGGCTG CTGATCTATGGAGCATCCACCAGAGCCACAGGAATCCCCGTGAGGTTC TCCGGAGGAGGATCTGGAACCGAGTTTACCCTGA CAATCTCCTCTCTG CAGAGCGAGGACTTTGCCATCTACTCCTGCCAGCAGTACATCTATTGG CCCTTCACATTTGGCCCTGGCACCACAGTGGATATCAAGACCACAACC CCTGCACCAAGGCCACCAACCCCAGCACC TACAAT C G CAAG C CAG C CA CTGTCCCTGAGACCAGAGGCATGTAGGCCTGCAGCAGGAGGAGCCGTG CACACCAGAGGCCTGGACTTTGCCTGCGATATC TATATCTGGGCACCA CTGGCAGGAACCTGTGGCGTGCTGCTGCTGAGCCTGGTCATCACCCTG TACTGCAAGCGCGGCCGGAAGAAGCTGCTGTATATCTTCAAGCAGCCC TTCATGCGCCCCGTGCAGACAACCCAGGAGGAGGACGGCTGCAGCTGT CGGTTCCCTGAAGAGGAGGAGGGAGGATGTGAGCTGAGGGTGAAGTTT AGCCGGTCCGCCGATGCACCAGCATAC CAGCAGGGCCAGAACCAGCTG TATAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGAT AAGAGGAGGGGAAGAGACCCAGAGATGGGAGGCAAGCCACGGAGAAAG AACCCCCAGGAGGGCCTGTACAATGAGCTGCAGAAGGACAAGATGGCC GAGGCCTATAGCGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAG GGACACGATGGCCTGTACCAGGGCC TGTCCACCGCCACAAAGGACACC TATGATGCCCTGCATATGCAGGCACTGCCTCCAAGGTGA (SEQ ID NO: 241) CD8c< signal peptide; 20B9 VH (Table 1, SEQ ID NO: 13); GS linker; 20B9 VL (Table 1, SEQ ID NO: 14) ; CD8c<loop; CD8aTM; 4-IBB signaling domain; CD3zeta signaling domain 14C11 ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCACTGGCCCTGCTGCTG CACGCAGCAAGACCACAGGTGACCCTGAAGGAGAGCGGACCCGTGCTG GTGAAGCCTACAGAGACCCTGACACTGACCTGCACAGTGAGCGGCTTC TCCCTGAACAATGCAAGGATGGGCGTGTCCTGGATCAGGCAGCCCCCT GGCAAGGCCCTGGAGTGGTTCGCCCACATCTTTAGCACCGAC GAGAAG TCCTTTCGCACATCTCTGAGAAGCAGGCTGACCCTGAGCAAGGATACA AGCAAGTCCCAGGTGGTGCTGACCATGACAAACATGGACCCCGTGGAT ACCGCCACATACTATTGCGCCAGACAGCTCCAATTACGAGGGCTAT TTCGATTACTGGGGCCAGGGCATCCTGGTGACCGTGTCTAGCGGCGGC GGCGGCTCTGGAGGAGGAGGAAGCGGAGGAGGAGGATCCGGCGG CGGC GGCTCTGAGATCGTGATGACCCAGTCTCCCGCCACACTGTCTGTGAGC CCTGGCGAGAGAGCCACACTGAGCTGTAGGGCCTCCCAGTCTGTGAGC AACAATCTGGCCTGGTATCAGCAGAAGCCAGGCCAGGCACCAAGGCTG CTGATCTACGGAGCATCCACCAGAGCCACAGGAGTGCCAGCAAGGTTC TCCGGATCTGACAGCGGCACCGAGTTTAGCCTGACAAT CTCCTCTCTG CAGTCCGAGGACTTCGCCGTGTATTTTTGCCAGCAGTACAAGGATTGG CCATTCACCTTTGGCCCCGGCACAAAGGTGGAGATCAAGACCACAACC CCTGCACCAAGACCACCAACCCCAGCACCTACAATCGCATCCCAGCCT CTGTCTCTGAGACCAGAGGCATGTAGGCCAGCAGCAGGAGGAGCAGTG CACACCAGGGGCCTGGACTTTGCCTGCGATATCTATATCTG GGCACCT CTGGCAGGAACCTGTGGCGTGCTGCTGCTGAGCCTGGTCATCACCCTG TATTGCAAGCGCGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCT TTTATGCGCCCAGTGCAGACAACCCAGGAGGAGGACGGCTGCTCCTGT CGGTTCCCTGAAGAGGAGGAGGGAGGATGTGAGCTGAGGGTGAAGTTT TCCCGGTCTGCCGATGCCCCAGCCTATCAGCA GGGCCAGAACCAGCTG TACAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGAT AAGAGGAGGGGAAGAGATCCAGAGATGGGAGGCAAGCCTCGGAGAAAG CD8c< signal peptide; 14С11 VH (Table 1, SEQ ID NO: 15); GS linker; 14C11 VL (Table 1, SEQ ID NO: 16); CD8c<loop; CD8c<TM; 4-IBB signaling domain; CD3zeta signaling domain

- 30 044329

AACCCACAGGAGGGCCTGTATAATGAGCTGCAGAAGGACAAGATGGCC GAGGCCTACTCCGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAG GGACACGATGGCCTGTATCAGGGCCTGTCTACCGCCACAAAGGACACC TACGATGCCCTGCATATGCAGGCACTGCCTCCAAGGTGA (SEQ ID NO: 242) 20E12 ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCACTGGCCCTGCTGCTG CACGCAGCAAGGCCAGAGGTGAACCTGGTGGAGTCCGGCGGCGGCCTG GTGAAGCCTGGCGGATCCCTGAGGCTGTCTTGCGAGGCAAGCGGCTTC ACCTTCAGCTACGCCTGGATGTCCTGGGTGCGCCAGGCCCCCGGCAAG GGACTGGAGTGGGTGGGACGGATCAAGTCCATCGCAGACGGAGG AGCA ACCGATTACGCAGCCCCTGTGAGAAACAGGTTCACAATCTCCAGAGAC GAT T C TAG GAATAC C C T G TAT C T GGAGAT G CAC T C T C T GAAGACAGAG GACACCGCCGTGTACTATTGCACCACAATCCCTGGCAACGACGCCTTT GATATGTGGGGCCAGGGCACAATGGTGACCGTGAGCTCCGGCGGCGGC GGCTCTGGAGGAGGAGGAAGCG GAGGAGGAGGAAGCGGGGGCGGCGGC TCTGACATCGTGCTGACACAGTCCCCACTGTCCCTGTCTGTGACCCCC GGCGAGCCTGCAAGCATCTCCTGTAGATCTAGCCAGAGCCTGCTGTAC TCCAACGGCAAGAATTATCTGGATTGGTTCCTGCACAAGCCAGGCCAG TCTCCCCAGCTGCTGATCTACCTGGGATCTAATAGGGCAAGCGGAGTG CCAGACCGGTTCTCTGGAAGC GGATCCGGCATCGACTTCATCCTGAAG ATCAGCAGGGTGGAGGCCGAGGATGTGGGCGTGTACTATTGCATGCAG G CC CAGCAGAAC CCATCACCTTC G GC CAG GG CACAAGAC T GGAGAT C AAGAC CAC AAC C C C AGC AC CAAGGC CAC C T ACAC C T G CAC C AAC CATC GCATCCCAGCCACTGTCTCTGAGGCCTGAGGCATGTCGGCCAGCAGCA GGAGGAGCAGTGCACACCCGCGGCCTGGACTTTGCCTGCGATTCTAC ATCTGGGCACCACTGGCAGGAACATGTGGCGTGCTGCTGCTGAGCCTG GTCATCACCCTGTACTGCAAGCGCGGCCGGAAGAAGCTGCTGTATA TC TTCAAGCAGCCTTTTATGAGACCAGTGCAGACAACCCAGGAGGAGGAC GGCTGCTCCTGTAGGTTCCCTGAAGAGGAGGAGGGCGGCTGTGAGCTG AGAGTGAAGTTTTCTAGGAGCGCCGATGCACCAGCATACCAGCAGGGA CAGAATCAGCTGTATAACGAGCTGAATCTGGGCCGGAGAGAGGAGTAT GACGTGCTGGATAAGAGGAGGGGAAGGGACCCTGAGATGGGA GGCAAG CCCCGGAGAAAGAACCCTCAGGAGGGCCTGTACAATGAGCTGCAGAAG GACAAGATGGCCGAGGCCTATAGCGAGATCGGCATGAAGGGAGAGAGG CGCCGGGGCAAGGGACACGATGGCCTGTACCAGGGCCTGTCCACAGCC ACCAAGGACACCTATGATGCCCTGCATATGCAGGCACTGCCTCCAAGG TGA (SEQ ID NO: 243) CD8a signal peptide; 20E12 VH (Table 1, SEQ ID NO: 39); GS linker; 20E12 VL (Table 1, SEQ ID NO: 40); CD8a loop; CD8aTM; 4-IBB signaling domain; CD3zeta signaling domain 32G8 ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCACTGGCCCTGCTGCTG CACGCAGCAAGGCCAGAGGTGAACCTGGTGGAGTCCGGCGGCGGCCTG GTGAAGCCTGGCGGATCCCTGAGGCTGTCTTGCGAGGCAAGCGGCTTC ACCTTCAGCTACGCCTGGATGTCCTGGGTGCGCCAGGCCCCCGGCAAG GGACTGGAGTGGGTGGGCCGGATCAAGTCCATCACCGACGGAGG CGTG ATCGATTACGCAGCACCTGTGAGAAACAGGTGCACAATCTCCAGAGAC GAT T C TAG GAATAC C C T G T AT C T GGAGAT G CAC T C T C T GAAGACAGAG GACACCGCCGTGTACTATTGTACCACAATCCCTGGCAACGACGATTTC GATATGTGGGGCCAGGGCAGAATGGTGACCGTGAGCTCCGGCGGCGGC GGCTCTGGAGGAGGAGGAAG CGGAGGAGGAGGAAGCGGGGGCGGCGGC TCTGACATCGTGCTGACACAGTCCCACTGTCCCTGTCTGTGACCCCC GGCGAGCCTGCAAGCATCTCCTGTAGGTCTAGCCAGAGCCTGCTGTAC TCCAACGGCAAGAATTATCTGGATTGGTTTCTGCACAAGCCAGCCAG TCTCCCCAGCTGCTGATCTACCTGGGATCTAATAGGGCAAGCGGAGTG CCAGACCGGTTCTCTGGAA GCGGATCCGGCATCGACTTCATCCTGAAG ATCAGCCGCGTGGAGGCAGAGGACGTGGGCGTGTACTATTGCATGCAG G CC CAGCAGAAC CCATCACCTTC G GC CAG GG CACAAGAC T GGAGAT C AAGAC CACAAC C C CAGCAC CAAGGC CAC C TACAC C T G CAC CAAC CATC GCATCCCAGCCACTGTCTCTGAGGCCTGAGGCATGTAGGCCAGCAGCA GGAGGAGCAGTGCACACCAGAGGCCTGGACTTTGCCTGCGATATCTAC ATCTGGGCACCACTGGCAGGAACATGTGGCGTGCTGCTGCTGAGCCTG GTCATCACCCTGTACTGCAAGCGCGGCCGGAAGAAGCTGCTGTATATC TTC AAGCAGCCTTTTATGAGACCAGTGCAGACAACCCAGGAGGAGGAC GGCTGCTCCTGTAGGTTCCCTGAAGAGGAGGAGGGCGGCTGTGAGCTG AGAGTGAAGTTTTCTAGGAGCGCCGATGCACCAGCATACCAGCAGGGA CAGAATCAGCTGTATAACGAGCTGAATCTGGGCCGGAGAGAGGAGTAT GACGTGCTGGATAAGAGGAGGGGAAGGGATCCTGAGATGGGAGGCAAG CCCCGGAGAAAGAACCCTCAGGAGGGCCTGTACAATGAGCTGCAGAAG GACAAGATGGCCGAGGCCTATAGCGAGATCGGCATGAAGGGAGAGAGG CD8a signal peptide; 32G8 VH (Table 1, SEQ ID NO: 43) ; GS linker; 32G8 VL (Table 1, SEQ ID NO: 40) ; CD8a loop; CD8aTM; 4-IBB signaling domain; CD3zeta signaling domain

- 31 044329

CGCCGGGGCAAGGGACACGATGGCCTGTACCAGGGCCTGTCCACAGCC ACCAAGGACACCTATGATGCCCTGCATATGCAGGCACTGCCTCCAAGG TGA (SEQ ID NO: 244) 26B9 ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCACTGGCCCTGCTGCTG CACGCAGCAAGGCCAGAGGTGCAGCTGGTGGAGTCTTGGGGCGTGCTG GTGAAGCCTGGCGGATCTCTGAGGCTGAGCTGCGCAGCATCCGGCTTC ATCTTTAACAATGCCTGGATGTCCTGGGTGCGCCAGGCCCCCGGCAAG GGACTGGAGTGGATCGGCCGGATCAAGAGCAAGTCCGACGGAG GAACC ACAGATTACGCAGCACCTGTGAAGGACCGCTTCACAATCTCTCGGGAC GATAG CAAGGATAS S T G TAT S T GCAGAT GAAC G GC S T GAAGACAGAG GACACCGCCGTGTACTTCTGCACCACAGCCCCTGGCGGCCCTTTTGAT TATTGGGGCCAGGGCACACTGGTGACCGTGAGCTCCGGAGGAGGAGGA AGCGGCGGAGGAGGCAGCGGCG GCGGCGGCTCTGGCGGCGGCGGCAGC GACATCGTGCTGACACAGAGCCCTCTGTCCCTGCCAGTGACCCCGGC GAGCCTGCCTCTATCAGCTGTCGCTCTAGCCAGAGCCTGCTGCACCGG GACGGCTTCAATTACCTGGATTGGTTTCTGCAGAAGCCAGGCCAGTCC CCCCAGCTGCTGATCTATCTGGCCTCCTCTAGAGCCTCTGGCGTGCCA GACAGGTTCTCCGGCT CTGACAGCGGCACAGACTTCACCCTGAAGATC AGCAGAGTGGAGGCCGAGGATGTGGGCGTGTACTATTGCATGCAGGCC CTGCAGACACCCATCACCTTTCGGCCAGGGCACAAGACTGGAGATCAAG ACCACAACCCCAGCACCAAGGCCACCTACACCTGCACCAACCATCGCA TCCCAGCCACTGTCTCTGAGACCTGAGGCCTGTAGGCCAGCAGCAGGA GGAGCAGTGCACACCAGGG GCCTGGACTTTGCCTGCGATATCTACATC TGGGCACCTCTGGCAGGAACATGTGGCGTGCTGCTGCTGAGCCTGGTC ATCACCCTGTACTGCAAGAGAGGCAGGAAGAAGCTGCTGTATATCTTC AAGCAGCCTTTTATGAGACCAGTGCAGACAACCCAGGAGGAGGACGGC TGCTCCTGTAGGTTCCCTGAAGAGGAGGAGGGAGGATGTGAGCTGAGG GTGAAGTTTTTCCCGGTC TGCCGATGCACCAGCATACCAGCAGGGACAG AACCAGCTGTATAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGAC GTGCTGGATAAGAGGCGCGGCAGAGATCCAGAGATGGGCGGCAAGCCC CGGAGAAAGAACCCTCAGGAGGGCCTGTACAATGAGCTGCAGAAGGAC AAGATGGCCGAGGCCTATAGCGAGATCGGCATGAAGGGAGAGGCGC CGGGGCAAGGGACACGAT GGCCTGTACCAGGGCCTGTCCACAGCCACC AAGGACACCTATGATGCCCTGCATATGCAGGCACTGCCTCCAAGGTGA (SEQ ID NO: 245) CD8a signal peptide; 26B9 VH (Table 1, SEQ ID NO: 41) ; GS linker; 26B9 VL (Table 1, SEQ ID NO: 42); CD8a loop; CD8aTM; 4-IBB signaling domain; CD3zeta signaling domain 30D8 ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCACTGGCCCTGCTGCTG CACGCAGCAAGGCCTGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTG GTGAAGCCTGGCGGATCCCTGAGGCTGTCTTGCGAGGCAAGCGGCTTC ACCTTTAGCGACGCATGGATGTCCTGGGTGCGCCAGGCCCCTGGCAAG GGACTGGAGTGGGTGGGACGGATCAAGAGCAAGACAGACGGCGG CACC ACAGATTACGTGGTGCCACTGAACGGCCGCTTCATCATCTCCCGCGAC GAT T C T C G GAATAC C C T G T AT C T GC AGC T GAACAAT C T GAAGACAGAG GATACCGCCGTGTACTATTGCACCACAGTGCCAGGCTCCTACGGCTAT TGGGGCCAGGGCACACTGGTGACCGTGAGCTCCGGCGGCGGCGGCTCT GGAGGAGGAGGAAGCGGAGGAGGA GGAAGCGGGGGCGGCGGCTCTGAC ATCGTGATGACACAGTCTCCACTGAGCCTGCCAGTGACCCCTGGCGAG CCAGCCTCCATCTCTTGTCGCTCTAGCCAGAGCCTGCTGCACAACAAG CGGAACAATTACCTGGATTGGTTTCTGCAGAAGCCTGGCCAGTCCCCT CAGCTGCTGATCTATCTGGCCAGCAATAGAGCCTCCGGAGTGCCAGAC AGGTTCTCTGGAGGAGGAAGCGGA ACAGACTTCACCCTGAAGATCAGC AGAGTGGAGGCCGAGGACGTGGGCGTGTACTATTGCATGCAGGCCCAG CAGACACCTATCACCTTCGGCCAGGGCACAAGACTGGAGATCAAGACC ACAACCCCAGCACCAAGGCCACCTACACCTGCACCAACCATCGCCTCC CAGCCTCTGTCTCTGAGACCAGAGGCATGTAGGCCAGCAGCAGGAGGA GCAGTGCACACCAGGGGCCTGGACT TTGCCTGCGATATCTACATCTGG GCACCTCTGGCAGGAACATGTGGCGTGCTGCTGCTGAGCCTGGTCATC ACCCTGTACTGCAAGAGAGGCAGGAAGAAGCTGCTGTATATCTTCAAG CAGCCCTTCATGAGACCCGTGCAGACAACCCAGGAGGAGGACGGCTGC TCTTGTAGGTTCCCAGAAGAGGAGGAGGGAGGATGTGAGCTGAGGGTG AAGTTTAGCCGGTCCGCC GATGCACCAGCATACCAGCAGGGACAGAAC CAGCTGTATAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTG CTGGATAAGAGGAGGGGAAGGGATCCAGAGATGGGAGGCAAGCCTCGG AGAAAGAACCCACAGGAGGGCCTGTACAATGAGCTGCAGAAGGACAAG ATGGCCGAGGCCTATTCTGAGATCGGCATGAAGGGAGAGAGGCGCCGG GGCAAGGGACACGATGG CCTGTACCAGGGGCCTGAGCACAGCCACCAAG GACACCTATGATGCCCTGCATATGCAGGCACTGCCTCCAAGGTGA CD8a signal peptide; 30D8 VH (Table 1, SEQ ID NO: 37); GS linker; 30D8 VL (Table 1, SEQ ID NO: 38); CD8a loop; CD8aTM; 4-IBB signaling domain; CD3zeta signaling domain

- 32 044329

(SEQ ID NO: 246) C6 ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCTCTGGCCCTGCTGCTG CACGCAGCAAGGCCACAGGTGCAGCTGGTGCAGTCCGGAGCAGAGGTG AAGAAGCCTGGCAGCTCCGTGAAGGTGAGCTGCAAGGCCTCCGGCGAC ACATTCTCTAGCAACGCAATCAGCTGGGTGCGCCAGGCCCCTGGCCAG GGACTGGAGTGGATGGGCGTGATCATCCCTATCTTCGGCACCGC CGAC TATGCCCAGAAGTTTCAGGGCCGGGTGACAATCACCGCCGATGAGTCT ACAAGCACCGCCTACATGGAGCTGTCCTCTCTGAGATCCGAGGACACA GCCGTGTACTATTGTGCCAGGCACACCTATCACGAGTACGCAGGAGGA TACTATGGAGGAGCAATGGATCCTTGGGGACAGGGCACACTGGTGACC GTGAGCTCCGGCGGCGGCGGCTCTGGAGGAGGAGGAAGCGGAGG agga CTCT GCCAGCCTGGCAATCTCCGGCCTGAGGTCTGAGGACGAGGCCGATTAC TATTGCGCCGCCTGGGACGATAACCTGAGCGGCTGGGTGTTTGGCACA G GCAC CAAG C TGACAGTGCTGACCA СAAC CCCTGCAC CAAGAC SASSA ACACCAGCACSTACCATCGCAAGCCAGCCASTGTGTCCCTGAGACCGAG GCCTGTAGGCCTGCAGCAGGAGGAGCAGTG CACACCAGGGGCCTGGAC TTTGCCTGCGATATCTATATCTGGGCACCACTGGCAGGAACATGTGGC GTGCTGCTGCTGAGCCTGGTCATCACCCTGTATTGCAAGAGAGGCAGG AAGAAGCTGCTGTACATCTTCAAGCAGCCCTTTATGCGCCCTGTGCAG ACAACCCAGGAGGAGGACGGCTGCAGCTGTCGGTTCCCAGAAGAGGAG GAGGGAGGATGTGAGCTGAGGGTGAAG TTTTCCCGGTCTGCCGATGCA CCAGCATATCAGCAGGGACAGAATCAGCTGTACAACGAGCTGAATCTG GGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGGGAC CCTGAGATGGGAGGCAAGCCACGGAGAAAAACCCCCAGGAGGGCCTGT ATAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACTCTGAGATCG GCATGAAGGGAGAGGCGCCGGGGCAA GGGACACGATGGCCTGTATC AGGGCCTGAGCACAGCCACCAAGGACACCTACGATGCCCTGCATATGC AGGCACTGCCTCCAAGGTGA (SEQ ID NO: 247) CD8a signal peptide; C6 VH (Table 1, SEQ ID NO: 48); GS linker; C6 VL (Table 1, SEQ ID NO: 49) ; CD8a loop; CD8aTM; 4-IBB signaling domain; CD3zeta signaling domain

Down-regulation or mutation of target antigens is commonly observed in cancer cells, creating cell escape variants with loss of antigen. Thus, to counteract tumor escape and impart greater target specificity to the immune cell, EGFRvIII specific CARs may contain one or more additional extracellular ligand-binding domains to simultaneously bind various elements in the target, thereby enhancing immune cell activation and function. In one embodiment, the extracellular ligand binding domains may be located sequentially on the same transmembrane polypeptide, and may optionally be separated by a linker. In some embodiments, these different extracellular ligand binding domains can be placed on different transmembrane polypeptides that make up the CAR. In some embodiments, the invention relates to a population of CARs, where each CAR contains a different extracellular ligand binding domain. In particular, the invention relates to methods for engineering immune cells, comprising providing an immune cell and expressing a population of CARs on the cell surface, where each CAR contains a different extracellular ligand binding domain. In another preferred embodiment, the invention relates to a method for engineering an immune cell, comprising providing the immune cell with and introducing into the cell polynucleotides encoding polypeptides consisting of a population of CARs, wherein each CAR contains different extracellular ligand binding domains. By CAR population, we mean at least two, three, four, five, six or more CARs, each of which contains different extracellular ligand binding domains. The different extracellular ligand binding domains of the invention can preferentially simultaneously bind different elements in a target, thereby enhancing immune cell activation and function. The invention also relates to an isolated immune cell that contains a population of CARs, each of which contains different extracellular ligand binding domains.

In another aspect, the invention provides polynucleotides encoding any of the CARs and polypeptides described in the present invention. Polynucleotides can be produced and expressed using procedures known in the art.

In another aspect, the invention provides compositions (such as pharmaceutical compositions) containing any of the cells of the invention. In some embodiments, the composition comprises a cell containing a polynucleotide encoding any of the CARs described in the present invention.

Expression vectors and administration of polynucleotide compositions are further described in the present invention.

In another aspect, the invention provides a method for producing any of the polynucleotides described in the present invention.

Polynucleotides complementary to any such sequences are also covered.

- 33 044329 invention. Polynucleotides can be single-stranded (coding or antisense) or double-stranded, and can be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules include hnRNA molecules, which contain introns and correspond to a DNA molecule in a one-to-one ratio, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may, but not necessarily, be present within the polynucleotide of the invention, and the polynucleotide may optionally be associated with other molecules and/or support materials.

Polynucleotides may contain a native sequence (ie, an endogenous sequence that encodes an antibody or part thereof) or may contain a variant of such a sequence. Polynucleotide variants contain one or more substitutions, additions, deletions and/or insertions such that the immunoreactivity of the encoded polypeptide is not reduced relative to the native immunoreactive molecule. The effect on the immunoreactivity of the encoded polypeptide can generally be assessed as described in the present invention. The variants preferably exhibit at least about 70% identity, more preferably at least about 80% identity, even more preferably at least about 90% identity, and most preferably at least about 95% identity to the polynucleotide sequence that encodes a native antibody or part thereof.

Two polynucleotide or polypeptide sequences are designated as identical if the nucleotide or amino acid sequence in the two sequences is the same when aligned for maximum consistency as described below. Comparisons between two sequences are typically made by comparing the sequences through a comparison window to identify and compare local regions of sequence similarity. A comparison window, as used in the present invention, refers to a segment of at least about 20 adjacent positions, typically 30 to about 75, or 40 to about 50, in which the sequence can be compared to a reference sequence with the same number of adjacent positions after optimal alignment of the two sequences.

Optimal sequence alignment can be performed using the Megalign program in the Lasergene bioinformatics software suite (DNASTAR, Inc., Madison, WI) using default parameters. This program implements several alignment schemes, described in the following references: Dayhoff, M.O., 1978, A model of evolutionary change in proteins - Matrices for detecting distant relationships. In Dayhoff, M.O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC vol. 5, suppl. 3, cc. 345-358; Hein J., 1990, Unified Approach to Alignment and Phylogenes cc. 626-645 Methods in Enzymology volume 183, Academic Press, Inc., San Diego, CA; Higgins, D. G. and Sharp, P.M., 1989, CABIOS 5:151-153; Myers, E.W. and Muller W., 1988, CABIOS 4:11-17; Robinson, E.D., 1971, Comb. Theor. 11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4:406-425; Sneath, P.H.A. and Sokal, R.R., 1973, Numerical Taxonomy the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, W.J. and Lipman, D.J., 1983, Proc. Natl. Acad. Sci. USA 80:726-730.

Preferably, the percentage of sequence identity is determined by comparing two optimally aligned sequences through a comparison window of at least 20 positions, where a portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) of 20 percent or less, typically 5 to 15 percent, or 10 to 12%, compared to reference sequences (which contain no additions or deletions) for optimal alignment of two sequences. The percentage is calculated by determining the number of positions at which identical nucleic acid bases or amino acid residues occur in both sequences, obtaining the number of matching positions, and dividing the number of matching positions by the total number of positions in the reference sequence (i.e., window size) and multiplying the result by 100 to obtain percent sequence identity.

Variants may also, or alternatively, be substantially homologous to the native gene, or a portion or complement thereof. Such polynucleotide variants are capable of hybridizing under moderately stringent conditions to a naturally occurring DNA sequence encoding the native antibody (or complementary sequence).

Suitable moderately stringent conditions include prewash in a solution of 5X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridization at 50-65°C, 5 X SSC, overnight; followed by washing twice at 65°C for 20 min with each of 2X, 0.5X, and 0.2X SSC containing 0.1% SDS.

As used in the present invention, high stringency conditions or high stringency conditions are those: (1) apply low ionic strength and high temperature for washing, for example, 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50°C; (2) hybridization uses a denaturing agent such as formamide, e.g. 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolido

- 34 044329 nom/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or (3) use 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated Salmon milk DNA (50 µg/ml), 0.1% SDS, and 10% dextran sulfate at 42°C, with washes and at 42°C in 0.2 x SSC (sodium chloride/sodium citrate) and 50% formamide at 55°C, followed by a high stringency wash containing 0.1 x SSC containing EDTA at 55°C. One skilled in the art will understand how to adjust temperature, ionic strength, etc., to accommodate factors such as probe length, etc.

It will be clear to one skilled in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide as described in the present invention. Some of these polynucleotides have minimal homology to the nucleotide sequence of any native gene. However, polynucleotides that differ due to differences in the codons used are specifically covered by the invention. In addition, alleles of genes containing polynucleotide sequences provided in the present invention fall within the scope of the invention. Alleles are endogenous genes that are altered by one or more mutations, such as deletions, additions and/or nucleotide substitutions. The resulting mRNA and protein may, but need not, have altered structure or function. Alleles can be identified using standard techniques (such as hybridization, amplification and/or comparison of sequence databases).

The polynucleotides of the present invention can be produced using chemical syntheses, recombinant methods or PCR. Methods for the chemical synthesis of polynucleotides are well known in the art and need not be described in more detail in the present invention. One skilled in the art can use the sequences provided in the present invention and a commercial DNA synthesizer to obtain the desired DNA sequence.

To produce polynucleotides using recombinant methods, a polynucleotide containing the desired sequence can be inserted into a suitable vector, and the vector, in turn, can be introduced into a suitable host cell for replication and amplification, as discussed in more detail in the present invention. Polynucleotides can be inserted into host cells using any methods known in the art. Cells are transformed by introducing an exogenous polynucleotide through direct uptake, endocytosis, transfection, Fcrossing, or electroporation. Once introduced, the exogenous polynucleotide may be maintained in cells as a non-integrated vector (such as a plasmid) or integrated into the genome of the host cell. Thus, the amplified polynucleotide can be isolated from the host cell using methods well known in the art. See, for example, Sambrook et al., 1989.

Alternatively, PCR provides the ability to reproduce DNA sequences. PCR technology is well known in the art and is described in US Patent Nos. 4,683,195, 4,800,159, 4,754,065 and 4,683,202, as well as PCR: The Polymerase Chain Reaction, Mullis et al. eds., Birkauswer Press, Boston, 1994.

RNA can be produced by using isolated DNA in a suitable vector and inserting it into a suitable host cell. Once the cell has replicated and the DNA is transcribed into RNA, the RNA can then be isolated using methods well known to one of ordinary skill in the art, as described in Sambrook et al., 1989, supra, for example.

Suitable cloning vectors can be constructed according to standard techniques, or can be selected from the large number of cloning vectors available in the art. While the cloning vector chosen may vary according to the host cell in which it will be used, suitable cloning vectors generally have the ability to self-replicate, can capture single mice for a preferred restriction endonuclease, and/or can carry genes for a marker that can used for selection of clones containing the vector. Suitable examples include plasmids and bacterial viruses, e.g. pUC18, pUC19, Bluescript (e.g. pBS SK+) and derivatives thereof, mpl8, mpl9, pBR322, pMB9, ColE1, pCR1, RP4, DNA phages, and shuttle vectors such as pSA3 and pAT28. These and many other cloning vectors are available from commercial suppliers such as BioRad, Strategene, and Invitrogen.

Expression vectors are typically replicable polynucleotide constructs that contain a polynucleotide in accordance with the invention. It is understood that the expression vector must be replicable in host cells either as episomes or as an integral part of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, cosmids, and the expression vector(s) described in PCT Publication No. WO 87/04462. Vector components may generally include, but are not limited to, one or more of the following structures: a signal sequence; replication origin point; one or

- 35 044329 several marker genes; suitable transcriptional control elements (such as promoters, enhancers and terminator). Expression (i.e., translation) also typically requires one or more translational control elements, such as ribosome binding sites, translation initiation sites, and stop codons.

Vectors containing polynucleotides of interest can be introduced into a host cell using any of a variety of suitable methods, including electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microparticle bombardment; lipofection; and infection (eg, if the vector is an infectious agent such as vaccinia virus). The choice of vectors or polynucleotides introduced will often depend on the characteristics of the host cell.

The polynucleotide encoding the EGFRvIII specific CARs disclosed in the present invention may exist as an expression cassette or expression vectors (e.g., a plasmid for introduction into a bacterial host cell, or a viral vector, such as a baculovirus vector for transfecting an insect host cell, or a plasmid or a virus vector, such as a lentivirus, for transfecting a mammalian host cell). In some embodiments, the polynucleotide or vector may include a nucleotide sequence encoding ribosomal hopping sequences, such as, for example, but not limited to, a sequence encoding a 2A peptide. 2A peptides, which are identified in the Afthovirus subgroup of picornaviruses, cause the ribosome to jump from one codon to the next without forming a peptide bond between the two amino acids encoded by the codons (see (Donnelly and Elliott 2001; Atkins, Wills et al. 2007; Doronina, Wu et al. . 2008)). A codon refers to three nucleotides on mRNA (or on the sense strand of a DNA molecule) that are translated by the ribosome into one amino acid residue. Thus, two polypeptides can be synthesized from a single, contiguous open reading frame within an mRNA if the polypeptides are separated by a 2A oligopeptide sequence that is in frame. Such ribosome hopping mechanisms are well known in the art, and the use of multiple vectors to express multiple proteins encoded by a single messenger RNA is known.

To target transmembrane polypeptides to the secretory pathway of a host cell, in some embodiments, a secretory signal sequence (also known as a signal peptide, leader sequence, prepro sequence, or presequence) is provided in the polynucleotide sequence or vector sequence. Secretory signal sequences are operably linked to a transmembrane nucleotide sequence, that is, the two sequences are joined in the correct reading frame and oriented to target the newly synthesized polypeptide into the secretory pathway of the host cell. Secretory signal sequences are typically located 5' to the nucleotide sequence encoding the polypeptide of interest, although certain secretory signal sequences may be located anywhere in the nucleotide sequence of interest (see, for example, Welch et al., US Pat. No. 5,037,743; Holland et al., US Patent No. 5,143,830). In some embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 206 or 214. One skilled in the art will recognize that, given the degeneracy of the genetic code, significant sequence variations are possible for these polynucleotide molecules. In some embodiments, the nucleotide sequences of the invention are codon-optimized for expression in mammalian cells, preferably for expression in human cells. Codon optimization refers to the exchange in a sequence of interest of codons that are typically rare in highly expressed genes of a given species for codons that are typically found frequently in highly expressed genes of such species, such codons encoding identical amino acids as the codons which have been replaced.

Methods for constructing an immune cell

The present invention provides methods for preparing immune cells for use in immunotherapy. In some embodiments, the methods include introducing a CAR in accordance with the invention into immune cells, and spreading the cells. In some embodiments, the invention relates to a method of constructing an immune cell, comprising: providing the cell with and expressing on the surface of the cell at least one CAR as described above. Methods for engineering immune cells are described, for example, in PCT Published Patent Application Nos. WO/2014/039523, WO/2014/184741, WO/2014/191128, WO/2014/184744, and WO/2014/184143, each of which is included incorporated into this application in its entirety by reference. In some embodiments, the method includes: transforming a cell with at least one polynucleotide encoding a CAR as described above, and expressing the polynucleotides in the cells.

In some embodiments, the polynucleotides are present in lentiviral vectors for stable expression in cells.

- 36 044329

In some embodiments, the method may further include the step of genetically modifying the cell by inactivating at least one gene expressing, for example, but not limited to, a TCR component, a target of an immunosuppressive agent, an HLA gene, and/or an immune checkpoint protein, such as, for example, PDCD1 or CTLA-4. By inactivating a gene, it is assumed that the gene of interest is not expressed in the form of a functional protein. In some embodiments, the gene to be inactivated is selected from the group including, for example, but not limited to, TCRa, TCRe, dCK, CD52, GR, PD-1, and CTLA-4. In some embodiments, the method includes inactivating one or more genes by introducing into cells a cleavage endonuclease capable of selectively inactivating a gene by selectively cleaving DNA. In some embodiments, the endonuclease that cleaves at more sparse sites may be, for example, a transcription activator-like effector nuclease (TALE nuclease) or a Cas9 endonuclease.

In some embodiments, an additional catalytic domain is used with an endonuclease that cleaves at more sparse sites to enhance its ability to inactivate target genes. For example, the additional catalytic domain may be a DNA end-processing enzyme.

Non-limiting examples of DNA end-processing enzymes include 5-3' exonucleases, 3-5' exonucleases, 5-3' alkaline exonucleases, 5' displaceable endonucleases, helicases, phosphatases, hydrolases and template-independent DNA polymerases. Non-limiting examples of such a catalytic domain include a protein domain or a catalytically active derivative of a protein domain selected from the group consisting of hExoI (EXO1_HUMAN), yeast ExoI (EXO1_YEAST), E. coli ExoI, human TREX2, mouse TREX1, human TREX1, bovine TREX1, rat TREX1 , TdT (terminal deoxynucleotidyl transferase) human DNA2, yeast DNA2 (DNA2_YEAST). In some embodiments, the additional catalytic domain may have 3'-5' exonuclease activity, and in some embodiments, the additional catalytic domain is a TREX, more preferably a TREX2 catalytic domain (WO 2012/058458). In some embodiments, said catalytic domain is encoded by a single chain TREX polypeptide. The additional catalytic domain may be fused to a nuclease fusion protein or a chimeric protein. In some embodiments, the additional catalytic domain is fused using, for example, a peptide linker.

In some embodiments, the method further includes the step of introducing into the cells an exogenous nucleic acid comprising at least a sequence homologous to a portion of the target nucleotide sequence such that homologous recombination occurs between the target nucleic acid sequence and the exogenous nucleic acid. In some embodiments, said exogenous nucleic acid contains first and second portions that are homologous to the 5' and 3' region of the target nucleotide sequence, respectively. The exogenous nucleic acid may also contain a third portion located between the first and second portions that does not contain homology between the 5' and 3' regions of the target nucleotide sequence. Following cleavage of the target nucleotide sequence, a homologous recombination event is stimulated between the target nucleotide sequence and the exogenous nucleic acid. In some embodiments, homologous sequences of at least about 50 bp, greater than about 100 bp, or greater than about 200 bp can be used within the donor matrix. The exogenous nucleic acid may have, for example, but not limited to, from about 200 bp to about 6000 bp, more preferably from about 1000 bp to about 2000 bp. The shared nucleic acid homologies are located in regions flanking the upstream and downstream of the break site, and the introduced nucleotide sequence is located between the two arms.

In some embodiments, the nucleic acid sequentially comprises a first region of homology to sequences upstream of said cleavage; a target gene inactivation sequence selected from the group consisting of TCRa, TCRe, CD52, glucocorticoid receptor (GR), deoxycytidine kinase (dCK), and an immune checkpoint protein such as, for example, programmed death-1 (PD-1); and a second region of homology to sequences along the course of cleavage. The polynucleotide introduction step can be simultaneous, before or after introduction or expression of an endonuclease that performs cleavage at more sparsely located sites. Depending on the location of the target nucleotide sequence where the breakage event occurs, such exogenous nucleic acid can be used to silence a gene, for example, if the exogenous nucleic acid is located within the open reading frame of the gene, or to introduce new sequences or genes of interest. Insertion of sequences by using such exogenous nucleino

- 37 044329 voic acid, can be used to modify a target existing gene, by correcting or replacing a gene (replacing alleles as a non-limiting example), or to up- or down-regulate the expression of a target gene (exchanging promoters as a non-limiting example), correcting or replacing a target gene. In some embodiments, inactivation of genes selected from the group consisting of TCRa, TCRe, CD52, GR, dCK, and immune checkpoint proteins can be accomplished at a precise location in the genome targeted by a specific TALE nuclease, wherein the specific TALE nuclease catalyzes the cleavage and wherein the exogenous nucleic acid sequentially comprises at least a region of homology and a sequence for inactivating one target gene selected from the group consisting of TCRa, TCRe, CD52, GR, dCK, immune checkpoint proteins, which is integrated by homologous recombination. In some embodiments, multiple genes can be, sequentially or simultaneously, inactivated by using multiple TALE nucleases, respectively, and specifically targeting one specific gene and multiple specific polynucleotides to inactivate a specific gene.

In some embodiments, the method includes inactivating one or more additional genes selected from the group consisting of TCRa, TCRe, CD52, GR, dCK, and immune checkpoint proteins. In some embodiments, gene inactivation can be accomplished by introducing into the cells at least one lower-site cleavage endonuclease, such as a lower-site cleavage endonuclease that specifically catalyzes cleavage at a target sequence in the cellular genome; and optionally, introducing into the cells an exogenous nucleic acid sequentially containing a first region of homology to upstream sequences, a sequence that is inserted into the genome of the cell, and a second region of homology to upstream sequences; wherein the introduced exogenous nucleic acid inactivates the gene and integrates at least one exogenous polynucleotide sequence encoding at least one recombinant protein of interest. In some embodiments, the exogenous polynucleotide sequence is integrated within a gene encoding a protein selected from the group consisting of TCRa, TCRe, CD52, GR, dCK, and immune checkpoint protein.

In another aspect, the step of genetically modifying cells may include: modifying T cells by inactivating at least one gene expressing a target of an immunosuppressive agent, and; cell proliferation, optionally in the presence of an immunosuppressive agent. An immunosuppressive agent is an agent that suppresses immune function through one of several mechanisms of action. An immunosuppressive agent may reduce the propagation and/or voraciousness of the immune response. Non-limiting examples of immunosuppressive agents include calcineurin inhibitors, target of rapamycin, interleukin-2 a-chain blockers, inosine monophosphate dehydrogenase inhibitors, dihydrofolic acid reductase inhibitors, corticosteroids and immunosuppressive antimetabolites. Some cytotoxic immunosuppressants act by inhibiting DNA synthesis. Others may act by activating T cells or by activating helper cells. The methods of the invention provide the ability to impart immunosuppressive resistance to T cells for immunotherapy by inactivating the immunosuppressive agent in the T cells. By way of non-limiting examples, targets for an immunosuppressive agent may be a receptor for an immunosuppressive agent, such as, for example, but not limited to, CD52, the glucocorticoid receptor (GR), members of the FKBP gene family, and members of the cyclophilin gene family.

In some embodiments, the genetic modification of the method involves the expression, in the engineered cells provided, of a single lower-site cleavage endonuclease, such as a higher-frequency cleavage endonuclease that specifically catalyzes cleavage at one target gene, thus inactivating the target gene. In some embodiments, a method of constructing cells includes at least one of the following steps: providing a T cell, such as from a cell culture or from a blood sample; selection of a gene in a T cell expressing a target for an immunosuppressive agent; introduction into T cells of an endonuclease that cleaves at more sparsely located sites, capable of being selectively inactivated by DNA cleavage, preferably by double-strand break of the gene encoding the target of the immunosuppressive agent, and cell proliferation, optionally in the presence of the immunosuppressive agent.

In some embodiments, the method includes: providing a T cell, such as from a cell culture or from a blood sample; selecting a gene in a T cell, where the gene expresses a target for an immunosuppressive agent; transformation of a T cell with a nucleic acid encoding an endonuclease that cleaves at more sparsely located sites, capable of being selectively inactivated by DNA cleavage, preferably by double-strand break in the gene encoding the target of an immunosuppressive agent, and the expression of endonucleases that cleave at more sparse sites sparsely located sites, in T cells; and cell proliferation, optionally in the presence of an immunosuppressive agent.

In some embodiments, the endonuclease that cleaves at more sparse sites specifically targets CD52 or GR. In some embodiments, the gene selected for inactivation encodes CD52, and the immunosuppressive therapy includes a humanized antibody targeting the CD52 antigen. In some embodiments, the gene selected for inactivation encodes GR, and the immunosuppressive therapy includes a corticosteroid such as dexamethasone. In some embodiments, the gene selected for inactivation is a member of the FKBP gene family or a variant thereof and the immunosuppressive therapy includes FK506, also known as Tacrolimus or fujimycin. In some embodiments, the FKBP gene family member is FKBP12 or a variant thereof. In some embodiments, the gene selected for inactivation is a member of the cyclophilin gene family or a variant thereof, and the immunosuppressive therapy includes cyclosporine.

In some embodiments, the endonuclease that cleaves at more sparse sites may be, for example, a meganuclease, a zinc finger nuclease, or a TALE nuclease. In some embodiments, the endonuclease that cleaves at more sparsely located sites is a TALE nuclease.

Also provided by the present invention are methods for constructing T cells suitable for immunotherapy, wherein the methods include: genetically modifying the T cells by inactivating at least one immune checkpoint protein. In some embodiments, the immune checkpoint protein is, for example, PD-1 and/or CTLA-4. In some embodiments, methods for genetically modifying a cell include: modifying T cells by inactivating at least one immune checkpoint protein; and cell proliferation. Immune checkpoint proteins include, but are not limited to, programmed death 1 (PD-1, also known as PDCD1 or CD279, accession number: NM_005018), Cytotoxic T lymphocyte antigen 4 (CTLA-4, also known as CD152, accession number GenBank accession AF414120.1), LAG3 (also known as CD223, accession number: NM_002286.5), Tim3 (also known as HAVCR2, GenBank accession number: JX049979.1), BTLA (also known as CD272, accession number: NM_181780. 3), BY55 (also known as CD160, GenBank accession number: CR541888.1), TIGIT (also known as VSTM3, accession number: NM_173799), B7H5 (also known as C10orf54, homologue of the mouse vista gene, accession number: NM_022153.1 ), LAIR1 (also known as CD305, GenBank accession number: CR542051.1), SIGLEC10 (GeneBank accession number: AY358337.1), 2B4 (also known as CD244, accession number: NM_001166664.1), which directly inhibit immune cells. For example, CTLA-4 is a cell surface protein expressed on certain CD4 and CD8 T cells; when, when captured by its ligands (B7-1 and B7-2) on antigen-presenting cells, the activation and effector function of T cells is inhibited.

In some embodiments, said method of constructing cells includes at least one of the following steps: providing a T cell, such as from a cell culture or from a blood sample; introduction into T cells of an endonuclease that cleaves at more sparsely located sites and is capable of being selectively inactivated by DNA cleavage, preferably by a double-strand break in one gene encoding an immune checkpoint protein; and cell proliferation. In some embodiments, the method includes: providing a T Cell, such as from a cell culture or from a blood sample; cleavage at more sparsely located sites capable of being selectively inactivated by DNA cleavage, preferably by double-strand breaks in the gene encoding the immune checkpoint protein; expression of endonucleases that perform cleavage at more sparsely located sites in T cells; cell proliferation. In some embodiments, the endonuclease that cleaves at more sparse sites specifically targets a gene selected from the group consisting of: PD-1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, 2B4 , TCRa, and TCRe. In some embodiments, the endonuclease that cleaves at more sparse sites may be a meganuclease, zinc finger nuclease, or TALEnuclease. In some embodiments, the endonuclease that cleaves at more sparsely located sites is a TALE nuclease.

In some embodiments, the present invention may be particularly useful for allogeneic immunotherapy. In such embodiments, the cells may be modified by a method comprising: inactivating at least one gene encoding a T cell receptor (TCR) component in T cells; and proliferation of T cells. In some embodiments, the genetic modification of the method relies on the expression, in the cells provided for construction, of a single endonuclease that cleaves at more sparse sites, such that the endonuclease that cleaves at more sparse sites specifically catalyzes cleavage at one target site. gene, thus inactivating the target

- 39 044329 gen. In some embodiments, the method of constructing cells includes at least one of the following steps: providing a T cell, such as from a cell culture or from a blood sample; introduction into T cells of an endonuclease that cleaves at more sparsely located sites, capable of being selectively inactivated by DNA cleavage, preferably by a double-strand break in at least one gene encoding a component of the T cell receptor (TCR), and cell proliferation.

In some embodiments, the method includes: providing a T cell, such as from a cell culture or from a blood sample; transfecting said T cell with a nucleic acid encoding a cleavage endonuclease at more sparse sites, capable of being selectively inactivated by DNA cleavage, preferably by double-strand break of at least one gene encoding a T cell receptor (TCR) component; expression of endonucleases that perform cleavage at more sparsely located sites in T cells; sorting transfected T cells that do not express TCR on their cell surface; and cell proliferation.

In some embodiments, the endonuclease that cleaves at more sparse sites may be a meganuclease, a zinc finger nuclease, or a TALE nuclease. In some embodiments, the endonuclease that cleaves at more sparsely located sites is a TALE nuclease. In some embodiments, TALE nucleases recognize and cleave the coding sequence for TCRa or TCRe. In some embodiments, the TALE nuclease comprises a polypeptide sequence selected from the amino acid sequence set forth in SEQ ID NO: 218, 219, 220, 221, 222, 223, 224, or 225.

TALE nuclease polypeptide sequences.

Repeat TRACT01-L

LTPQQVVAIASNGGGKQALE TVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALE TVQRL LPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGK QALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVV AIASHDGGKQ ALE TVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALE TVQALLPVLCQA HGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQ ALLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIG GKQALE TVQALLPVLCQAHGLTPQQ VVAIASNGGGKQALE TVQRLLPVLCQAHGLTPEQ VVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLC QAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALE (SEQ ID NO: 218)

Repeat TRAC T01-R

LTPEQVVAIASHDGGKQALE TVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALE TVQRL LPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGK QALETVQALLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVV AIASHDGGKQA LETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQA HGLTPQQVVAIASNNGGKQALE TVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALE TVQ RLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIG GKQALETVQALLPVLCQAHGLTPEQ VVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQ VVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLC QAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALE (SEQ ID NO: 219)

Repeat TRBC T01-L

LTPQQVVAIASNNGGKQALE TVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALE TVQRL LPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGK

- 40 044329

QALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVV AIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQA HGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGGKQALETVQ RLLPVLCQAHG LTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDG GKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQ VVAIASNGGGKQALE TVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALE TVQRLLPVLC QAHGLTPEQVVAIASNIGGKQALE TVQALLPVLCQAHGLTPQQVVAIASNGGGRPALE (SEQ ID NO: 220)

Repeat TRBC T01-R

HGLTPEQ VVAIASHDGGKQALE TVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALE TVQ RLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGG GKQALE TVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALE TVQRLLPVLCQAHGLTPQQ VVAIASNNGGKQALE TVQRL LPVLCQAHGLTPQQVVAIASNNGGKQALE TVQRLLPVLC QAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALE T VQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASN GGGRPALE (SEQ ID NO: 221)

Repeat TRBCT02-L

LTPEQVVAIASHDGGKQALE TVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALE TVQRL LPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGK QALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVV AIASHDGGKQA LETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQA HGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQ ALLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNG GKQALE TVQRLLPVLCQAHGLTPQ QVVAIASNGGGKQALE TVQRLLPVLCQAHGLTPEQ VVAIASHDGGKQALE TVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALE TVQRLLPVLC QAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALE (SEQ ID NO: 222)

Repeat TRBC T02-R

LTPQQVVAIASNNGGKQALE TVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALE TVQRL LPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGK QALETVQALLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVV AIASNIGGKQA LETVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQA HGLTPEQVVAIASHDGGKQALE TVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALE TVQ RLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGG GKQALE TVQRLLPVLCQAHGLTPQQ VVAIASNNGGKQALE TVQRLLPVLCQAHGLTPEQ VVAIASHDGGKQALE TVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALE TVQRLLPVLC QAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALE (SEQ ID NO: 223)

Repeat CD52 T02-L

LTPQQVVAIASNGGGKQALE TVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALE TVQRL LPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGK QALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVV AIASHDGGKQ ALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQA HGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQ RLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDG GKQALETVQRLLPVLCQAHGLT PEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQ VVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLC QAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGRPALE (SEQ ID NO: 224)

Repeat CD52 T02-R

LTPQQVVAIASNGGGKQALE TVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALE TVQRL LPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGK QALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVV AIASNGGGKQ ALE TVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALE TVQRLLPVLCQA HGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQ RLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGG GKQALE TVQRLLPVLCQAHGLTPQ QVVAIASNNGGKQALE TVQRLLPVLCQAHGLTPQQ VVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLC QAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALE (SEQ ID NO: 225)

In another aspect, another stage of genetic modification of the cell may be a

- 41 044329 means of spreading TCRa deficient T cells, including the introduction into T cells of pTa (also known as preTCRa) or a functional variant thereof and the spreading of the cells, optionally by stimulation of the CD3 complex. In some embodiments, the method includes: a) transforming cells with a nucleic acid encoding at least a fragment of pTa to maintain CD3 surface expression; b) expressing said pTa in cells; and c) cell proliferation, optionally by stimulation of the CD3 complex.

Also provided are methods for producing T cells for immunotherapy, including the steps of the method provided in the present invention for distributing the T cells. In some embodiments, the pTa polynucleotide sequence may be introduced randomly or by homologous recombination. In some embodiments, the insertion may be associated with inactivation of the TCRa gene.

Various functional variants of pTa can be used. A functional peptide variant refers to a molecule substantially similar to either the entire peptide or a fragment thereof. A pTa fragment or functional variant thereof refers to any subset of the molecule that is a shorter peptide than the full-length pTa. In some embodiments, the pTa or functional variants may be, for example, a full-length pTa or a C-terminal truncated pTa version. In the C-terminal truncated pTa, one or more residues are missing from the C-terminal region. By way of non-limiting examples, the C-terminal truncated pTa version lacks 18, 48, 62, 78, 92, 110, or 114 residues from the C-terminus of the protein. Variants of the amino acid sequence of a peptide can be prepared by mutations in the DNA that encodes the peptide. Such functional variants include, for example, deletions from, or insertions or substitutions of residues within an amino acid sequence. Any combination of deletion, insertion and substitution can also be carried out to produce the final construct, provided that the final construct has the desired activity, especially restoring the functionality of the CD3 complex. In a preferred embodiment, at least one mutation is introduced into different pTa versions as described above to affect dimerization. As a non-limiting example, the mutated residue may be at least W46R, D22A, K24A, R102A or R117A of a human pTa protein or aligned positions using the CLUSTALW method on a pTa family or homologous member. Preferably, pTa or a variant thereof as described above contains the mutated residue W46R or the mutated residues D22A, K24A, R102A and R117A. In some embodiments, the pTa or variants are also fused to a signal transduction domain, such as CD28, OX40, ICOS, CD27, CD137 (4-1BB) and CD8 as non-limiting examples. The extracellular domain of pTa or variants as described above can be fused to a fragment of the TCRa protein, particularly the transmembrane and intracellular domain of TCRa. pTa variants can also be fused to the intracellular domain of TCRa.

In some embodiments, pTa versions may be fused to an extracellular ligand binding domain. In some embodiments, pTa or a functional variant thereof is fused to a single chain antibody fragment (scFv) containing a light and heavy variable fragment and a target antigen of a specific monoclonal antibody, connected by a flexible linker.

The term TCRa-deficient T cell refers to an isolated T cell that lacks expression of a functional TCRa chain. This can be accomplished by various methods, but by way of non-limiting examples, by engineering the T cell such that it does not express any functional TCRa on its cell surface or by engineering the T cell such that it produces very little functional TCRa chain on its surface or by constructing a T cell that expresses a mutated or truncated form of the TCRa chain. TCRa deficient cells can no longer proliferate via the CD3 complex. Therefore, to overcome this problem and allow proliferation of TCRa-deficient cells, pTa or a functional variant thereof is introduced into the cells, thereby restoring a functional CD3 complex. In some embodiments, the method further comprises introducing into said T cells endonucleases that cleave at more sparsely located sites capable of being selectively inactivated by DNA cleavage of one gene encoding one component of the T cell receptor (TCR). In some embodiments, the endonuclease that cleaves at more sparsely located sites is a TALE nuclease.

In another aspect, engineered T cells obtained using the methods described in the present invention can be contacted with bispecific antibodies. For example, T cells may be contacted with bispecific antibodies ex vivo before administration to a patient, or in vivo after administration to a patient. Bispecific antibodies contain two variable regions with different antigenic properties, which makes it easier to target engineered cells directly

- 42 044329 proximity to the target antigen. As a non-limiting example, a bispecific antibody may target a tumor marker and lymphocyte antigen, such as, for example, but not limited to, CD3, and has the ability to redirect and activate any circulating T cells on the tumor.

In some embodiments, the polynucleotides encoding the polypeptides of the present invention may be mRNA that is directly introduced into cells, for example by electroporation. In some embodiments, cytoPulse technology can be used to temporarily permealize living cells to deliver material into the cells. Parameters can be modified to determine conditions for higher transfection efficiency with minimal mortality.

Also provided by the present invention are methods for transforming T cells. In some embodiments, the method includes: contacting a T cell with RNA and applying to the T cell a rapid pulse sequence comprising: (a) an electrical pulse with a voltage ranging from about 2250 to 3000 V per centimeter; (b) pulse width 0.1 ms; (c) a pulse interval of approximately 0.2 to 10 ms between the electrical pulses of steps (a) and (b); (d) an electrical pulse with a voltage ranging from approximately 2250 to 3000 V with a pulse width of approximately 100 ms and a pulse interval of approximately 100 ms between the electrical pulse of stage (b) and the first electrical pulse of stage (c); and (e) four electrical pulses of approximately 325 V with a pulse width of approximately 0.2 ms and a pulse interval of 2 ms between each of the 4 electrical pulses. In some embodiments, a method of transforming a T cell, comprising contacting said T cell with RNA and applying to the T cell a rapid pulse sequence comprising: (a) an electrical pulse at a voltage of approximately 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2400, 2450, 2500, 2600, 2700, 2800, 2900 or 3000V per centimeter; (b) pulse width 0.1 ms; (a) and a pulse interval of approximately 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 ms between the electrical pulses of steps (a) and (b); (d) One electric impulse with a voltage in the range of approximately 2250, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2400, 2450, 2500, 2600, 2700, 2800, 2900 or 3000 V with a pulse width of 100 ms and 100 ms width and 3000 a pulse interval of 100 ms between the electrical pulse of stage (b) and the first electrical pulse of stage (c); and (e) 4 electrical pulses of approximately 325 V with a pulse width of approximately 0.2 ms and a pulse interval of approximately 2 ms between each of the 4 electrical pulses. Any values included in the range of values described above are disclosed in the present invention. The electroporation medium can be any suitable medium known in the art. In some embodiments, the electroporation medium has a conductivity in the range of about 0.01 to about 1.0 millisiemens.

In some embodiments, by way of non-limiting examples, the RNA encodes a cleavage site endonuclease, one monomer of a cleavage site endonuclease such as a hemi-TALEnuclease, a CAR, at least one component of a multistranded chimeric antigen a receptor, pTa or a functional variant thereof, an exogenous nucleic acid, and/or one additional catalytic domain.

Engineered immune cells

The invention also provides engineered immune cells containing any of the CAR polynucleotides described in the present invention. In some embodiments, the CAR can be introduced into an immune cell as a transgene using a plasmid vector. In some embodiments, the plasmid vector may also contain, for example, a selectable marker that allows for the identification and/or selection of cells receiving the vector.

CAR polypeptides can be synthesized in situ in a cell after the introduction of polynucleotides encoding the CAR polypeptides into the cell. Alternatively, CAR polypeptides can be produced outside of cells and then introduced into cells. Methods for introducing a polynucleotide construct into cells are known in the art. In some embodiments, suitable transfection techniques can be used to integrate the polynucleotide construct into the genome of the cell. In other embodiments, transient transfection techniques can be used to transiently express a polynucleotide construct, and the polynucleotide construct is not integrated into the genome of the cell. In other embodiments, virus-mediated methods can be used. Polynucleotides can be introduced into the cell by any suitable means, such as, for example, recombinant viral vectors (eg, retroviruses, adenoviruses), liposomes, etc. Transient transfection methods include, for example, but are not limited to, microinjection, electroporation or bombardment particles.

The polynucleotides can be included in vectors, such as, for example, plasmid vectors or viral vectors.

Also provided by the present invention are isolated cells and cell lines obtained using the above-described cell engineering methods provided by the present invention. In some embodiments, the isolated cell contains at least one

CAR as described above. In some embodiments, the isolated cell contains a population of CARs, where each CAR contains different extracellular ligand binding domains.

Also provided in the present invention are isolated immune cells obtained in accordance with any of the methods described above. Any immune cell capable of expressing heterologous DNA can be used for the purpose of expressing CARs of interest. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell may be derived from, for example, but not limited to, a stem cell. The stem cells may be adult stem cells, non-human fetal stem cells, more preferably non-human stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells or hematopoietic stem cells. Representative human cells are CD34+ cells. The isolated cell may also be a dendritic cell, a killer dendritic cell, a mast cell, an NK cell, a B cell, or a T cell selected from the group consisting of inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, memory lymphocytes, or helper T lymphocytes. In some embodiments, the cell may be derived from the group including CD4+ T lymphocytes and CD8+ T lymphocytes.

Before distribution and genetic modification, the cell source may be obtained from the subject using a variety of non-limiting methods. The cells can be obtained from a variety of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, peritoneal effusion, pleural effusion, splenic tissue and tumors. In some embodiments, any number of T cell lines available and known to one skilled in the art may be used. In some embodiments, the cells may be derived from a healthy donor, from a patient diagnosed with cancer, or from a patient diagnosed with an infection. In some embodiments, the cells may be part of a mixed population of cells in which different phenotypic characteristics are present.

Also provided in the present invention are cell lines derived from a transfected T cell in accordance with any of the above methods. The present invention also provides modified cells that are resistant to immunosuppressive therapy. In some embodiments, an isolated cell in accordance with the invention contains a polynucleotide encoding a CAR.

Immune cells according to the invention can be activated and expanded, either before or after genetic modification of T cells, using methods as generally described, for example, but not limited to, in US patent No. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and US Patent Application Published No. 20060121005. T cells can be expanded in vitro or in vivo. In general, the T cells of the invention can be propagated, for example, by contacting an agent that stimulates the CD3 TCR complex and a costimulatory molecule on the surface of the T cells to create an activation signal for the T cell. For example, chemicals such as calcium ionophore A23187, phorbol 12-myristate 13 acetate (PMA), or mitogenic lectins such as phytohemagglutinin (PHA) can be used to create an activation signal for the T cell.

In some embodiments, T cell populations can be stimulated in vitro by contacting, for example, an anti-CD3 antibody, or an antigen-binding fragment thereof, or a surface-immobilized anti-CD2 antibody, or by contacting a protein kinase C activator (e.g. bryostatin) in combination with a calcium ionophore. To costimulate an accessory molecule on the surface of T cells, a ligand that binds the accessory molecule can be used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions suitable to stimulate T cell proliferation. Conditions suitable for culture of T cells include a suitable culture medium (eg, minimal essential medium or RPMI medium 1640 or X-Vivo 15, (Lonza)), which may contain factors necessary for proliferation and viability, including serum (eg , fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-2, IL-15, TGFp, and TNF, or any other cell growth additives known to one skilled in the art. Other cell growth additives include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetylcysteine and 2-mercaptoethanol. Culture media may include RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 10, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either without serum or supplemented with a suitable amount of serum (or plasma) or a certain set of hormones and/or amount of cytokine(s) sufficient for the growth and proliferation of T cells. Antibiotics, such as penicillin and streptomycin, are included only in the experimental cultures and not in the cell cultures that will be infused into the subject. The target cells are maintained under conditions necessary to support growth, such as suitable temperature (eg, 37°C) and atmosphere (eg, air plus 5% CO2). T cells stimulated for different times may exhibit different characteristics.

In some embodiments, cells of the invention may be propagated by co-culture with tissues or cells. The cells can also spread in vivo, for example, in the blood of a subject after the cell is introduced into the subject.

In some embodiments, an isolated cell in accordance with the present invention contains one inactivated gene selected from the group consisting of CD52, dCK, GR, PD-1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, 2B4, HLA, TCRa and TCRe and/or, expresses CAR, multi-chain CAR and/or pTa transgene. In some embodiments, the isolated cell contains polynucleotides encoding polypeptides containing multi-chain CARs. In some embodiments, an isolated cell in accordance with the present invention contains two inactivated genes selected from the group consisting of: CD52 and GR, CD52 and TCRa, CDR52 and TCRe, GR and TCRa, GR and TCRe, TCRa and TCRe, PD-1 and TCRa, PD-1 and TCRe, CTLA-4 and TCRa, CTLA-4 and TCRe, LAG3 and TCRa, LAG3 and TCRe, Tim3 and TCRa, Tim3 and TCRe, BTLA and TCRa, BTLA and TCRe, BY55 and TCRa, BY55 and TCRe, TIGIT and TCRa, TIGIT and TCRe, B7H5 and TCRa, B7H5 and TCRe, LAIR1 and TCRa, LAIR1 and TCRe, SIGLEC10 and TCRa, SIGLEC10 and TCRe, 2B4 and TCRa, 2B4 and TCRe and/or expresses CAR, multichain CAR and pTa transgene.

In some embodiments, the TCR imparts non-functionality to cells in accordance with the invention by inactivating the TCRa gene and/or TCRe gene(s). In some embodiments, a method is provided for producing modified cells originating from an individual where the cells can proliferate independently of the major histocompatibility complex (MHC) signaling pathway. Modified cells that can proliferate independently of the MHC signaling pathway and that can be obtained using this method are within the scope of the present invention. The modified cells disclosed in the present invention can be used to treat patients in need with respect to host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD); Therefore, within the scope of the present invention is a method of treating patients in need thereof with respect to host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD), comprising treating said patients by administering to said patient an effective amount of modified cells containing inactivated TCRa and/or TCRe genes.

In some embodiments, immune cells are engineered to be resistant to one or more chemotherapy drugs. The chemotherapeutic drug may be, for example, a purine nucleotide analog (PNA), thereby making the immune cell suitable for the treatment of cancer by combining adoptive immunotherapy and chemotherapy. Typical PNAs are, for example, clofarabine, fludarabine, and cytarabine, alone or in combination. PNAs are metabolized by deoxycytidine kinase (dCK) into mono-, di-, and tri-phosphate PNAs. Their tri-phosphate forms compete with ATP for DNA synthesis, acting as pro-apoptotic agents, and are effective inhibitors of ribonucleotide reductase (RNR), which is involved in the production of trinucleotides. The present invention provides EGFRvIII specific CAR-T cells containing an inactivated dCK gene. In some embodiments, dCK silenced cells are produced by transfecting T cells using polynucleotides encoding a specific TAL nuclease targeting dCK genes, for example, by electroporation of mRNA. dCK silenced EGFRvIII specific CAR-T cells are resistant to PNAs, including, for example, clofarabine and/or fludarabine, and maintain cytotoxic T cell activity towards EGFRvIII-expressing cells. In another example, the chemotherapeutic drug may be, for example, a CD52-targeting molecule such as an anti-CD52 monoclonal antibody (eg, alemtuzumab). CD52 is a protein present on the surface of lymphocytes, and anti-CD52 antibodies can induce apoptosis and lysis of immune cells through antibody- and complement-dependent cytotoxicity, leading to lymphatic depletion. The present invention provides EGFRvIII specific CAR-T cells containing an inactivated CD52 gene. In some embodiments, CD52 silenced cells are produced by transfecting T cells using polynucleotides encoding a specific TAL nuclease targeting the CD52 gene, for example, by electroporation of mRNA. CD52 silenced EGFRvIII specific CAR-T cells are resistant to anti-CD52 molecules, including, for example, alemtuzumab, and maintain cytotoxic T cell activity against EGFRvIII-expressing cells in the presence of anti-CD52 molecules.

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In some embodiments, isolated cells or cell lines according to the invention may contain pTa or a functional variant thereof. In some embodiments, the isolated cell or cell line may be further genetically modified by inactivating the TCRa gene.

In some embodiments, the CAR-T cell comprises a polynucleotide encoding a suicide polypeptide, such as, for example, RQR8. See, for example, WO 2013153391 A, which is hereby incorporated by reference in its entirety. In CAR-T cells containing a polynucleotide encoding a suicide polypeptide, the suicide polypeptide is expressed on the surface of the CAR-T cell. In some embodiments, the suicide polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 226.

CPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTTACPYSNPSLCSGGGGSP

APRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVL

LLS LVITLYCNHRNRRRVCKCPRPW (SEQ ID NO. 226).

In some embodiments, the suicide polypeptide may also contain a signal peptide at the amino terminus. If a suicide polypeptide is expressed on the surface of a CAR-T cell, binding of rituximab to the R epitopes (ie, the epitope recognized by rituximab - CPYSNPSLC (SEQ ID NO: 256)) of the polypeptide causes cell lysis. More than one rituximab molecule can bind to a polypeptide expressed on the cell surface. Each R epitope of the polypeptide can bind a separate rituximab molecule. Deletion of EGFRvIII specific CAR-T cells can occur in vivo, for example by administering rituximab to a patient. The decision to delete the transferred cells may be driven by undesirable effects found in the patient that are associated with the transferred cells, such as when unacceptable levels of toxicity are found.

In some embodiments, the suicide polypeptide may contain one, two, three, or more epitopes that are recognized by an antibody (eg, rituximab).

In some embodiments, the suicide polypeptide may be provided to an EGFRvIII specific CAR T cell in a polypeptide that is separate from the CAR-containing polypeptide. In some embodiments, the suicide polypeptide may be provided to an EGFRvIII specific CAR T cell on the same polypeptide chain as the CAR polypeptide. In a CAR-containing suicide polypeptide, typically the suicide polypeptide is provided in the extracellular portion of the CAR.

In a CAR-containing suicide polypeptide, the suicide polypeptide may contain, for example, one or more copies of the amino acid sequence of the epitope recognized by rituximab (CPYSNPSLC (SEQ ID NO: 256)). The suicide polypeptide can be located at various positions in the CAR. For example, the suicide polypeptide may be N-terminal to the scFv or it may be C-terminal to the scFv in the CAR. In some embodiments, the suicide peptide may be located between the scFv and the loop region of the CAR. In some embodiments, the CAR may contain more than one suicide polypeptide. For example, a CAR may contain a suicide polypeptide N-terminal to the scFv and a suicide polypeptide C-terminal to the scFv. Each of these polypeptides may contain one or more copies of an epitope recognized by rituximab. For example, a CAR may contain a first suicide polypeptide at a position N-terminal to the scFv, where the first suicide polypeptide contains one copy of an epitope recognized by rituximab, and a second suicide polypeptide at a position C-terminal to the scFv, where the second suicide polypeptide contains two copies of the epitope recognized by rituximab.

Also provided by the present invention are nucleic acids encoding EGFRvIII-specific CARs that contain a suicide polypeptide sequence in the CAR.

In an example, a suicide polypeptide on the same polypeptide chain as the EGFRvIII specific CARs may have a sequence designated herein as the R2 suicide sequence. The R2 suicide sequence contains two copies of the epitope recognized by rituximab (CPYSNPSLC (SEQ ID NO: 256)). Such EGFRvIII-specific CARs may be referred to in the present invention as EGFRvIII-R2 CARs. In table 5D shows the amino acid sequences of exemplary EGFRvIII-R2 CARs according to the present invention. In table 5D, the signal/leader peptide sequence is in bold, the GS linker ((GGGGS) ₄ (SEQ ID NO: 202)) is underlined, and the R2 suicide sequence is in bold and underlined. In table 5E shows exemplary nucleotide sequences encoding exemplary EGFRvIII-R2 CARs of the present invention.

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Table 5D

Amino acid sequences of typical EGFRvIII specific CARs with R2 suicide sequence

CAR Amino acid sequence of CAR Components (in order, N-terminus to C-terminus) 14С11- R2 MALPVTALLLPLALLLHAARPQVTLKESGPVLVKPTETLTLTCTVSGF SLNNARMGVSWIRQPPGKALEWFAHIFSTDEKSFRTSLRSRLTLSKDT SKSQVVLMTTNMDPVDTATYYCARDSSNYEGYFDYWGQGILVTVSSGG GGSGGGGSGGGGSGGGGSEIVM T Q S PAT L SVS P G E RA T L S S RAS Q SV S NNLAWYQQKPGQAPRLLIYGASTRATGVPARFSGSDSGTEFSLTISSL Q SEDFAVYFСQQYКDWРФТFGРГТКВЕI КGSGGGGSCPYSNPSLCSGG GGSCPYSNPSLCSGGGGSTTTRARRRRRTARTIASQРLSLRREACRRА AGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKL LY IFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQ GQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP R (SEQ ID NO: 248) CD8a signal peptide; 14C11 VH (Table 1, SEQ ID NO: 15); GS linker; 14С11 VL (Table 1, SEQ ID NO: 16) ; R2 suicide sequence; CD8a loop; CD8aTM; 4-IBB signaling domain; CD3zeta signaling domain 32А10R2 MALPVTALLLPLALLLHAARPQVTLKESGPVLVKPTETLTLTCTVSGF SLSNARMGVSWIRQPPGKALEWLAHIFSTDEKSIRRSLRSRLTLSKDT SKSQVVLTMTNMDPVDTATYFCARDSSNYEGYFDYWGQGTLVTVSSGG GGSGGGGSGGGGSGGGGSEV VM T Q S P A T L S V S P G E R V T L S C R A S Q S V S SNFAWYQQRPGQAPRLLLYGATTRATGLPGRFSGSGSGTENILTISSL QSEDFAIYFCQQYКDWPFTFGGSКVDI КGSGGGGSCPYSNPSLCSGG GGSCPYSNPSLCSGGGGSTTTPAPRPPTPAPTIASQPLSLRPEACRPA AGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLY IFKQPFMR PVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQ GQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP R(SEQ ID NO: 249) CD8a signal peptide; 32A10 VH (Table 1 SEQ ID NO: 11); GS linker; 32A10 VL (Table 1 SEQ ID NO: 12); R2 suicide sequence; CD8a loop; CD8aTM; 4-IBB signaling domain; CD3zeta signaling domain 26B9-R2 MALPVTALLLPLALLLHAARPEVQLVESWGVLVKPGGSLRLSCAASGF IFNNAWMSWVRQAPGKGLEWIGRIKSKSDGGTTDYAAPVKDRFTI3RD DSKDTLYLQMNGLKTEDTAVYFCTTAPGGPFDYWGQGTLVTVSSGGGG SGGGGSGGGGSGGGGSDIVLTQSPLSLPVTPGEPASISCRSSQSLLHR DGFNYLDWFLQK PGQSPQLLIYLASSRASGVPDRFSGSDSGTDFTLKI SRVEAEDVGVYYCMQALQTPITFGQGTRLEIK GSGGGGSCPYSNPSLCSGGGGSCPYSNPSLCSGGGGS TTTPAPRPPTP APTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVL LLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEG GCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPE MGGKPRRKNPQEG LYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQG LSTATKDTYDALHMQALPPR (SEQ ID NO: 250) CD8a signal peptide; 26B9 VH (Table 1, SEQ ID NO: 41) ; GS linker; 26B9 VL (Table 1, SEQ ID NO: 42); R2 suicide sequence; CD8a loop; CD8aTM; 4-IBB signaling domain; CD3zeta signaling domain 30D8-R2 MALPVTALLLPLALLLHAARPEVQLVESGGGLVKPGGSLRLSCEASGF TFSDAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYVVPLNGRFII 3RD DSRNTLYLQLNNLKTEDTAVYYCTTVPGSYGYWGQGTLVTVSSGGGGS GGGGSGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSSQSLLHNK RNNYLDW FLQKPGQSPQLLIYLASNRASGVPDRFSGGGSGTDFTLKI3 RVEAEDVGVYYCMQAQQTPITFGQGTRLEIKGSGGGGSCPYSNPSLCS CD8a signal peptide; 30D8 VH (Table 1, SEQ ID NO: 37); GS linker; 30D8 VL (Table 1, GGGGSCPYSNPSLCSGGGGSTTTPAPRPPTPAPTIASQPLSLRPEACR PAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKL LYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAY QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNE LQKDKMAEAY SEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR (SEQ ID NO: 251) SEQ ID NO: 38); R2 suicidal subsequence; CD8a loop; CD8aTM; 4-IBB signaling domain; CD3zeta signal domain

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Table 5E

Nucleotide sequences of typical EGFR VIII specific CARs with R2 suicide sequence

CAR Nucleotide sequence of CAR Components 14С11- R2 ATGGCACTGCCAGTGACCGCCCTGCTGCTGCCTCTGGCCCTGCTGCTG CACGCAGCCAGACCCCAGGTGACACTGAAGGAGAGCGGCCCCGTGCTG GTGAAGCCTACAGAGACACTGACCCTGACCTGCACAGTGAGCGGCTTC TCCCTGAACAATGCAAGGATGGGCGTGTCCTGGATCAGGCAGCCACCT GGCAAGGCCCTGGAGTGGTTCGCCCACATCTTTAGCACCGAC GAGAAG TCCTTTCGCACATCTCTGAGAAGCAGGCTGACCCTGAGCAAGGATACA AGCAAGTCCCAGGTGGTGCTGACCATGACAAACATGGACCCTGTGGAT ACCGCCACATACTATTGTGCCCGGGACAGCTCCAATTACGAGGGCTAT TTCGATTACTGGGGCCAGGGCATCCTGGTGACCGTGTCTAGCGGCGGC GGCGGCTCTGGAGGAGGAGGAAGCGGAGGAGGAGGATCCGGCGG CGGC GGCTCTGAGATCGTGATGACCCAGTCCCCAGCCACACTGTCTGTGAGC CCAGGAGAGAGAGCCACCCTGTCTTGCAGGGCCTCCCAGTCTGTGAGC AACAATCTGGCCTGGTATCAGCAGAAGCCTGGCCAGGCCCCAAGGCTG CTGATCTACGGAGCAAGCACCAGAGCAACAGGAGTGCCTGCAAGGTTC TCCGGATCTGACAGCGGCACCGAGTTTTCTCTGACA ATCTCCTCTCTG CAGAGCGAGGACTTCGCCGTGTATTTTTGTCAGCAGTACAAGGATTGG CCATTCACCTTTGGCCCCGGCACAAAGGTGGAGATCAAGGGCTCCGGA GGAGGAGGATCCTGCCCCTATTCCAACCCTTCTCTGTGCAGCGGAGGA GGAGGAAGCTGTCCATACTCCAATCCCTCCCTGTGCTCCGGCGGCGGA G GAT C CAC CACAAC C C CAG CAC C TAGAC SASSAAS C C CAGCAC CAASA ATCGCATCCCAGCCTCTGTCTCTGCGGCCCGAGGCATGCAGGCCAGCA GCAGGCGGCGCCGTGCACACCAGGGGCCTGGACTTTGCCTGCGATATC TATATCTGGGCACCACTGGCAGGAACCTGTGGCGTGCTGCTGCTGAGC CTGGTCATCACCCTGTATTGCAAGCGCGGCCGGAAGAAGCTGCTGTAC ATCTTCAAGCAGCCTTTTATGCGC CCAGTGCAGACAACCCAGGAGGAG GACGGCTGCTCCTGTCGGTTCCCTGAAGAGGAGGAGGGAGGATGTGAG CTGCGCGTGAAGTTTTTCCCGGTCTGCCGATGCCCCAGCCTATCAGCAG GGCCAGAACCAGCTGTACAACGAGCTGAATCTGGGCCGGAGAGAGGAG TACGACGTGCTGGATAAGAGGAGGGGAAGAGATCCCGAGATGGGAGGC AAGCCTCGGAGAAAGAACCCCA CAGGAGGGCCTGTAATAATGAGCTGCAG AAGGACAAGATGGCCGAGGCCTACTCTGAGATCGGCATGAAGGGAGAG AGGCGCCGGGGCAAGGGACACGATGGCCTGTATCAGGGCCTGTCCACC GCCACAAAGGACACCTACGATGCCCTGCACATGCAGGCCCTGCCTCCA AGGTGA (SEQ ID NO: 252) CD8c< signal peptide; 14C11 VH (Table 1, SEQ ID NO: 15); GS linker; 14C11 VL (Table 1, SEQ ID NO: 16); R2 suicidal subsequence; CD8c<loop; CD8c<TM; 4-IBB signaling domain; CD3zeta signaling domain 32А10R2 ATGGCACTGCCAGTGACCGCCCTGCTGCTGCCTCTGGCCCTGCTGCTG CACGCAGCCAGACCCCAGGTGACACTGAAGGAGTCCGGCCCCGTGCTG GTGAAGCCTACAGAGACACTGACCCTGACCTGCACAGTGAGCGGCTTC TCTCTGAGCAACGCAAGGATGGGCGTGTCCTGGATCAGGCAGCCACCT GGCAAGGCCCTGGAGTGGCTGGCCCACATCTTTTCCACCGAC GAGAAG TCTATCCGGAGAAGCCTGCGCTCCCGGCTGACCCTGAGCAAGGATACA TCCAAGTCTCAGGTGGTGCTGACCATGACAAACATGGACCCTGTGGAT ACCGCCACATACTTCTGTGCCCGGGACAGCTCCAATTACGAGGGCTAT TTTGATTACTGGGGCCAGGGCACCCTGGTGACAGTGTCTAGCGGAGGA GGAGGAAGCGGAGGAGGAGGATCAGGCGGCGGCGGCTCTGGCGG CGGC GGCAGCGAGGTGGTCATGACCCAGTCTCCAGCCACACTGAGCGTGTCC CCAGGAGAGCGCGTGACCCTGAGCTGCCGGGCCTCTCAGAGCGTGTCC TCTAACTTCGCCTGGTATCAGCAGCGGCCCGGACAGGCACCAAGGCTG CTGCTGTACGGAGCAACCACAAGAGCAACAGGCCTGCCTGGCAGGTTT CD8c< signal peptide; 32A10 VH (Table 1 SEQ ID NO: 11); GS linker; 32A10 VL (Table 1 SEQ ID NO: 12) ; R2 suicide sequence; CD8c<loop; CD8c<TM; 4-IBB signaling domain; CD3zeta signal

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G GAT C TAC CACAAC C C CAG CAC C TAGAC CACCAAC C C CAGCAC C CACA ATCGCCTCTCAGCCTCTGAGCCTGCGCCCAGAGGCATGCAGGCCAGCA GCAGGAGGAGCAGTGCACACCAGGGGCCTGGACTTCGCCTGCGATATC TATATCTGGGCACCACTGGCAGGAACCTGTGGCGTGCTGCTGCTGTCC CTGGTCATCACCCTGTATTGCAAGAGAGGCAGG AAGAAGCTGCTGTAC ATCTTCAAGCAGCCTTTTATGCGCCCAGTGCAGACAACCCAGGAGGAG GACGGCTGCAGCTGTCGGTTCCCTGAAGAGGAGGAGGGCGGCTGTGAG CTGAAGTGAAGTTTTCCAGGTCTGCCGATGCCCCAGCCTATCAGCAG GGCCAGAATCAGCTGTACAACGAGCTGAATCTGGGCAGGCGCGAGGAG TACGACGTGCTGGATAAGAGGAGAGGACGC GATCCCGAGATGGGAGGC AAGCCTAGGCGCAAGAACCCAGGAGGGCCTGTATAATGAGCTGCAG AAGGACAAGATGGCCGAGGCCTACTCTGAGATCGGCATGAAGGGAGAG CGGAGAAGGGGCAAGGGACACGATGGCCTGTATCAGGGCCTGAGCACC GCCACAAAGGACACCTACGATGCCCTGCACATGCAGGCCCTGCCTCCA AGGTGA (SEQ ID NO: 253) domain 26B9-R2 ATGGCCCTGCCAGTGACCGCCCTGCTGCTGCCACTGGCCCTGCTGCTG CACGCCGCCAGACCTGAGGTGCAGCTGGTGGAGAGCTGGGGCGTGCTG GTGAAGCCAGGAGGCTCTCTGAGGCTGAGCTGCGCAGCATCCGGCTTC ATCTTTAACAATGCCTGGATGTCCTGGGTGAGACAGGCACCAGGCAAG GGCCTGGAGTGGATCGGCAGGATCAAGAGCAAGTCCGACGGAG GAACC ACAGATTACGCAGCACCCGTGAAGGACCGCTTCACAATCTCTCGGGAC GATAGCAAGGATACCCTGTATCTGCAGATGAACGGCCTGAAGACAGAG GACACCGCCGTGTACTTCTGCACCACAGCCCCAGGCGGCCCCTTTGAT TATTGGGGCCAGGGCACACTGGTGACCGTGAGCTCCGGAGGAGGAGGA AGCGGCGGAGGAGGCAGCGGCGGCGGCGGCTCTGGCGGCGGC GGCAGC GACATCGTGCTGACACAGAGCCCACTGTCCCTGCCTGTGACCCCAGGA GAGCCCGCCTCTATCAGCTGTCGCTCTAGCCAGAGCCTGCTGCACCGG GACGGCTTCAATTACCTGGATTGGTTTCTGCAGAAGCCTGGCCAGAGC CCACAGCTGCTGATCTATCTGGCCTCCTCTAGAGCATCCGGAGTGCCT GACAGGTTCTCCGGATCTGACAGCGGCACAGACTTCACCC TGAAGATC TCCCGCGTGGAGGCAGAGGATGTGGGCGTGTACTATTGCATGCAGGCC CTGCAGACACCAATCACCTTCGGCCAGGGCACACGGCTGGAGATCAAG GGATCCGGAGGAGGAGGATCTTGCCCCTACTCTAACCCTAGCCTGTGC AGCGGCGGAGGAGGATCTTGTCCATATTCTAATCCAAGCCTGTGCAGC G GG GGAGGAG GAAG CAC CACAAC C C C T G CACCAAGAC C C C C TACAC CA GCACCTACCATCGCATCCCAGCCACTGTCTCTGCGGCCCGAGGCATGT AGGCCAGCAGCAGGAGGAGCAGTGCACACCAGGGGCCTGGACTTTGCC TGCGATATCTACATCTGGGCACCACTGGCAGGAACATGTGGCGTGCTG CTGCTGAGCCTGGTCATCACCCTGTACTGCAAGAGAGGCAGGAAGAAG CTGCTGTATATCTTCA AGCAGCCTTTTATGCGCCCAGTGCAGACAACC CAGGAGGAGGACGGCTGCTCCTGTAGGTTCCCAGAAGAGGAGGAGGGA GGATGTGAGCTGCGCGTGAAGTTTTTCCCGGTCTGCCGATGCACCTGCA TACCAGCAGGGACAGAACCAGCTGTATAACGAGCTGAATCTGGGCCGG AGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGACGCGATCCTGAG ATGGGAGGCA AGCCCCGGAGAAAGAACCCTCAGGAGGGCCTGTACAAT GAGCTGCAGAAGGACAAGATGGCCGAGGCCTATTCCGAGATCGGCATG AAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTACCAGGGC CTGTCTACAGCCACCAAGGACACCTATGATGCCCTGCACATGCAGGCC CTGCCACCAAGGTGA (SEQ ID NO: 254) CD8a signal peptide; 26B9 VH (Table 1, SEQ ID NO: 41); GS linker; 26B9 VL (Table 1, SEQ ID NO: 42) ; R2 suicide sequence; CD8a loop; CD8aTM; 4-IBB signaling domain; CD3zeta signaling domain 30D8-R2 ATGGCACTGCCAGTGACAGCCCTGCTGCTGCCTCTGGCCCTGCTGCTG CACGCAGCCAGACCAGAGGTGCAGCTGGTGGAGTCCGGAGGAGGCCTG GTGAAGCCAGGAGGCTCCCTGAGGCTGTCTTGCGAGGCCAGCGGCTTC ACCTTTAGCGACGCCTGGATGTCCTGGGTGAGACAGGCACCAGGCAAG GGCCTGGAGTGGGTGGGCAGGATCAAGAGCAAGACAGACGGC GGCACC ACAGATTACGTGGTGCCTCTGAACGGCCGGTTCATCATCTCCCGCGAC GATTCTCGGAATACCTGTATCTGCAGCTGAACAATСTGAAGACAGAG GATACCGCCGTGTACTATTGCACCACAGTGCCTGGCTCCTACGGCTAT TGGGGCCAGGGCACACTGGTGACCGTGAGCTCCGGCGGCGGCGGCTCT GGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGGGGGC GGCGGCTCTGAC ATCGTGATGACACAGTCTCCACTGAGCCTGCCAGTGACCCCAGGAGAG CD8a signal peptide; 30D8 VH (Table 1, SEQ ID NO: 37); GS linker; 30D8 VL (Table 1, SEQ ID NO: 38); R2 suicide sequence; CD8a loop; CD8aTM;

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CCTGCCTCCATCTCTTGTCGCTCTAGCCAGTCCCTGCTGCACAACAAG CGGAACAATTACCTGGATTGGTTCCTGCAGAAGCCAGGCCAGTCTCCC CAGCTGCTGATCTATCTGGCCAGCAATAGAGCCTCCGGAGTGCCAGAC AGGTTCTCTGGAGGAGGAAGCGGAACAGACTTCACCCTGAAGATCAGC CGCGTGGAGGCAGAGGACGTGGGCGTGTACTATTGCATGCAGGCCC AG CAGACACCCATCACCTTTGGCCAGGGAACCCGGCTGGAGATCAAGGGC TCCGGAGGAGGAGGATCCTGCCCTTACTCCAACCCATCTCTGTGCAGC GGAGGAGGAGGATCTTGTCCATATTCCAATCCTTCCCTGTGCTCCGGA G GAG GAG GAAG CAC CACAACCCCTGCACCAAGACCCCCTACACCAGCA CCTACCATCGCATCCCAGCCTCTGTCTCTGCGGCCCGAGGCATG TAGG CCAGCAGCAGGCGGCGCCGTGCACACCAGGGGCCTGGACTTTGCCTGC GATATCTACATCTGGGCACCACTGGCAGGAACATGTGGCGTGCTGCTG CTGTCTCTGGTCATCACCCTGTACTGCAAGAGAGGCAGGAAGAAGCTG CTGTATATCTTCAAGCAGCCCTTCATGCGGCCCGTGCAGACAACCCAG GAGGAGGACGGCTGCAGCTGTCGGTTCCCTGAAGAGGAGGAGGG AGGA TGTGAGCTGCGCGTGAAGTTTAGCCGGTCCGCCGATGCACCAGCATAC CAGCAGGGCCAGAACCAGCTGTATAACGAGCTGAATCTGGGCCGGAGA GAGGAGTACGACGTGCTGGATAAGAGGAGGGGACGCGATCCTGAGATG GGAGGCAAGCCCTGGAGAAAGAACCACAGGAGGGCCTGTACAATGAG CTGCAGAAGGACAAGATGGCCGAGGCCTATAGCGA GATCGGCATGAAG GGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTACCAGGGCCTG TCCACAGCCACCAAGGACACCTATGATGCCCTGCACATGCAGGCCCTG CCACCAAGGTGA (SEQ ID NO: 255) 4-1BB signaling domain; CD3zeta signaling domain

Therapeutic Applications

Isolated cells obtained by the methods described above, or cell lines derived from such isolated cells, can be used as a medicine. In some embodiments, such a drug can be used to treat cancer, including solid tumors and liquid tumors. In some embodiments, the cancer is an EGFRvIII-associated cancer (e.g., any cancer expressing EGFRvIII) including, but not limited to, glioblastoma (e.g., glioblastoma multiforme), anaplastic astrocytoma, giant cell glioblastoma, gliosarcoma, anaplastic oligodendroglioma, anaplastic ependymoma, anaplastic oligoastrocytoma, choroid plexus carcinoma, anaplastic ganglioglioma, pineoblastoma, pineocytoma, meningioma, medulloepithelioma, ependymoblastoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, atypical teratoma oid-rhabdoid tumor, mixed glioma, head and neck cancer, non-small cell lung cancer , breast cancer, ovarian cancer, prostate cancer, medullobastomy, colorectal cancer, anal cancer, cervical cancer, kidney cancer, skin cancer, pancreatic cancer, liver cancer, bladder cancer, stomach cancer, thyroid cancer glands, mesothelioma, uterine cancer, lymphoma, or leukemia.

In some embodiments, a drug as described in the present invention can be used to 1) inhibit the growth or progression of a tumor in a subject who has cancer cells expressing EGFRvIII; 2) inhibiting metastasis of cancer cells expressing EGFRvIII in a subject; and 3) inducing tumor regression in a subject that has cancer cells expressing EGFRvIII.

In some embodiments, an isolated cell in accordance with the invention, or a cell line derived from the isolated cells, can be used to prepare a medicament for the treatment of cancer in a patient in need thereof.

Also provided by the present invention are methods of treating patients. In some embodiments, the method includes providing an immune cell according to the invention to a patient in need thereof. In some embodiments, the method includes the step of administering transformed immune cells according to the invention to a patient in need thereof.

In some embodiments, T cells of the invention can stably undergo in vivo T cell expansion and can persist for an increased period of time.

The methods of treatment according to the invention can be debilitating, curative or prophylactic. The method according to the invention can be either part of an autologous immunotherapy or part of an allogeneic immunotherapy treatment. The invention is particularly suitable for allogeneic immunotherapy. T cells from donors can be transformed into non-alloreactive cells using standard protocols and replicated as needed, thereby producing CAR-T cells that can be administered to one or more patients. Such CAR-T cell therapy may be provided as a ready-to-use therapeutic product.

Cells that can be used in the disclosed methods are described, for example, in the previous section. The treatment can be used to treat patients who have been diagnosed with, for example, a malignant neoplasm. Malignancies that can be treated include, for example, cancers that are classified as EGFRvIII, including any of the above malignancies. Types of malignant neoplasms,

- 50 044329 treatable cells with CAR and CAR-T cells according to the invention include, but are not limited to, certain liquid and solid tumors such as glioblastoma multiforme, head and neck cancer, non-small cell lung cancer, breast cancer, ovarian cancer and prostate cancer. Tumors/malignancies in adults and tumors/malignancies in children are also covered.

In some embodiments, treatment may be administered in combination with one or more therapies against a malignancy selected from the group: antibody therapy, antibody-drug conjugate therapy, chemotherapy, targeted therapy, cytokine therapy, vaccine therapy, oncolytic virus therapy, dendritic therapy cell therapy, gene therapy, nanoparticle therapy, hormonal therapy, surgical therapy, laser therapy, tumor site treatment, and radiation therapy. For example, the CAR and CAR-T cells of the invention may be administered to a patient in combination with (e.g., before, concurrently, or after) 1) standard treatment, including radiation, surgical resection, chemotherapy (e.g., temozolomide, procarbazine, carmustine, lomustine, vincristine, and etc.), antibody therapy such as bevacizumab, anti-angiogenic therapy, and/or tumor site therapy; 2) vaccines, including EGFRvIII vaccine; 3) antibody-drug conjugate therapy, including, but not limited to, drug conjugates that target the HER family of receptors, such as EGFR, HER2, HER3, and HER4; 4) targeted therapy such as kinase inhibitors (eg, everolimus); and 5) immunotherapies, including, but not limited to, anti-PD-1, anti-PD-L1, anti-PDL2, anti-41BB, anti-TIM3, anti-LAG3, anti-TIGIT, anti-OX40, anti -HVEM, anti-BTLA, anti-CD40, anti-CD47, anti-CSF1R, anti-CSF1, anti-MARCO, anti-IL8, anti-CXCR4, and anti-CTLA4 antibodies.

In some embodiments, the treatment may be administered to patients undergoing immunosuppressive therapy. Indeed, the invention is preferably based on cells or a population of cells that are conferred resistance to at least one immunosuppressive agent (eg, cyclosporine, azathioprine, methotrexate, mycophenolate and FK506) due to inactivation of the gene encoding the receptor for such immunosuppressive agent. In this aspect, immunosuppressive therapy will assist in the selection and expansion of T cells in accordance with the invention in the patient. Administration of cells or populations of cells in accordance with the invention can be accomplished by any suitable means, including aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described in the present invention can be administered to a patient subcutaneously, intradermally, intratumorally, intracranially, intranodally, intramedullary, intramuscularly, by intravenous or intralymphatic injection, or intraperitoneally. In one embodiment, the cellular compositions of the invention are preferably administered by intravenous injection.

In some embodiments, the treatment may be administered to patients undergoing a lymphatic depletion regimen. Depletion of immune regulatory elements by cytotoxic agents or whole body irradiation may enhance the antitumor activity of the CAR and CAR-T cells of the present invention. For example, a cytotoxic agent in a lymphatic depletion regimen includes, but is not limited to, fludarabine, cyclophosphamide, and/or alemtuzumab.

In some embodiments, administration of cells or population of cells may include administration of, for example, about 10 ⁴ to about 10 ⁹ cells per kg of body weight, including all integer cell numbers within these ranges. In some embodiments, administration of cells or population of cells may include administration of from about 105 to 106 cells per kg of body weight, including all integer cell counts within these ranges. Cells or populations of cells can be administered in one or more doses. In some embodiments, said effective number of cells may be administered as a single dose. In some embodiments, said effective number of cells may be administered in more than one dose over a period of time. Regulation of the moment of administration is within the competence of the attending physician and depends on the clinical condition of the patient. The cells or population of cells can be obtained from any source, such as a blood bank or donor. Since individual needs differ, determining the optimal ranges of effective amounts of a given cell type for a particular disease or condition is within the skill of one skilled in the art. An effective amount means an amount that provides a therapeutic or prophylactic benefit. The dose administered will depend on the age, health and weight of the recipient, the type of concomitant treatment, if any, the frequency of treatment and the nature of the desired effect. In some embodiments, an effective amount of cells or a composition containing these cells is administered parenterally. In some embodiments, administration may be via intravenous administration. In some embodiments, administration may be by direct injection into the tumor.

Sets

The invention also provides kits for use in the methods of the present invention. The kits of the invention include one or more containers containing a polynucleotide encoding an EGFRvIII specific CAR, or an engineered immune cell containing a polynucleotide encoding an EGFRvIII specific CAR, as described in the present invention, and instructions for use in accordance with any of the methods according to the invention described in the present invention. In general, these instructions describe the administration of an engineered immune cell for the above-described therapeutic treatments.

Instructions regarding the use of engineered immune cells as described in the present invention will typically include information regarding the dosage, dosage regimen, and route of administration for the intended treatment. Containers may be unit doses, bulk packages (eg, multi-dose packages), or subunit doses. The instructions provided in the kits of the invention are typically written instructions on a label or insert (eg, a piece of paper included in the kit), but machine readable instructions (eg, instructions located on a magnetic or optical storage disk) are also acceptable.

Kits according to the present invention are provided in suitable packages. Suitable packaging includes, but is not limited to, vials, bottles, containers, flexible packaging (eg, sealed Mylar or plastic bags), and others. Also covered are packages for use in combination with a specific device, such as an inhaler, a nasal administration device (eg, a nebulizer), or an infusion device such as a minipump. The kit may have a sterile access port (eg, the container may be an intravenous solution bag or a vial having a stopper that can be pierced with a hypodermic needle). The container may also have a sterile access port (eg, the container may be a bag of intravenous solution or a vial having a stopper that can be pierced with a hypodermic needle). At least one active agent in the composition is an EGFRvIII antibody. The container may further contain a second pharmaceutically active agent.

Sets may optionally provide additional components such as buffers and interpretable information. Typically, the kit contains a container and a label or sheet(s) insert(s) on or associated with the container.

Incorporated by reference in this application for all purposes is the contents of U.S. Provisional Patent Application Nos. 62/281,533 (filed Jan. 21, 2016) and 62/431,758 (filed Dec. 8, 2016).

Biological deposition

Representative materials of the present invention have been deposited with the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209, USA, in . A vector___having an ATCC accession number.____is a polynucleotide encoding a ____light chain variable region, and a vector___having an ATCC accession number no. is a polynucleotide encoding the _____ variable region of the heavy chain. The deposits were made in accordance with the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and its secondary legislation (Budapest Treaty). This ensures that a viable deposit culture is maintained for 30 years from the date of deposit. The deposit will be submitted in accordance with the ATCC under the terms of the Budapest Treaty, and as agreed by Pfizer, Inc. and ATCC, which guarantees the continued and unrestricted availability of the progeny of the deposit culture to an unlimited number of persons upon the issuance of an appropriate U.S. patent or upon the presentation to an unlimited number of persons of any patent application of the United States or another country, whichever occurs first, and guarantees the availability of the progeny to a specialist, as determined as prescribed by the Office of the United States Patent and Trademark Office, having authority thereto, pursuant to 35 U.S.C. § 122 and official rules thereunder (including 37 C.F.R. § 1.14 with particular reference to 886 OG 638).

The applicants of this application agree that if the culture of materials during deposit dies or is lost or destroyed during cultivation under suitable conditions, then the materials will be promptly replaced upon notification with others of the same kind. The availability of material deposited shall not be construed as a license to carry out the invention in violation of the rights guaranteed under any government under its patent laws.

The following examples are presented for purposes of illustration only and are not intended to limit the scope of the invention in any way. Moreover, various modifications according to the invention in addition to those presented and described in the present invention will be apparent to one skilled in the art based on the foregoing description and are covered by the scope of the appended claims.

Examples

Example 1 Affinity determination for recombinant Ahtu-EGFRvIII mouse-human chimeric antibody and humanized antibodies.

In this example, the affinity of chimeric and humanized anti-EGFRvIII antibodies was determined

- 52 044329 at 25°C and 37°C.

Ahtu-EGFRvIII mouse (m) antibody, m62G7, generated from hybridomas, was sequenced and subcloned into suitable vectors for expression as mouse-human chimeric antibodies. The murine m62G7 antibody CDR was grafted onto a human scaffold and expressed as a human IgG1 recombinant antibody, h62G7. Affinity variants of h62G7 were generated by introducing mutations into the CDRs of the heavy and light chains. The affinities of the recombinant anti-EGFRvIII chimeric antibody m62G7 and humanized h62G7 antibodies were measured on a Biacore™ T200 surface plasmon resonance biosensor equipped with a high purity CM4 sensor chip coupled to anti-human Fc (GE Healthcare Inc., Piscataway, NJ). Anti-EGFRvIII antibodies were then captured by anti-human Fc. Monomeric 8-histidine tagged humEGFRvIII extracellular domain was then injected as the analyte in a tenfold dilution series with a maximum concentration at 1000 nM. The affinity of anti-EGFRvIII antibodies for humEGFRvIII was measured at both temperatures of 25°C and 37°C (Table 6). None of these antibodies showed detectable binding to 1000 nM 8-histidine tagged recombinant wild-type EGFRwt protein under similar assay conditions.

In table 6, variations are described with reference h62G7 to heavy chain variations, then light chain variations. For example, the antibody clone h62G7-EQ/L6 refers to the h62G7 clone containing the EQ variation in the heavy chain (also referred to herein as h62G7-EQ) and the L6 variation in the light chain (also referred to herein as h62G7-L6). These heavy chain and light chain amino acid sequences are presented in Table. 2. Also, in the present invention, the h62G7 variant may be referred to with either the heavy chain or light chain variant written first - thus, for example, h62G7-EQ/L6 and h62G7-L6/EQ both refer to an antibody that contains h62G7-EQ heavy chain and h62G7-L6 light chain.

Table 6

Antibody 25°C 37°C k _a (l/Mc) k _d (l/c) _Kd (hM) k _a (l/Mc) k _d (l/c) _Kd (hM) m62G7 7.30E+05 6.40E-02 88.7 8.00E+05 l,70E-01 207.0 h62G7-EQ/L6 2.40E+05 1.00E-02 43.8 6.60E+05 7.40E-02 112.8 h62G7-EQ/Ll-DV 2.00E+05 l,20E-05 59.9 3.70E+05 6.90E-02 185.8 h62G7-H14/Ll-DV l,80E+04 2.00E-02 1087.9 6.60E+04 1.00E-01 1539.6 h62G7-H14/L6 l,30E+04 l,30E-02 992.2 4.30E+04 6.80E-02 1583.3

Example 2 Affinity Determination for Human Anti-EGFRvIII Antibodies.

In this example, the affinity of various human anti-EGFRvIII antibodies was determined at 37°C.

To generate human antibodies to EGFRvIII, AlivaMab transgenic mice (Ablexis LLC, San Francisco, CA) were immunized in an alternating schedule with a paraformaldehyde-fixed rat glioblastoma cell line expressing EGFRvIII, F98-npEGFRvIII (American Type Culture Collection, Manassas, VA) and peptides (SEQ ID NO: 227: CGSGSGLEEKKGNYVVTDH) directed to the docking region in EGFRvIII. Hybridomas were prepared using standard techniques. To determine the binding affinity and specificity of these hybridomas for EGFRvIII, antibodies in culture supernatants were captured with anti-mouse Fc using a Biacore™ T200 biosensor equipped with CM4 sensor chips coupled to anti-mouse Fc (Biacore™ AB, Uppsala, Sweden - now GE Healthcare ). Monomeric 8-histidine tagged humEGFRvIII extracellular domain was then injected as the analyte in a tenfold dilution series with a maximum concentration at 1000 nM. The affinity of anti-EGFRvIII antibodies to humEGFRvIII was measured at 37°C (Table 7). None of these hybridoma antibodies showed detectable binding to 1000 nM 8-histidine tagged recombinant wild-type EGFRwt protein under similar assay conditions.

Table 7

Antibody EGFRvIII binding at 37°C k _a (l/Mc) k _d (l/c) _Kd (hM) 42G9 6.88E+04 5.63E-04 8.2 32A10 6.54E+04 6.26E-04 9.6 21E11 6.66E+04 6.32E-04 9.5 49B11 7.64E+04 6.95E-04 9D 46E10 5.97E+04 7.16E-04 12.0 12H6 5.93E+04 7.33E-04 12.4

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19A9 5.58E+04 1.04E-03 18.6 11В11 5.21E+04 1.13E-03 21.7 21E7 6.52E+04 1,ZOE-OZ 19.9 20В9 4.67E+04 1.50E-03 32.1 12V2 7.38E+04 1.79E-03 24.3 11F10 6.63E+04 2.81E-03 42.4 17G11 5.61E+04 3.00E-03 53.5 29D5 1.02E+05 4.24E-03 41.6 14S11 7.55E+04 5.93E-03 78.5 20E12 3.99E+04 1.41E-02 353.4 20G5 1.25E+05 2.89E-02 231.2 26V9 1.31E+05 3.20E-02 244.3 30D8 1.61E+05 2.77E-02 172.0 32G8 6.82E+03 1.22E-02 1788.9 34E7 3.77E+04 1.28E-02 339.5

Example 3 Binding Specificity of Ahtu-EGFRvIII Antibodies to Cell Lines Expressing EGFRvIII by Flow Cytometry.

This example demonstrates the cell specificity of binding of anti-EGFRvIII antibodies to cells expressing EGFRvIII.

To evaluate the cellular binding specificity of anti-EGFRvIII antibodies derived from AlivaMab mice, three isogenic rat glioblastoma cell lines and a human cancer cell line were used: F98 (does not express any form of humEGFR), F98-EGFRwt (expresses wild-type EGFR), F98-npEGFRvIII (expressing EGFRvIII) and A431 (an epidermoid carcinoma cell line overexpressing wild-type EGFR), all obtained from the American Type Culture Collection (Manassas, VA). For cell staining, 500 thousand cells were incubated with 50 μl of hybridoma supernatants for 45 min at 4°C, washed with binding buffer (PBS + 0.5% BSA), with followed by incubation with FITC-conjugated goat anti-mouse Fc specific antibody from Jackson ImmunoResearch Laboratories (West Grove, PA). In table 8A and 8B, the mean fluorescence intensities (MFIs) of EGFRvIII antibodies (excluding clone 20G5) on the EGFRvIII expressing cell line were at least 10-fold higher than on non-expressing cell lines. In fig. 1A, fig. 1B, and FIG. 1C shows examples of FACS binding histograms of three EGFRvIII specific clones that were cloned and expressed as recombinant human IgG1 antibodies, 42G9 (Fig. 1A), 32A10 (Fig. 1B) and 32G8 (Fig. 1C), to three F98 cell lines.

Table 8A

F98 F98-EGFRwt F98-EGFRvIII A431 Antibody MFI % positive MFI % positive MFI % positive MFI % positive Only 2nd AB 170 0.6 202 1.7 258 2.3 592 0.4 aHTH-EGFR(wt (dt) and νΙΠ) 163 0.5 9608 98.3 5329 99.4 55240 100.0 42G9 159 0.4 185 1.6 3247 98.5 538 0.3 32A10 159 0.5 185 1.4 3349 98.3 531 0.2 21E11 159 0.3 184 1.3 3105 98.5 555 0.5 49В11 156 0.6 185 1.3 2980 98.5 599 0.8 46E10 158 0.4 187 1.6 2986 98.7 560 0.5 12N6 157 0.5 188 1.9 3445 98.3 569 0.8 19A9 158 0.5 168 1.6 3100 98.1 578 1.0 11В11 161 0.6 187 1.7 3391 98.2 589 1.2 21E7 159 0.3 184 1.3 3105 98.5 603 1.1 20В9 157 0.3 189 1.8 3418 98.3 558 0.7 12V2 156 0.4 185 1.5 2749 97.9 571 0.8 11F10 155 0.5 187 1.6 3283 98.0 582 1.1 17G11 157 0.6 184 1.5 3357 98.1 556 0.7 29D5 155 0.3 185 1.3 2829 97.9 531 0.4 14S11 157 0.4 185 1.3 3213 98.2 580 0.8

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Table 8B

F98 F98-EGFRwt F98-EGFRvIII A431 Antibody MFI % positive MFI % positive MFI % positive MFI % positive Only 2nd Ab 235 0.2 252 0.2 322 1.3 185 0.7 aHTH-EGFR(wt (dt) and vIII) 245 0.3 6857 97.2 5827 99.4 44493 100.0 20E12 381 6.0 348 3.4 3976 97.9 302 2.6 20G5 1248 16.8 1070 12.6 4639 98.5 391 2.0 26V9 310 4.1 298 2.3 5405 98.6 276 1.7 30D8 296 4.0 280 1.7 5165 98.6 269 1.3 32G8 329 4.9 301 1.6 3734 98.6 271 1.2 34E7 485 6.9 371 4.0 4128 98.5 294 1.1

These results demonstrate the binding specificity of anti-EGFRvni antibodies to cells that express EGFRvIII.

Example 4 Affinity Determination of Fully Human Anti-EGFRvIII Antibodies from a Phage Library.

In this example, the affinity of various human anti-EGFRvIII antibodies was determined at 25°C.

Human anti-EGFRvIII antibodies were sequenced and subcloned into suitable vectors for expression as recombinant human IgG1 antibodies. Antibody affinities were measured at 25°C (Table 9) on a Biacore™ T200 surface plasmon resonance biosensor equipped with a CM4 anti-human Fc sensor chip (GE Healthcare Inc., Piscataway, NJ). Anti-EGFRvIII antibodies were captured using anti-human Fc. Monomeric 8-histidine tagged humEGFRvIII extracellular domain was then injected as the analyte in 10-fold serial dilutions starting at 1000 nM. Of the two antibodies, only C6 showed very weak but detectable binding to 1000 nM 8-histidine tagged recombinant wild-type EGFRwt protein at 25°C.

Table 9

Antibody EGFRvIII binding at 25°C k _a (1/Ms) k _d (l/c) K _o (nM) AT 5 2.08E+04 1.41E-02 677.9 C6 1.68E+04 8.94E-03 532.1

Example 5: Affinity Determination for Recombinant Single Chain Fv Formatted Anti-EGFRvIII Antibodies.

In this example, the affinity of various recombinant single chain Fv formatted anti-EGFRvIII antibodies was determined at 37°C.

To convert a conventional antibody into a single chain Fv (scFv) fragment, the heavy chain variable domain and the light chain variable domain were joined together using a (GGGGS)4 (SEQ ID NO: 202) linker. The scFv fragment was then fused to the human IgG1 Fc component to facilitate recombinant expression and purification. The affinities of the scFv-Fc fusion proteins were measured at 37°C on a Biacore™ T200 surface plasmon resonance biosensor equipped with a CM4 anti-human Fc sensor chip (GE Healthcare Inc., Piscataway, NJ) as described above. scFv-Fc proteins were captured using anti-human Fc. Monomeric 8-histidine tagged humEGFRvIII extracellular domain was then injected as the analyte in 10-fold serial dilutions starting at 1000 nM. In table 10 shows that the scFv reformatted fusion proteins retained binding to EGFRvIII and that the affinities of the scFv-Fc proteins in both HL (heavy chain variable domain at the N-terminus) and LH (light chain variable domain at the N-terminus) orientations were comparable to their corresponding parts of a conventional antibody, listed in table. 6, 7 and 9. These scFv-Fc proteins were also tested for binding to 1000 nM 8-histidine tagged recombinant wild-type EGFRwt protein at 25°C, but none showed significant binding.

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Table 10

scFv-Fc protein EGFRvIII binding at 37°C ka(1/Ms) kd(l/c) KD(hM) h62G7-L6/EQ 7.5E+05 7.6E-02 100.9 C6.HL 1.9E+04 2.0E-02 1063.8 14C11.HL 4.2E+04 4, ZE-OZ 104.3 14C11.LH 4.8E+04 6.5E-03 136.0 20B9.HL 2.4E+04 1.8E-03 73.8 20B9.LH 3.0E+04 2.5E-03 82.7 32A10.HL 4.7E+04 1.2E-03 24.5 32A10.LH 4DE+04 1.2E-03 29.8 42G9.HL 5.6E+04 1DE-03 18.8 42G9.LH 4.0E+04 8.8E-04 22.1 26B9.HL 5.7E+04 4.0E-02 704.9 26B9.LH 6.9E+04 3.4E-02 494.2 30D8.HL 2,ЗЭ+05 5,ZE-02 224.6 30D8.LH 1DE+05 3.8E-02 348.6 20E12.HL 2DE+04 1.9E-02 893.7 20E12.LH 1.6E+04 1.5E-02 932.5 32G8.HL 8.7E+03 1,ZE-02 1490.0

Example 6. Production and detection of T cells with EGFRvIII specific chimeric antigen receptor (CAR).

In this example, the expression and binding specificity of EGFRvIII antigen by specific CAR T cells was determined.

PBMCs from healthy donors obtained from AllCells (Alameda, CA) were thawed according to the manufacturer's instructions and cultured overnight in X-Vivo™-15 medium (Lonza, Walkersville, MD) supplemented with 5% human serum. T cells were activated for 3 days in X-Vivo™15 medium (Lonza) supplemented with 20 ng/ml human IL-2, 5% human serum, and Dynabeads Human CD3/CD28 T activator at a particle:cell ratio of 1:1 (Life Technologies, Carlsbad, CA). T cells were then transduced with a bicistronic lentiviral vector into which the BFP-T2A-EGFRvIII specific CAR expression cassette was anchored under the control of the EFla promoter at a multiplicity of infection (MOI) of 5. In this construct, the EGFRvIII specific CAR was co-expressed with BFP (blue fluorescent protein) , to facilitate the detection of EGFRvIII specific CARs. The EGFRvIII specific CARs contained the VH and VL sequences of various anti-EGRRvIII clones (h62G7-L6/EQ, 14C11, 20B9, 32A10, 42G9, C6, 20E12, 26B9, 30D8, and 32G8; described elsewhere in the present invention). CAR T cells were maintained in culture until 14 days after transduction. The percentage of cells expressing EGFRvIII specific CARs was monitored over time (at days 4, 9, and 14 after lentiviral transduction of primary T cells) by flow cytometry using BFP (blue fluorescent protein) for detection (Fig. 2A) (determined by percentage of BFP positive viable cells).

To determine the target binding specificity of CAR, recombinant proteins EGFRvIIImFc and EGFR-mFc, which contain the extracellular domain of either humEGFRvIII or wild-type human EGFR, respectively, fused to the mouse IgGl-Fc domain, were produced in HEK293 cells. Target binding specificity was determined by incubating various EGFRvIII specific CAR T cells with either EGFRvIII-mFc or EGFR-mFc protein, then using a PE-labeled goat anti-mouse Fc secondary antibody (Jackson ImmunoResearch) and analyzed by flow cytometry on Day 4 post-T transduction cells with vectors encoding CARs containing various EGFRvIII specific clones (h62G7-L6/EQ, 14C11, 20B9, 32A10, 42G9, C6, 20E12, 26B9, 30D8, and 32G8). As shown in FIG. 2B, CAR T cells containing VH and VL sequences of clones h62G7-L6/EQ, 14C11, 20B9, 32A10, 42G9, 20E12, 26B9, and 30D8 bind to EGFRvIII-mFc, but do not significantly bind to EGFR-mFc. At Day 14 post-transduction, the correlation of CAR expression, as detected by BFP, in cells and the binding of recombinant EGFRvIII-mFc to cells was determined. A representative data set is shown in FIG. 2C, which shows the expression of CARs containing ten different EGFRvIII specific clones (h62G7-L6/EQ, 14C11, 20B9, 32A10, 42G9, C6, 20E12, 26B9, 30D8, and 32G8) in CAR T cells at Day 14 post-transduction. as determined by BFP detection in cells and cell binding of recombinant EGFRvIII-mFc.

These results indicate that CAR T cells containing CARs including the VH and VL sequences of clones h62G7-L6/EQ, 14C11, 20B9, 32A10, 42G9, 20E12, 26B9, and 30D8 bind to EGFRvIII-mFc, but do not significantly bind to EGFR-mFc.

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Example 7 Target-dependent and -independent degranulation of EGFRvIII specific CAR T cells.

In this example, the degranulation activity of EGFRvIII specific CAR T cells was determined in the presence and absence of targeted expression of malignant cell lines.

Five cell lines were used to evaluate the degranulation activity of CAR T cells. The human lung cancer cell line NCI-H522 and the glioblastoma cell line U87MG were obtained from the American Type Culture Collection (Manassas, VA) and cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Mediatech Inc., Manassas, VA). Because most malignant cell lines do not express EGFRvIII, NCI-H522 (which does not express detectable levels of wild-type EGFR or EGFRvIII) was transduced with a lentiviral vector encoding the full-length EGFRvIII gene (SEQ ID NO: 201) to generate NCI-H522-EGFRvIII, which expresses low EGFRvIII level. To generate isogenic human glioblastoma cell lines that express various EGFR proteins, the endogenous wild-type EGFR gene U87MG was first knocked out to create the EGFR-null U87KO cell line. U87-KO cells were then transduced with a lentiviral vector encoding the full-length wild-type EGFR gene (accession number NP_005219) to generate an EGFR-overexpressing cell line, U87-KO-EGFRwt. Similarly, U87-KO-EGFRvIII (which overexpresses EGFRvIII) was generated from U87-KO by lentiviral transduction using a vector encoding the full-length EGFRvIII gene (SEQ ID NO: 201).

For the degranulation assay, 100K T cells expressing various EGFRvIII specific CARs (containing clones h62G7-L6/EQ, 14C11, 20B9, 32A10, 42G9, C6, 20E12, 26B9, 30D8, and 32G8) were incubated in 96-well plates together with an equal number of malignant cells expressing varying levels of EGFRvIII protein or EGFRwt protein. Co-cultures were maintained in a final volume of 100 μl of X-Vivo™-15 medium (Lonza, Walkersville, MD) for 6 h at 37°C with 5% CO ₂ . CD107a staining was performed upon cell stimulation by adding a fluorescent anti-CD107a antibody (Miltenyi Biotec, San Diego, CA) to the coculture start, along with a final concentration of 1 μg/ml anti-CD49d (BD Pharmingen, San Diego, CA), 1 μg/ml anti-CD28 (Miltenyi Biotec), and 1x Monensin (eBioscience, San Diego, CA) solution. After an incubation period of 6 h, cells were stained with reducible viable dye and fluorochrome-conjugated anti-CD8 antibody (Miltenyi Biotec) and analyzed by flow cytometry. Degranulation activity was determined as % viable /CD8+/BFP+/CD107a+ cells, and by determining the mean fluorescence intensity (MFI) signal for CD107a staining for CD8+/BFP+ cells. Degranulation studies were performed on Day 14 after CAR transduction. In fig. Figure 3 shows results from two donor PBMCs for each EGFRvIII specific CAR. With the exception of CAR T cells containing clones C6 and 32G8, increased degranulation activity (more CAR-T alone) was observed only when EGFRvIII specific CAR T cells were co-cultured with cell lines that express EGFRvIII, such as NCIH522-EGFRvIII and U87-KO-EGFRvIII.

These results indicate that EGFRvIII specific CAR T cells exhibit degranulation activity in the presence of EGFRvIII-expressing tumor cells, but do not exhibit degranulation activity in the presence of tumor cells that do not express EGFRvIII.

Example 8. Secretion of interferon gamma (IFNy) EGFRvIII by specific CAR T cells.

This example shows the level of IFNy secretion of EGFRvIII specific CAR T cells when EGFRvIII specific CAR T cells are co-cultured with cell lines expressing the target protein.

EGFRvIII specific CAR T cells expressing CARs containing various EGFRvIII specific clones (h62G7-L6/EQ, 14C11, 20B9, 32a1o, 42G9, C6, 20E12, 26B9, 30D8, and 32G8) were incubated in 96-well plates (100 thousand . cells/well), together with equal numbers of cells expressing varying levels of EGFRvIII or EGFRwt proteins (NCI-H522, NCI-H522-EGFRvIII, U87-KO, U87-KO-EGFRwt, and U87-KO-EGFRvIII cells; each were described above in Example 7). Co-cultures were maintained in a final volume of 100 μl of X-Vivo™-15 medium (Lonza) for 18 h at 37°C with 5% CO2. The supernatant was then collected and frozen. IFNy in the supernatant was measured using a Human IFN-gamma Quantikine ELISA Kit (R&D systems, Minneapolis, MN) according to the manufacturer's specifications. As shown in Fig. 4, for most CAR T cells, the level of IFNy secretion correlates with the amount of EGFRvIII expressed by co-cultured cells: the low NCI-H522-EGFRvIII expressor induces a small amount of IFNy, the high U87-KOEGFRvIII expressor induces a large amount of IFNy. T cells expressing the EGFRvIII specific 32G8 CAR clone did not secrete significant levels of IFNy under all conditions tested. On the other hand, EGFRvIII T cells, a specific C6 CAR clone, secrete high levels of IFNy when cocultured with either U87-KO-EGFRwt or U87-KO-EGFRvIII cells.

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These results suggest that for most EGFRvIII specific CAR T cells, the level of IFNy secretion correlates with the amount of EGFRvIII expressed by the cocultured cells

Example 9. Cytotoxicity of EGFRvIII specific CAR T cells.

In this example, the cytotoxicity of EGFRvIII specific CAR T cells was determined by co-cultivating EGFRvIII specific CAR T cells with cell lines expressing the target protein.

EGFRvIII specific CAR T cells expressing CARs containing various EGFRvIII specific clones (h62G7-L6/EQ, 14C11, 20B9, 32A10, 42G9, C6, 20E12, 26B9, 30D8, and 32G8) were seeded in 96-well plates (400 thousand . cells/well), together with 20 thousand target cells expressing various levels of EGFRvIII protein. The target cells were: U87-KO-EGFRwt, NCI-H522EGFRvIII, and U87-KO-EGFRvIII described above. Target (EGFRvIII positive: NCI-H522EGFRvIII and U87-KO-EGFRvIII) and control (EGFRvIII negative: U87-KO-EGFRwt) cells were plated and labeled with the fluorescent intracellular dye CFSE before being cocultured with EGFRvIII specific CAR T cells . Cocultures were incubated for 4 h at 37°C with 5% CO ₂ . After this incubation period, cells were labeled with a reducible viable dye and analyzed by flow cytometry. The viability of each cell population (EGFRvIII positive target cells or EGFRvIII negative control cells) was determined and the % lysis of specific cells was calculated. Cytotoxicity studies were performed 14 days after CAR T cell transduction. All EGFRvIII specific CAR T cells, with the exception of 32G8, were able to lyse both target low-expressing cells (NCI-H522-EGFRvIII) and target high-expressing cells (U87KO-EGFRvIII) to varying degrees. Additionally, EGFRvIII-specific C6 CAR clone T cells lysed both wild-type EGFR and EGFRvIII-expressing cells (Fig. 5).

These results indicate that EGFRvIII-specific CAR T cells effectively kill cells that express EGFRvIII.

Example 10. In vivo studies of EGFRvIII specific CAR T cells using the U87-KOEGFRvIII model.

This example demonstrates the antitumor activity of EGFRvIII specific CAR T cells in a subcutaneous U87-KO-EGFRvIII GBM xenograft model.

Three million U87-KO-EGFRvIII GBM tumor cells (described above) were implanted subcutaneously into 5- to 6-week-old NSG mice (Jackson Laboratory, Sacramento, CA). Tumor volume was measured once a week using a caliper and calculated according to the following formula:

Tumor volume = (length x width ² )/2.

On day 8 after tumor implantation, animals were randomized according to tumor size with five animals per group and administered a single dose of 6 million CAR positive EGFRvIII specific CAR T cells (expressing EGFRvIII specific clone 14C11, 32A10 or 26B9; clones are described elsewhere in the present invention) or an equivalent total number of non-transduced T cells by bolus injection into the tail vein. In fig. 6 shows that 14C11, 32A10 and 26B9 EGFRvIII specific CAR T cells, but not untransduced T cells, inhibit the growth of U87-KO-EGFRvIII tumor xenograft in vivo.

These results indicate that EGFRvIII-specific CAR T cells effectively inhibit tumor growth of EGFRvIII-expressing cells in vivo.

Example 11 Surface expression of EGFRvIII specific CARs containing the R2 suicide sequence.

This example shows the expression of EGFRvIII specific CARs containing the R2 suicide sequence in T cells.

The R2 suicide sequence is a suicide sequence based on the CD20 epitope and contains two tandem copies of the rituximab recognition epitope. The R2 sequence was inserted between the scFv and CD8a loop sequences of the various EGFRvIII specific CARs described elsewhere in the present invention (14C11, 32A10, 30D8, and 26B9) to create the various EGFRvIII-R2 CARs (referred to herein as 14C11-R2, 32A10- R2, 30D8-R2, and 26B9-R2, respectively). T cells were transduced with a lentiviral vector that anchors various EGFRvIII-R2 CAR expression cassettes under the control of the EF1a promoter at a multiplicity of infection (MOI) of 5 or 25 (for clones 26B9-R2 and 30D8R2). CAR T cells were maintained in culture until 14 or 15 days after transduction. CAR expression was monitored over time (at Day 4, Day 9/10, and Day 14/15 after T cell transduction) by flow cytometry using biotinylated recombinant proteins: EGFRvIII-mFc or rituximab to cells (conjugated to EZ-Link™ Sulfo -NHS-SS-Biotin; ThermoFisher, Waltham, MA), then with PE conjugated streptavidin (BD Biosciences, San Diego, CA). Percentages of CAR positive cells as detected with biotinylated EGFRvIII-mFc and biotinylated-

Claims (13)

rituximab are presented in Fig. 7 and 8, respectively. Binding to non-transduced T cells (NTD) was used as a control. These results demonstrate the expression in T cells of EGFRvIII-specific CARs containing the R2 suicide sequence. Example 12. Cytotoxicity of EGFRvIII-specific R2 CAR T cells. This example demonstrates the cytotoxicity of EGFRvIII-specific R2 CAR T cells when co-cultured with target expressing cell lines. Cytotoxicity assays were performed as described in Example 9 using EGFRvIII-R2 CAR 14C11-R2, 32A10-R2, 30D8-R2, and 26B9-R2, which are described in Example 11. All EGFRvIII-specific R2 CAR T cells tested were able to lyse as low-level target expressing cells (NCI-H522-EGFRvIII) and high-level target expressing cells (U87KO-EGFRvIII) to varying degrees (Fig. 9). These results indicate that EGFRvIII-specific R2 CAR T cells effectively kill cells that express EGFRvIII. Although the described principles have been described with reference to various applications, methods, kits and compositions, it should be appreciated that various changes and modifications may be made without deviating from the principles disclosed in the present invention and the invention claimed below. The above examples are presented to better illustrate the principles disclosed and are not intended to limit the scope of the principles presented in the present invention. Although the principles of the present invention have been described in the context of these exemplary embodiments, one skilled in the art will readily appreciate that various variations and modifications of these exemplary embodiments are possible without undue experimentation. All such variations and modifications are within the scope of the claimed invention. All references cited in the present invention, including patents, patent applications, documents, manuals, etc., and references are cited in these sources to the extent that they are incorporated herein by reference in their entirety. In the event that one or more of the included references and related materials differ from or are inconsistent with this application, including, but not limited to, terms defined, terms used, techniques described, or others, then the terms of this application shall apply. The foregoing description and examples describe in detail certain specific embodiments of the invention and describe the best embodiment contemplated by the inventors. However, it should be appreciated that, regardless of how much detail the foregoing may appear in the specification, the invention may be practiced in various ways and the invention should be construed in accordance with the appended claims and any equivalents thereof. CLAIM 1. Chimeric antigen receptor (CAR) specific for epidermal growth factor receptor variant III (EGFRvIII), containing an EGFRvIII-binding domain, a transmembrane domain and an intracellular signaling domain, where the EGFRvIII-binding domain contains: (a) a heavy chain variable region (VH) containing three complementarity determining regions: VH complementarity determining region 1 (VH CDR1), VH complementarity determining region 2 (VH CDR2) and VH complementarity determining region 3 (VH CDR3) of 42G9, located sequentially from N-terminus to C-terminus VH CDR1, VH CDR2 and VH CDR3, where VH CDR1 contains the amino acid sequence of one of SEQ ID NO: 74-76, VH CDR2 contains the amino acid sequence of one of SEQ ID NO: 77-78 and VH CDR3 contains the amino acid sequence SEQ ID NO: 79; and a light chain variable region (VL) containing three complementarity determining regions: VL complementarity determining region 1 (VL CDR1), VL complementarity determining region 2 (VL CDR2) and VL complementarity determining region 3 (VL CDR3) of 42G9, located sequentially from N-terminus to C-terminus of VL CDR1, VL CDR2 and VL CDR3, wherein VL CDR1 contains the amino acid sequence of SEQ ID NO: 156, VL CDR2 contains the amino acid sequence of SEQ ID NO: 157 and VL CDR3 contains the amino acid sequence of SEQ ID NO: 158; or (b) a VH region containing three complementarity determining regions VH CDR1, VH CDR2 and VH CDR3 from 32A10, located sequentially from the N-terminus to the C-terminus of VH CDR1, VH CDR2 and VH CDR3, where VH CDR1 contains the amino acid sequence of one of SEQ ID NO: 80-82, VH CDR2 contains the amino acid sequence of one of SEQ ID NO: 83-84 and VH CDR3 contains the amino acid sequence of SEQ ID NO: 85; and a VL region containing three complementarity determining regions VL CDR1, VL CDR2 and VL CDR3 from 32A10, located sequentially from the N-terminus to the C-terminus of VL CDR1, VL CDR2 and VL - 59 044329 CDR3, wherein VL CDR1 contains the amino acid sequence of SEQ ID NO: 159, VL CDR2 contains the amino acid sequence of SEQ ID NO: 160 and VL CDR3 contains the amino acid sequence of SEQ ID NO: 161; or (c) a VH region containing three complementarity determining regions VH CDR1, VH CDR2 and VH CDR3 from 20B9, located sequentially from the N-terminus to the C-terminus of VH CDR1, VH CDR2 and VH CDR3, wherein VH CDR1 contains the amino acid sequence of one of SEQ ID NO: 80-82, VH CDR2 contains the amino acid sequence of one of SEQ ID NO: 86-87 and VH CDR3 contains the amino acid sequence of SEQ ID NO: 79; and a VL region containing three complementarity determining regions VL CDR1, VL CDR2 and VL CDR3 from 20B9, located sequentially from the N-terminus to the C-terminus of VL CDR1, VL CDR2 and VL CDR3, where VL CDR1 contains the amino acid sequence of SEQ ID NO: 162 , VL CDR2 contains the amino acid sequence of SEQ ID NO: 163 and VL CDR3 contains the amino acid sequence of SEQ ID NO: 164; or (d) a VH region containing three complementarity determining regions VH CDR1, VH CDR2 and VH CDR3 from 14C11, located sequentially from the N-terminus to the C-terminus of VH CDR1, VH CDR2 and VH CDR3, where VH CDR1 contains the amino acid sequence of one of SEQ ID NO: 88-90, VH CDR2 contains the amino acid sequence of one of SEQ ID NO: 91-92 and VH CDR3 contains the amino acid sequence of SEQ ID NO: 85; and a VL region containing three complementarity determining regions VL CDR1, VL CDR2 and VL CDR3 from 14C11, located sequentially from the N-terminus to the C-terminus of VL CDR1, VL CDR2 and VL CDR3, where VL CDR1 contains the amino acid sequence of SEQ ID NO: 165 , VL CDR2 contains the amino acid sequence of SEQ ID NO: 163 and VL CDR3 contains the amino acid sequence of SEQ ID NO: 161; or (e) a VH region containing the three complementarity determining regions VH CDR1, VH CDR2 and VH CDR3 from 30D8, located sequentially from the N-terminus to the C-terminus of VH CDR1, VH CDR2 and VH CDR3, wherein VH CDR1 contains the amino acid sequence of one of SEQ ID NO: 109-111, VH CDR2 contains the amino acid sequence of one of SEQ ID NO: 112-113 and VH CDR3 contains the amino acid sequence of SEQ ID NO: 114; and a VL region containing three complementarity determining regions VL CDR1, VL CDR2 and VL CDR3 from 30D8, located sequentially from the N-terminus to the C-terminus of VL CDR1, VL CDR2 and VL CDR3, where VL CDR1 contains the amino acid sequence of SEQ ID NO: 182 , VL CDR2 contains the amino acid sequence of SEQ ID NO: 183 and VL CDR3 contains the amino acid sequence of SEQ ID NO: 184; or (f) a VH region containing three complementarity determining regions VH CDR1, VH CDR2 and VH CDR3 from 20E12, located sequentially from the N-terminus to the C-terminus of VH CDR1, VH CDR2 and VH CDR3, wherein VH CDR1 contains the amino acid sequence of one of SEQ ID NO: 115-117, VH CDR2 contains the amino acid sequence of one of SEQ ID NO: 118-119 and VH CDR3 contains the amino acid sequence of SEQ ID NO: 120; and a VL region containing three complementarity determining regions VL CDR1, VL CDR2 and VL CDR3 from 20E12, located sequentially from the N-terminus to the C-terminus of VL CDR1, VL CDR2 and VL CDR3, where VL CDR1 contains the amino acid sequence of SEQ ID NO: 185 , VL CDR2 contains the amino acid sequence of SEQ ID NO: 186 and VL CDR3 contains the amino acid sequence of SEQ ID NO: 184; or (g) a VH region containing three complementarity determining regions VH CDR1, VH CDR2 and VH CDR3 from 26B9, located sequentially from the N-terminus to the C-terminus of VH CDR1, VH CDR2 and VH CDR3, wherein VH CDR1 contains the amino acid sequence of one of SEQ ID NO: 121-123, VH CDR2 contains the amino acid sequence of one of SEQ ID NO: 124-125 and VH CDR3 contains the amino acid sequence of SEQ ID NO: 126; and a VL region containing three complementarity determining regions VL CDR1, VL CDR2 and VL CDR3 from 26B9, located sequentially from the N-terminus to the C-terminus of VL CDR1, VL CDR2 and VL CDR3, wherein VL CDR1 contains the amino acid sequence of SEQ ID NO: 187 , VL CDR2 contains the amino acid sequence of SEQ ID NO: 188 and VL CDR3 contains the amino acid sequence of SEQ ID NO: 189; or (h) a VH region containing three complementarity determining regions VH CDR1, VH CDR2 and VH CDR3 from C6, located sequentially from the N-terminus to the C-terminus of VH CDR1, VH CDR2 and VH CDR3, wherein VH CDR1 contains the amino acid sequence of one of SEQ ID NO: 137-139, VH CDR2 contains the amino acid sequence of one of SEQ ID NO: 140-141 and VH CDR3 contains the amino acid sequence of SEQ ID NO: 142; a VL region containing three complementarity determining regions VL CDR1, VL CDR2 and VL CDR3 from C6, located sequentially from the N-terminus to the C-terminus of VL CDR1, VL CDR2 and VL CDR3, where VL CDR1 contains the amino acid sequence of SEQ ID NO: 195, VL CDR2 contains the amino acid sequence of SEQ ID NO: 196 and VL CDR3 contains the amino acid sequence of SEQ ID NO: 197; and (i) a VH region containing three complementarity determining regions VH CDR1, VH CDR2 and VH CDR3 from 12B2, located sequentially from the N-terminus to the C-terminus of VH CDR1, VH CDR2 and VH CDR3, where VH CDR1 contains the amino acid sequence of one of SEQ ID NO: 74-76, VH CDR2 contains the amino acid sequence of one of SEQ ID NO: 102 or 103 and VH CDR3 contains the amino acid sequence of SEQ ID NO: 104; and a VL region containing three complementarity determining regions VL CDR1, VL CDR2 and VL CDR3 from 12B2, located sequentially from the N-terminus to the C-terminus of VL CDR1, VL CDR2 and VL CDR3, wherein VL CDR1 contains the amino acid sequence of SEQ ID NO: 176 , VL CDR2 contains the amino acid sequence of SEQ ID NO: 172 and VL CDR3 contains the amino acid sequence of SEQ ID NO: 177. 2. A chimeric antigen receptor (CAR) specific for epidermal growth factor receptor variant III (EGFRvIII) and containing an EGFRvIII-binding domain, a transmembrane domain and an intracellular signaling domain, wherein the EGFRvIII-binding domain contains a single chain variable fragment (scFv) containing a VH region and a VL region selected from the group consisting of: i) a VH region containing the amino acid sequence of SEQ ID NO: 9, and a VL region containing the amino acid sequence of SEQ ID NO: 10 (42G9); ii) a VH region containing the amino acid sequence of SEQ ID NO: 11, and a VL region containing the amino acid sequence of SEQ ID NO: 12 (32A10); iii) a VH region containing the amino acid sequence of SEQ ID NO: 13, and a VL region containing the amino acid sequence of SEQ ID NO: 14 (20B9); iv) a VH region containing the amino acid sequence of SEQ ID NO: 15, and a VL region containing the amino acid sequence of SEQ ID NO: 16 (14C11); v) a VH region containing the amino acid sequence of SEQ ID NO: 37, and a VL region containing the amino acid sequence of SEQ ID NO: 38 (30D8); vi) a VH region containing the amino acid sequence of SEQ ID NO: 39, and a VL region containing the amino acid sequence of SEQ ID NO: 40 (20E12); vii) a VH region containing the amino acid sequence of SEQ ID NO: 41, and a VL region containing the amino acid sequence of SEQ ID NO: 42 (26B9); and viii) a VH region containing the amino acid sequence of SEQ ID NO: 48, and a VL region containing the amino acid sequence of SEQ ID NO: 49 (C6); and ix) a VH region containing the amino acid sequence of SEQ ID NO: 30, and a VL region containing the amino acid sequence of SEQ ID NO: 31 (12B2). 3. A chimeric antigen receptor (CAR) specific for epidermal growth factor receptor variant III (EGFRvIII) and comprising an EGFRvIII-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the EGFRvIII-specific chimeric antigen receptor (CAR) contains an amino acid sequence selected from group consisting of SEQ ID NOs: 53-57 and 59-61. 4. The EGFRvIII-specific CAR according to one of claims 1 to 3, wherein the intracellular signaling domain is a first intracellular signaling domain and optionally the CAR further comprises a second intracellular signaling domain. 5. The EGFRvIII-specific CAR of claim 4, wherein the first intracellular signaling domain comprises a CD3zeta signaling domain and the optional second intracellular signaling domain comprises a 41BB signaling domain. 6. The EGFRvIII-specific CAR according to one of claims 1 to 5, further comprising a stem domain between the extracellular ligand binding domain and the first transmembrane domain. 7. The EGFRvIII-specific CAR of claim 6, wherein the stem domain is selected from the group consisting of: a human CD8a hinge, a human CD28 hinge, an IgG1 hinge, and an FcyRIIIa hinge. 8. EGFRvIII-specific CAR according to one of claims 1 to 7, wherein the transmembrane domain comprises a CD8a chain transmembrane domain. 9. An isolated polynucleotide having a nucleotide sequence encoding an EGFRvIII-specific CAR according to one of claims 1 and 4-8. 10. An isolated polynucleotide having a nucleotide sequence encoding an EGFRvIII-specific CAR according to one of claims 2 and 4-8. 11. An isolated polynucleotide having a nucleotide sequence encoding an EGFRvIII-specific CAR according to one of claims 3-8. 12. An isolated expression vector containing a polynucleotide according to one of claims 9-11. 13. An isolated engineered immune cell of hematopoietic origin for the treatment of cancer, containing a polynucleotide according to one of claims 9-11, or an expression vector according to claim 12, where said immune cell expresses on its cell surface membrane the EGFRvIII-specific CAR according to one of claims 1 -8, while the immune cell is an inflammatory -