EGFRvIII binding proteins

Abstract

The present invention generally relates to proteins that bind to human epidermal growth factor receptor variant III (EGFRvIII). The present invention also relates to chimeric antigen receptors (CARs) that contain EGFRvIII binding proteins, nucleic acids and vectors that encode CARs, and cells that contain CARs. The present invention also relates to methods for treating and diagnosing diseases such as cancer.

Description

[Technical field]

[0001] This application claims priority to AU2021901227, filed April 26, 2021, and AU2021901226, filed April 26, 2021, the entire contents of each of which are incorporated by reference herein.

[0002] FIELD OF THEINVENTION The present invention generally relates to proteins that bind to human epidermal growth factor receptor variant III (EGFRvIII). The present invention also relates to chimeric antigen receptors (CARs) that contain EGFRvIII binding proteins, nucleic acids and vectors that encode CARs, and cells that contain CARs. The present invention also relates to methods for treating and diagnosing diseases such as cancer. [Background technology]

[0003] 2. Background of the Invention EGFRvIII is the most common mutated variant of EGFR observed in human tumors and can be detected in approximately 30% of newly diagnosed cases of glioblastoma multiforme (GBM). GBM is the most aggressive and malignant adult brain tumor with an expected 5-year survival rate of 5%. Current treatments of surgery, chemotherapy, and radiation therapy have not improved survival over the last few decades.

[0004] EGFRvIII mutations have also been shown to be present in murine fibroblasts and in breast, ovarian, colon, lung, and prostate cancer cells.

[0005] The mutation present in EGFRvIII is an in-frame deletion of 801 base pairs from exons 2 to 7, which splits the codon and creates a glycine residue at the junction, resulting in a tumor-specific and immunogenic neoepitope and a constitutively active form of the protein. This mutation also confers resistance to radiation and chemotherapy, further worsening the prognosis of patients with this mutation.

[0006] Therefore, there is a need for new EGFRvIII binding molecules that can be used in cancer therapy. Summary of the Invention

[0007] The present inventors have identified binding proteins that bind to EGFRvIII with high affinity. These binding proteins can be used both alone (e.g., as isolated antibodies or fragments thereof) or within chimeric antigen receptor (CAR) constructs, e.g., for CAR-T cell therapy.

[0008] Thus, in one aspect, the invention provides binding proteins comprising an antigen-binding site that binds to human epidermal growth factor receptor variant III (EGFRvIII) with an affinity of at least about 1 nM. In some embodiments, the binding protein binds to EGFRvIII with an affinity of at least about 0.75 nM, at least about 0.5 nM, at least about 0.25 nM, at least about 0.1 nM, at least about 0.075 nM, at least about 0.05 nM, at least about 0.025 nM, or at least about 0.01 nM.

[0009] In some embodiments, affinity is determined in an assay in which the binding protein is immobilized and soluble EGFRvIII is contacted with the binding protein. In one embodiment, the soluble EGFRvIII is the extracellular domain of EGFRvIII (e.g., an EGFRvIII construct in which the transmembrane and intracellular domains have been removed).

[0010] In one embodiment, the affinity of the binding protein for EGFRvIII is at least 20 times, at least 100 times, or at least 1000 times stronger than its affinity for wild-type epidermal growth factor receptor (EGFR). In one embodiment, the binding protein does not detectably bind to EGFR in an assay in which the binding protein is immobilized and soluble EGFR is contacted with the binding protein. In one embodiment, the affinity of the binding protein for EGFRvIII is at least 20 times, at least 40 times, at least 60 times, at least 80 times, at least 100 times, at least 200 times, at least 300 times, at least 400 times, at least 500 times, at least 600 times, at least 800 times, at least 1000 times, at least 5000 times, or at least 10000 times stronger than its affinity for wild-type epidermal growth factor receptor (EGFR). Advantageously, in such an embodiment, the binding protein has a higher tumor specificity as a result of its lower affinity for EGFR expressed in non-cancerous cells.

[0011] The inventors have discovered that in some embodiments, the binding proteins can also bind to EGFR when it is overexpressed on the surface of cancer cells. Thus, in some embodiments, the binding proteins bind to EGFR on cancer cells that overexpress EGFR. In some embodiments, the binding proteins bind to EGFR on cancer cells that overexpress EGFR due to amplification of the EGFR gene. In one embodiment, the binding proteins bind to an epitope that is exposed on cells (e.g., cancer cells) that express aberrant EGFR or overexpressed EGFR, but not on wild-type cells. In one embodiment, the binding proteins bind to EGFR that is present on cancer cells, but not detectable on wild-type cells. Advantageously, in these embodiments, the binding proteins can be used in therapy against cancers that overexpress EGFR (e.g., as a result of gene amplification) and cancers that express EGFRvIII mutants, thereby expanding the potential therapeutic indications for these binding proteins.

[0012] In some embodiments, the binding protein binds to EGFR on glioblastoma cells or epithelial cells.In one embodiment, the affinity of the binding protein to the EGFR that is overexpressed by cancer cells is at least 20 times, at least 100 times, or at least 1000 times stronger than the affinity of the EGFR that is expressed by wild-type cells, or the binding protein does not detectably bind to the EGFR that is expressed by wild-type cells.Affinity can be evaluated by preformed assays using cell lines such as U87, U251 or A431.

[0013] In some embodiments, the binding protein comprises a heavy chain variable region (V) comprising the amino acid sequence set forth in SEQ ID NO:3. H ) and a light chain variable region (V L ) binds to the same epitope in EGFRvIII as that bound by a protein containing

[0014] In one aspect of the invention, a human epidermal growth factor receptor variant III (EGFRvIII) binding protein is provided that comprises an antigen binding domain that binds EGFRvIII, which competitively inhibits binding of EGFRvIII to an antibody comprising: A heavy chain variable region (V) comprising CDR1, CDR2, and CDR3 comprising the sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively. H ); and A light chain variable region (V) comprising CDR1, CDR2, and CDR3 comprising the sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively. L );or A heavy chain variable region (V) comprising CDR1, CDR2, and CDR3 comprising the sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively. H ); and A light chain variable region (V) comprising CDR1, CDR2, and CDR3 comprising the sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively. L ).

[0015] In one embodiment, the binding protein competitively inhibits binding of EGFRvIII to an antibody comprising: V comprising the amino acid sequence set forth in SEQ ID NO:3 H and V comprising the amino acid sequence set forth in SEQ ID NO:4 L ;or V comprising the amino acid sequence set forth in SEQ ID NO:5 H and V comprising the amino acid sequence set forth in SEQ ID NO:6 L .

[0016] In one embodiment, the EGFRvIII binding protein binds to human EGFRvIII with an affinity of at least about 1 nM.

[0017] In one embodiment, the binding protein comprises: A heavy chain variable region (V) comprising CDR1, CDR2, and CDR3 comprising the sequences set forth in SEQ ID NOs:7, 8, and 9, respectively. H ); and / or A light chain variable region (V) comprising CDR1, CDR2, and CDR3 comprising the sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively. L );or A heavy chain variable region (V) comprising CDR1, CDR2, and CDR3 comprising the sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively. H ); and / or A light chain variable region (V) comprising CDR1, CDR2, and CDR3 comprising the sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively. L ).

[0018] In one embodiment, the binding protein comprises: A heavy chain variable region (V) comprising CDR1 comprising an amino acid sequence having no more than two amino acid substitutions relative to SEQ ID NO: 13, CDR2 comprising an amino acid sequence having no more than four amino acid substitutions relative to SEQ ID NO: 14, and CDR3 comprising an amino acid sequence having no more than three amino acid substitutions relative to SEQ ID NO: 15.H ), and / or A light chain variable region (V) comprising CDR1 comprising an amino acid sequence having no more than three amino acid substitutions relative to SEQ ID NO: 16, CDR2 comprising an amino acid sequence having no more than two amino acid substitutions relative to SEQ ID NO: 17, and CDR3 comprising an amino acid sequence having no more than three amino acid substitutions relative to SEQ ID NO: 18. L );or A heavy chain variable region (V) comprising CDR1 comprising an amino acid sequence having no more than two amino acid substitutions relative to SEQ ID NO:19, CDR2 comprising an amino acid sequence having no more than four amino acid substitutions relative to SEQ ID NO:20, and CDR3 comprising an amino acid sequence having no more than three amino acid substitutions relative to SEQ ID NO:21. H ), and / or A light chain variable region (V) comprising CDR1 comprising an amino acid sequence having no more than three amino acid substitutions relative to SEQ ID NO:22, CDR2 comprising an amino acid sequence having no more than two amino acid substitutions relative to SEQ ID NO:23, and CDR3 comprising an amino acid sequence having no more than three amino acid substitutions relative to SEQ ID NO:24. L ).

[0019] In one aspect, the above referred to CDR sequences have no more than four, no more than three, no more than two or no more than one amino acid substitution relative to the set forth SEQ ID NO.

[0020] In one embodiment, the binding protein comprises: A heavy chain variable region (V) comprising CDR1, CDR2, and CDR3 comprising the sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively. H ); and / or A light chain variable region (V) comprising CDR1, CDR2, and CDR3 comprising the sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively. L );or A heavy chain variable region (V) comprising CDR1, CDR2, and CDR3 comprising the sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively. H ); and / or A light chain variable region (V) comprising CDR1, CDR2, and CDR3 comprising the sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively. L ).

[0021] In one embodiment, the binding protein comprises: V comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively. H , and V comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively. L .

[0022] In one embodiment, the binding protein comprises: A heavy chain variable region (V) comprising an amino acid sequence at least about 70% identical, at least about 80% identical, at least about 90% identical, or at least about 95% identical to the sequence set forth in SEQ ID NO:3. H ), and / or A light chain variable region (V) comprising an amino acid sequence at least about 70% identical, at least about 80% identical, at least about 90% identical, or at least about 95% identical to the sequence set forth in SEQ ID NO:4. L );or A heavy chain variable region (V) comprising an amino acid sequence at least about 70% identical, at least about 80% identical, at least about 90% identical, or at least about 95% identical to the sequence set forth in SEQ ID NO:5. H ), and / or A light chain variable region (V) comprising an amino acid sequence at least about 70% identical, at least about 80% identical, at least about 90% identical, or at least about 95% identical to the sequence set forth in SEQ ID NO:6. L ).

[0023] In one embodiment, the binding protein comprises: A heavy chain variable region (V) comprising the amino acid sequence set forth in SEQ ID NO:3. H ) and / or a light chain variable region (V L );or A heavy chain variable region (V H ) and / or a light chain variable region (V L ).

[0024] In one embodiment, the binding protein comprises the amino acid sequence set forth in SEQ ID NO:3. H and V comprising the amino acid sequence set forth in SEQ ID NO:4 L Includes.

[0025] In one embodiment, the binding protein comprises the amino acid sequence set forth in SEQ ID NO:5. H and V comprising the amino acid sequence set forth in SEQ ID NO:6 L Includes.

[0026] In some embodiments, the binding protein comprises: (i) a CDR1 comprising an amino acid sequence having no more than two amino acid substitutions relative to SEQ ID NO: 41, a CDR2 comprising an amino acid sequence having no more than four amino acid substitutions relative to SEQ ID NO: 42, and a CDR3 comprising an amino acid sequence having no more than three amino acid substitutions relative to SEQ ID NO: 43; H and / or (ii) a CDR1 comprising an amino acid sequence having no more than three amino acid substitutions relative to SEQ ID NO: 44, a CDR2 comprising an amino acid sequence having no more than two amino acid substitutions relative to SEQ ID NO: 45, and a CDR3 comprising an amino acid sequence having no more than three amino acid substitutions relative to SEQ ID NO: 46; LIn one aspect, the above-referenced CDR sequences have no more than four, no more than three, no more than two, or no more than one amino acid substitution relative to the set forth SEQ ID NO.

[0027] In some embodiments, the binding protein comprises: (i) a V comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 41, 42, and 43, respectively; H and / or (ii) V comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 44, 45, and 46, respectively. L .

[0028] In some embodiments, the binding protein comprises: (i) a V comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 41, 42, and 43, respectively; H , and (ii) V comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 44, 45, and 46, respectively. L .

[0029] In some embodiments, the binding protein comprises: (i) a CDR1 comprising an amino acid sequence having no more than two amino acid substitutions relative to SEQ ID NO: 47, a CDR2 comprising an amino acid sequence having no more than four amino acid substitutions relative to SEQ ID NO: 48, and a CDR3 comprising an amino acid sequence having no more than three amino acid substitutions relative to SEQ ID NO: 49; H and / or (ii) a CDR1 comprising an amino acid sequence having no more than three amino acid substitutions relative to SEQ ID NO:50, a CDR2 comprising an amino acid sequence having no more than two amino acid substitutions relative to SEQ ID NO:51, and a CDR3 comprising an amino acid sequence having no more than three amino acid substitutions relative to SEQ ID NO:52; L .

[0030] In one aspect, the above referred to CDR sequences have no more than four, no more than three, no more than two or no more than one amino acid substitution relative to the set forth SEQ ID NO.

[0031] In one aspect, the above referred to CDR sequences have no more than four, no more than three, no more than two or no more than one amino acid substitution relative to the set forth SEQ ID NO.

[0032] In some embodiments, the binding protein comprises: (i) a V comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 47, 48, and 49, respectively; H and / or (ii) V comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 50, 51, and 52, respectively. L .

[0033] In some embodiments, the binding protein comprises: (i) a V comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 47, 48, and 49, respectively; H , and (ii) V comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 50, 51, and 52, respectively. L .

[0034] In some embodiments, the binding protein is V H and V L Including V H and V L combine to form an Fv which contains the antigen-binding site.

[0035] In some embodiments, V H and V L is in a single polypeptide chain.

[0036] In some embodiments, the binding protein comprises: (i) single chain Fv fragment (scFv); (ii) a dimeric scFv (di-scFv); or (iii) the constant region, fragment crystallizable (Fc) region, or heavy chain constant domain (C H )2 and / or C H At least one of (i) and / or (ii) linked to 3.

[0037] In some embodiments, the binding protein comprises an scFv.

[0038] In some embodiments, the binding protein comprises an amino acid sequence that is at least about 70% identical, at least about 80% identical, at least about 90% identical, or at least about 95% identical to the sequence set forth in SEQ ID NO:27.

[0039] In some embodiments, the binding protein comprises the amino acid sequence set forth in SEQ ID NO:27.

[0040] In some embodiments, the binding protein is a bispecific T cell engager (BiTE).

[0041] In some embodiments, V L and V H are present in separate polypeptide chains.

[0042] In some embodiments, the binding protein comprises: (i) Diabody; (ii) triabody; (iii) tetrabodies; (iv) Fab; (v) F(ab')2; (vi) Fv; or (vii) an antibody constant region, Fc region, or C H 2 and / or C H At least one of (i) to (vi) connected to 3.

[0043] In some embodiments, the binding protein comprises an Fc region. In one embodiment, the binding protein is an antibody.

[0044] In some embodiments, the binding protein is conjugated to another compound. In some embodiments, the binding protein is conjugated to a half-life extending moiety. In some embodiments, the binding protein is conjugated to a polymer (e.g., PEG). In some embodiments, the binding protein is conjugated to a cytotoxic agent.

[0045] In some embodiments, the binding protein is chimeric, deimmunized, humanized, human, or primatized, hi some embodiments, the binding protein is a human protein.

[0046] In another aspect, the invention provides a composition comprising a binding protein of the invention and a pharma- ceutically acceptable carrier.

[0047] In another aspect, the invention provides a cell comprising a binding protein of the invention.

[0048] Advantageously, the present inventors have discovered that the binding proteins of the present invention can be incorporated into chimeric antigen receptors (CARs) to generate CAR-expressing cells for targeting EGFRvIII-expressing cells. Thus, in another aspect, the present invention provides a CAR comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain of the present invention.

[0049] In some embodiments, the intracellular domain comprises a primary signaling domain and a costimulatory domain. In one embodiment, the intracellular domain comprises two or more costimulatory domains. In an alternative embodiment, the CAR does not comprise a costimulatory domain.

[0050] In some embodiments, the primary signaling domain comprises a primary signaling domain of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, common FcR gamma (FCER1G), FcR beta (Fc epsilon Rib), CD79a, CD79b, Fc gamma RIIa, DAP10, or DAP12.

[0051] In some embodiments, the primary signaling domain is a primary signaling domain of CD3 zeta. In some embodiments, the primary signaling domain comprises an amino acid sequence that is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95%, or about 100% identical to the sequence set forth in SEQ ID NO:35.

[0052] In some embodiments, the costimulatory domain is selected from the group consisting of CD28, 4-1BB (CD137), OX40, CD27, CD30, CD40, CD134, PD-1, ICOS, lymphocyte function associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFl), CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TRANCE / RANKL, DNAM1( CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSG Contains the costimulatory domains of L1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, or TNFR2.

[0053] In some embodiments, the costimulatory domain is the costimulatory domain of CD28.

[0054] In some embodiments, the costimulatory domain comprises an amino acid sequence that is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95%, or about 100% identical to the sequence set forth in SEQ ID NO:36.

[0055] In some embodiments, the transmembrane domain is selected from the group consisting of CD28, CD3 epsilon, CD3 zeta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD1 9, IL2R beta, IL2R gamma, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGBl, CD29, ITGB2, CD18, LFA-1, ITGB7, DNAMl (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT including the transmembrane domains of AM, Ly9 (CD229), CD160 (BY55), PSGLl, CDIOO (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, NKG2C, or TNFR2, or the alpha, beta, or zeta chains of the T cell receptor.

[0056] In some embodiments, the transmembrane domain is the transmembrane domain of CD28. In other embodiments, the transmembrane domain comprises an amino acid sequence that is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95%, or about 100% identical to the sequence set forth in SEQ ID NO:37.

[0057] In some embodiments, the antigen binding domain is connected to the transmembrane domain by a hinge region, hi some embodiments, the hinge region comprises a CD8 hinge, an IgG hinge, an IgD hinge, a CD28 hinge, a KIR2DS2 hinge, or a glycine-serine linker.

[0058] In some embodiments, the hinge region comprises the hinge region of CD8 alpha. In some embodiments, the hinge region comprises an amino acid sequence at least 70%, or at least 80%, or at least 90%, at least 95%, or about 100% identical to SEQ ID NO:38.

[0059] In some embodiments, the CAR further comprises a leader sequence. In some embodiments, the leader sequence is a human leader sequence. In some embodiments, the leader sequence is an N-terminal leader sequence. In some embodiments, the leader sequence is an IgG kappa leader sequence, an IgG2 heavy chain leader sequence, an IL2 leader sequence, or an IgK VIII leader sequence. In some embodiments, the leader sequence comprises an amino acid sequence that is at least 70%, or at least 80%, or at least 90%, at least 95%, or about 100% identical to SEQ ID NO:39. Other suitable leader sequences will be known to those of skill in the art. In some embodiments, the leader sequence, when expressed in a cell, is cleaved from the CAR during intracellular processing and localization of the CAR to the cell membrane.

[0060] In some embodiments, the CAR comprises more than one antigen-binding domain.Thus, in some embodiments, the CAR is a multispecific CAR that comprises two or more antigen-binding domains, and one of the antigen-binding domains comprises the binding protein of the present invention.The two or more antigen-binding domains can bind to the same or different targets.

[0061] In one embodiment, the CAR comprises an amino acid sequence that is at least about 70%, at least about 80%, at least about 90%, or at least about 95% identical to the sequence set forth in SEQ ID NO:25 or SEQ ID NO:26.

[0062] In one embodiment, the CAR comprises the amino acid sequence set forth in SEQ ID NO:25 or SEQ ID NO:26.

[0063] In another aspect, the invention provides a nucleic acid encoding a binding protein of the invention or a CAR of the invention.

[0064] In one aspect, the nucleic acid comprises a nucleotide sequence that is codon optimized for expression in a human cell.

[0065] In one embodiment, the nucleic acid comprises a nucleotide sequence that is at least about 70%, at least about 80%, at least about 90%, or at least about 95% identical to any one or more of the sequences provided in SEQ ID NOs:29, 30, 31, 32, 33, or 34.

[0066] In one embodiment, the nucleic acid comprises any one or more of the nucleotide sequences provided in SEQ ID NOs:29, 30, 31, 32, 33, or 34.

[0067] In one aspect, the nucleic acid comprises i) a nucleotide sequence that is at least about 70%, at least about 80%, at least about 90%, or at least about 95% identical to SEQ ID NO:29; and ii) a nucleotide sequence that is at least about 70%, at least about 80%, at least about 90%, or at least about 95% identical to SEQ ID NO:30.

[0068] In one embodiment, the nucleic acid comprises a nucleotide sequence that is at least about 70%, at least about 80%, at least about 90%, or at least about 95% identical to SEQ ID NO:31.

[0069] In one embodiment, the nucleic acid comprises a nucleotide sequence that is at least about 70%, at least about 80%, at least about 90%, or at least about 95% identical to SEQ ID NO:33.

[0070] In one embodiment, the nucleic acid comprises a nucleotide sequence that is at least about 70%, at least about 80%, at least about 90%, or at least about 95% identical to SEQ ID NO:34.

[0071] In another aspect, the present invention provides a vector comprising the nucleic acid of the present invention.In one embodiment, the vector is a DNA vector, an RNA vector, a plasmid, a lentivirus vector, an adenovirus or an adeno-associated virus vector, or a retrovirus vector.In some embodiments, the nucleic acid of the present invention is operably linked to promoter in the vector.

[0072] In another aspect, the present invention provides a cell comprising the binding protein of the present invention, the CAR of the present invention, the nucleic acid of the present invention, or the vector of the present invention. In a similar aspect, the present invention provides a cell expressing the binding protein or CAR of the present invention.

[0073] In some embodiments, the cell is an immune effector cell. In some embodiments, the immune effector cell is a T cell or a NK cell. In some embodiments, the immune effector cell is a CD8+ T cell. In some embodiments, the immune effector cell is a CD4+ T cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.

[0074] In one embodiment, the cells described herein can further comprise a second (or more) CAR, e.g., a second CAR that comprises a different antigen binding domain, e.g., to the same target (i.e., EGFRvIII) or to a different target. In one embodiment, the second CAR comprises an antigen binding domain that binds to a target expressed in the same cancer cell type as the target of the first CAR.

[0075] In one embodiment, the second CAR in the cell is an inhibitory CAR, and the inhibitory CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain of an inhibitory molecule. In one embodiment, the inhibitory CAR comprises an antigen-binding domain that binds to a target found in normal cells, but not in diseased cells, for example, normal cells that also express the target of the first CAR. The inhibitory molecule may be selected from one or more of the following: PD1, PD-L1, CTLA-4, TIM-3, LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, TGF beta, CEACAM-1, CEACAM-3, and CEACAM-5. In one embodiment, the second CAR comprises the extracellular domain of PD1 or a fragment thereof.

[0076] In another aspect, the present invention provides a composition comprising a nucleic acid of the invention, a vector of the invention, or a cell of the invention, and a pharma- ceutically acceptable carrier.

[0077] In another aspect, the present invention provides a method of producing a CAR-expressing cell, the method comprising introducing a nucleic acid of the present invention or a vector of the present invention into a cell under conditions such that the CAR is expressed.

[0078] In some cases, it may be advantageous to reduce the amount of or remove regulatory T cells before introducing a nucleic acid or vector into a cell. Thus, in some embodiments, the method of producing a CAR-expressing cell comprises the steps of: a) providing a population of cells comprising regulatory T cells; and b) removing regulatory T cells from the population, thereby providing a population of T regulatory-depleted cells; and c) introducing a nucleic acid or vector of the invention into the population of T regulatory depleted cells such that the CAR is expressed. Advantageously, such a method can be used to generate a population of CAR-expressing cells that are immune effector cells, e.g., CD4+ T cells and CD8+ T cells.

[0079] In some embodiments, regulatory T cells are removed from the population of cells using anti-CD25 or anti-GITR antibodies.

[0080] In certain embodiments, the method further comprises expanding the population of cells after introducing the nucleic acid molecule encoding the CAR.

[0081] In some embodiments, the population of cells is expanded by culturing the cells in the presence of an agent that stimulates a CD3 / TCR complex-associated signal and / or a ligand that stimulates a costimulatory molecule on the surface of the cells. The agent may be an anti-CD3 antibody or fragment thereof, and / or a bead conjugated with an anti-CD28 antibody or fragment thereof.

[0082] In some embodiments, the population of cells is expanded in a suitable medium containing one or more interleukins that increase the cells by at least 200-fold, 250-fold, 300-fold, or 350-fold over a 14 day expansion period as measured by flow cytometry.

[0083] In other embodiments, the population of cells is expanded in the presence of IL-15 and / or IL-7.

[0084] In certain aspects, the methods further comprise the step of cryopreserving the population of cells after expanding the cells.

[0085] In another aspect, the invention provides a method of treating a subject having a cancer associated with EGFRvIII expression, the method comprising administering a binding protein of the invention, a composition of the invention, or a cell of the invention to the subject.

[0086] In a related aspect, the invention provides a binding protein of the invention, a composition of the invention, or a cell of the invention for use in the treatment of a cancer associated with expression of EGFRvIII.

[0087] In another related aspect, the invention provides the use of a binding protein of the invention, a composition of the invention, or a cell of the invention in the manufacture of a medicament for the treatment of a cancer associated with expression of EGFRvIII.

[0088] In some embodiments, a population of cells of the present invention is administered.In some embodiments, the cells administered to the subject are allogeneic or autologous cells.In some embodiments, the cells lack or have low expression of functional T cell receptor (TCR) or functional human leukocyte antigen (HLA).The phrase "low expression" in this context refers to an expression amount that is insufficient to affect cell signaling, such as an expression amount of TCR that is insufficient to activate the cell in the presence of that antigen.Advantageously, such cells are specific for the antigen recognized by CAR.

[0089] In some embodiments, the treatment comprises administering an agent that enhances the efficacy of the cells. In some embodiments, the agent is selected from one or more of the following: a) protein phosphatase inhibitors; b) kinase inhibitors; c) cytokines; d) inhibitors of immune inhibitory molecules; or e) Agents that reduce the level or activity of regulatory T cells.

[0090] In some embodiments, the cancer associated with expression of EGFRvIII is lung cancer, breast cancer, colorectal cancer, prostate cancer, ovarian cancer, skin cancer, melanoma, gastric cancer, pancreatic cancer, liver cancer, brain cancer, glioblastoma, neuroblastoma, blood cancer, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative neoplasms, parathyroid cancer, kidney cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, T-cell lymphoma, pharyngeal cancer, thymoma, thymic carcinoma, Wilms' tumor, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Merkel cell carcinoma, esophageal cancer, bladder cancer, bile duct cancer, bone cancer, Ewing's sarcoma, osteosarcoma, malignant fibrous histiocytoma, or multiple myeloma.

[0091] In some embodiments, the cancer associated with EGFRvIII expression is glioma, medulloblastoma, breast cancer, ovarian cancer, colon cancer, lung cancer, or prostate cancer. In some embodiments, the cancer is glioblastoma; breast cancer, ovarian cancer, lung cancer; head and neck squamous cell carcinoma; medulloblastoma, colorectal cancer, prostate cancer, or bladder cancer.

[0092] In some embodiments, the cancer associated with EGFRvIII expression is glioblastoma (also known as glioblastoma multiforme, GBM).

[0093] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In other embodiments, the subject is a non-human mammal.

[0094] As described herein, the inventors have discovered that in some embodiments, the binding proteins or cells of the invention are also effective against cancer cells that overexpress EGFR, e.g., by gene amplification. Thus, in another aspect, the invention provides a method of treating a subject having a cancer associated with overexpression of EGFR, comprising administering to the subject a binding protein of the invention, a composition of the invention, or a cell of the invention.

[0095] In a related aspect, the invention provides a binding protein of the invention, a composition of the invention, or a cell of the invention for use in the treatment of a cancer associated with overexpression of EGFR.

[0096] In another related aspect, the invention provides the use of a binding protein of the invention, a composition of the invention, or a cell of the invention in the manufacture of a medicament for the treatment of a cancer associated with overexpression of EGFR.

[0097] In some embodiments, the cancer associated with overexpression of EGFR is lung cancer, breast cancer, colorectal cancer, prostate cancer, ovarian cancer, skin cancer, melanoma, gastric cancer, pancreatic cancer, liver cancer, brain cancer, glioblastoma, neuroblastoma, blood cancer, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative neoplasms, parathyroid cancer, kidney cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, T-cell lymphoma, pharyngeal cancer, thymoma, thymic carcinoma, Wilms' tumor, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Merkel cell carcinoma, esophageal cancer, bladder cancer, bile duct cancer, bone cancer, Ewing's sarcoma, osteosarcoma, malignant fibrous histiocytoma, or multiple myeloma.

[0098] In some embodiments, the cancer associated with overexpression of EGFR is glioma, pancreatic cancer, colorectal cancer, lung cancer, breast cancer, gastric cancer, renal cancer, cervical cancer, ovarian cancer, adenocarcinoma, bladder cancer, or head and neck cancer.

[0099] In some embodiments, the cancer associated with overexpression of EGFR is glioblastoma.

[0100] In another aspect, the invention provides a method for detecting a cell expressing EGFRvIII, the method comprising the steps of contacting a binding protein of the invention with a sample comprising the cell and detecting binding between the binding protein and EGFRvIII.

[0101] In a related aspect, the invention provides a method of diagnosing a subject as having a cancer associated with EGFRvIII expression, comprising the steps of: a) obtaining a sample comprising cells from a subject; b) determining whether the cells express EGFRvIII by contacting the sample with a binding protein of the invention and detecting binding between the binding protein and EGFRvIII; and c) diagnosing the subject as having a cancer associated with EGFRvIII expression if binding is detected in step b).

[0102] In another aspect, the invention provides a method for detecting cells that overexpress EGFR, the method comprising the steps of contacting a binding protein of the invention with a sample comprising the cells and detecting binding between the binding protein and EGFR.

[0103] In a related aspect, the invention provides a method of diagnosing a subject as having a cancer associated with overexpression of EGFR, comprising the steps of: a) obtaining a sample comprising cells from a subject; b) determining whether the cell overexpresses EGFR by contacting the sample with a binding protein of the invention and detecting binding between the binding protein and EGFR; and c) diagnosing the subject as having a cancer associated with overexpression of EGFR if binding is detected in step b).

[0104] In another aspect, the present invention provides a method of activating an immune effector cell comprising a CAR of the present invention, the method comprising contacting the immune effector cell with EGFRvIII.

[0105] In some embodiments, the immune effector cells are contacted with EGFRvIII in the subject. In some embodiments, the method further comprises administering the immune effector cells to the subject. In some embodiments, the immune effector cells are contacted with EGFRvIII in vitro.

[0106] Any embodiment herein should be construed as applying mutatis mutandis to any other embodiment unless otherwise indicated. For example, the binding protein examples outlined above apply equally to the CARs, nucleic acids, vectors, and methods of the present invention, as will be understood by those skilled in the art.

[0107] The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of illustration only. Functionally equivalent products, compositions, and methods are clearly within the scope of the invention as described herein.

[0108] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, a reference to a single step, composition of matter, group of steps, or group of compositions of matter should be interpreted as encompassing one and more (i.e., one or more) of that step, composition of matter, group of steps, or group of compositions of matter. [Brief description of the drawings]

[0109] [Figure 1] Figure 1 - Screening of initial anti-EGFRvIII scFv clones. Thirteen high affinity anti-EGFRvIII scFv clones identified using the display library were evaluated for specificity for EGFRvIII compared to wild type EGFR. Biotinylated scFvs were first bound to streptavidin Dynabeads, which were then washed and blocked with free biotin before binding to soluble EGFRvIII or EGFR labeled with ATTO488 (fluorescence on the y-axis; increasing fluorescence indicates increased binding). An irrelevant biotinylated scFv was used as a negative control (bars labeled "1" and "2"). Bars 3-28 correspond to 13 different anti-EGFRvIII scFv clones, with odd numbers corresponding to binding to EGFRvIII and even numbers corresponding to binding of WT EGFR. The two scFvs that showed the highest specificity for EGFRvIII compared to WT EGFR were GCT01 (bars 3 and 4) and GCT02 (bars 13 and 14). [Diagram 2] Figure 2 - Analysis of surface plasmon resonance data to determine specificity and affinity of GCT01 and GCT02. Surface plasmon resonance (SPR) data showing response units of GFP, GCT01 scFv, and GCT02 scFv against EGFR protein (top) and EGFRvIII protein (bottom). [Diagram 3] Figure 3 - Analysis of binding to EGFRvIII expressed on the surface of cells. Flow cytometry showing binding of recombinant GCT01 and GCT02 to EGFRvIII or EGFR protein expressed on the surface of a panel of human cell lines. [Figure 4] Figure 4 - Transduction efficiency of GCT01 and GCT02 on the surface of primary T cells. Transduced primary T cells were analyzed by flow cytometry 24 hours post-transduction to determine expression of the transgene construct (mCherry, X-axis) and by labeling with anti-MYC to determine cell surface expression (Y-axis). Representative flow cytometry plots showing expression of GCT01 and GCT02 CARs are shown. [Diagram 5] Figure 5 - GCT01 and GCT02 kill EGFRvIII expressing tumor cells in vitro. Chromium release assay of primary murine CD8+ T cells 8 days post-transduction expressing either GCT01 or GCT02 CAR when cultured for 24 hours with Cr-labeled target cells at an effector:target ratio of 10:1. Cytotoxicity was measured after 24 hours, except for cell lines marked with "**", which were measured after 4 hours. Mean + / - SD is shown. Data were measured in triplicate. Representative of 3 experiments. [Figure 6] Figure 6 - GCT01 CAR T cells induce rapid and complete elimination of subcutaneous lung cancer tumors in vivo. Tumor growth curves of subcutaneous HCC827 lung cancer tumors treated with IV adoptive transfer of 10e6 (1CD4:1CD8) GCT01 CAR T cells or PBS- / - on day 7 post-implantation. Tumor size is measured 3 times per week using digital calipers. N=5 NSG mice per group. Data are representative of 4 experiments. [Figure 7] Figure 7 - GCT01 and GCT02 CART cells induce tumor regression of intracranial glioma tumors. (A) Tumor growth curves of intracranial U87WT.EGFRvIII ns GFP_Luc tumors in NSG mice administered 1CD4:1CD8 CAR T cells as measured by weekly bioluminescence imaging. Tumor size is measured by radiance. N=5 mice per group. Each line represents one mouse. (B) Weekly IVIS imaging shows a reduction in tumor burden in mice treated with infusion of GCT01 and GCT02 CAR T cells. Weeks indicate post CAR T cell infusion. Data are representative of two experiments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0110] Description of sequence listing SEQ ID NO:1 - Human wild-type EGFR amino acid sequence SEQ ID NO:2-Human EGFRvIII amino acid sequence SEQ ID NO:3-GCT01 VH amino acid sequence SEQ ID NO:4-GCT01 VL amino acid sequence SEQ ID NO:5-GCT02 VH amino acid sequence SEQ ID NO:6-GCT02 VL amino acid sequence SEQ ID NO:7 - HCDR1 consensus amino acid sequence based on GCT01 and GCT02 SEQ ID NO:8 - HCDR2 consensus amino acid sequence based on GCT01 and GCT02 SEQ ID NO:9 - HCDR3 consensus amino acid sequence based on GCT01 and GCT02 SEQ ID NO:10 - LCDR1 consensus amino acid sequence based on GCT01 and GCT02 SEQ ID NO:11 - LCDR2 consensus amino acid sequence based on GCT01 and GCT02 SEQ ID NO:12 - LCDR3 consensus amino acid sequence based on GCT01 and GCT02 SEQ ID NO:13-GCT01 HCDR1 amino acid sequence SEQ ID NO:14-GCT01 HCDR2 amino acid sequence SEQ ID NO:15-GCT01 HCDR3 amino acid sequence SEQ ID NO:16-GCT01 LCDR1 amino acid sequence SEQ ID NO:17-GCT01 LCDR2 amino acid sequence SEQ ID NO:18-GCT01 LCDR3 amino acid sequence SEQ ID NO:19-GCT02 HCDR1 amino acid sequence SEQ ID NO:20-GCT02 HCDR2 amino acid sequence SEQ ID NO:21-GCT02 HCDR3 amino acid sequence SEQ ID NO:22-GCT02 LCDR1 amino acid sequence SEQ ID NO:23-GCT02 LCDR2 amino acid sequence SEQ ID NO:24-GCT02 LCDR3 amino acid sequence SEQ ID NO:25-GCT01 CAR amino acid sequence SEQ ID NO:26-GCT02 CAR amino acid sequence SEQ ID NO:27-Complete scFv amino acid sequence of GCT01 SEQ ID NO:28-Complete scFv amino acid sequence of GCT02 SEQ ID NO:29-GCT01 VH DNA sequence SEQ ID NO:30-GCT01 VL DNA sequence SEQ ID NO:31-GCT02 VH DNA sequence SEQ ID NO:32-GCT02 VL DNA sequence SEQ ID NO:33-GCT01 complete scFv DNA sequence SEQ ID NO:34-GCT02 complete scFv DNA sequence SEQ ID NO:35-CD3 zeta primary signaling domain amino acid sequence SEQ ID NO:36-CD28 costimulatory domain amino acid sequence SEQ ID NO:37-CD28 transmembrane domain amino acid sequence SEQ ID NO:38 - CD8 alpha hinge region amino acid sequence SEQ ID NO:39 - Exemplary CAR N-terminal leader amino acid sequence SEQ ID NO:40-Non-signaling EGFRvIII protein SEQ ID NO:41- Alternative GCT01 HCDR1 amino acid sequence SEQ ID NO:42-Alternative GCT01 HCDR2 amino acid sequence SEQ ID NO:43-Alternative GCT01 HCDR3 amino acid sequence SEQ ID NO:44- Alternative GCT01 LCDR1 amino acid sequence SEQ ID NO:45- Alternative GCT01 LCDR2 amino acid sequence SEQ ID NO:46- Alternative GCT01 LCDR3 amino acid sequence SEQ ID NO:47-Alternative GCT02 HCDR1 amino acid sequence SEQ ID NO:48-Alternative GCT02 HCDR2 amino acid sequence SEQ ID NO:49-Alternative GCT02 HCDR3 amino acid sequence SEQ ID NO:50- Alternative GCT02 LCDR1 amino acid sequence SEQ ID NO:51 - Alternative GCT02 LCDR2 amino acid sequence SEQ ID NO:52- Alternative GCT02 LCDR3 amino acid sequence

[0111] Detailed Description of the Invention Common Techniques and Definitions Unless otherwise defined, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., immunology, molecular biology, CAR-T design and therapy, cancer therapy, pharmacology, protein chemistry, and biochemistry).

[0112] Unless otherwise indicated, the techniques utilized in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), TA Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), DM Glover and BD Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and FM Ausubel et al., (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and JE Coligan et al., (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates to date), and other sources.

[0113] As used herein, the term about refers to + / - 10%, more preferably + / - 5% of the specified value, unless otherwise stated.

[0114] Throughout this specification, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated element, integer, or step, or a group of elements, integers, or steps, but not the exclusion of any other element, integer, or step, or group of elements, integers, or steps.

[0115] As used in this application, the term "or" is intended to mean an inclusive "or" rather than an exclusive "or." That is, unless otherwise stated or clear from the context, "X uses A or B" is intended to mean any of the natural inclusive permutations. That is, if X uses A; X uses B; or X uses both A and B, then "X uses A or B" is satisfied under any of the foregoing examples. Furthermore, at least one and / or the like of A and B generally means A or B, or both A and B. Furthermore, as used in this application and the appended claims, the articles "a" and "an" may generally be construed to mean "one or more," unless otherwise stated or clear from the context that the singular form is intended.

[0116] The term "protein" should be construed to include a single polypeptide chain, i.e., a series of consecutive amino acids linked by peptide bonds, or a series of polypeptide chains linked together covalently or non-covalently (i.e., a polypeptide complex). For example, a series of polypeptide chains can be covalently linked, for example, using a suitable chemical linker or disulfide bond. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions. In some embodiments, the protein is an isolated protein.

[0117] In other embodiments, the protein may be a binding protein. Typically, as used herein, a "binding protein" is understood to be a protein that contains an antigen-binding site useful for binding, for example, to EGFRvIII.

[0118] The term "isolated protein" or "isolated binding protein" refers to a protein that, because of its origin or source of origin, is not associated with naturally associated components that accompany it in its native state; it is substantially free of other proteins from the same source. Proteins can be rendered substantially free of naturally associated components using protein purification techniques known in the art, or can be substantially purified by isolation.

[0119] As used herein, the term "bind" refers to the interaction of a binding protein or its antigen-binding site with an antigen, meaning that the interaction is dependent on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the antigen. For example, an antibody recognizes and binds to a specific protein structure, not to the whole protein. If an antibody binds to epitope "A", the presence of a molecule containing epitope "A" (or free, unlabeled "A") in a reaction involving labeled "A" and a binding protein will reduce the amount of labeled "A" bound to the antibody.

[0120] As used herein, the term "specifically binds" or "binds specifically" should be interpreted to mean that the binding protein of the present disclosure reacts or binds to a particular antigen or a cell expressing a particular antigen more frequently, rapidly, and with a longer duration and / or greater affinity than it reacts or binds to another antigen or cell. For example, the binding protein binds to EGFRvIII with significantly greater affinity (e.g., 20-fold or 40-fold or 60-fold or 80-fold to 100-fold or 150-fold or 200-fold) than it binds to other complement components or to antigens commonly recognized by polyreactive natural antibodies (i.e., naturally occurring antibodies known to bind to a variety of antigens naturally found in humans). Generally, but not necessarily, reference to binding should be understood to mean specific binding, with each term providing explicit support for the other term.

[0121] As used herein, the term "not detectably bind" is understood to mean that the binding protein, e.g., antibody, binds to the candidate antigen at a level less than 10% or 8% or 6% or 5% above background. Background can be the level of binding signal detected in the absence of binding protein and / or in the presence of a negative control binding protein (e.g., an isotype control antibody), and / or the level of binding detected in the presence of a negative control antigen. The level of binding is detected using a biosensor analysis (e.g., Biacore) in which the binding protein is immobilized and contacted with the antigen, or vice versa.

[0122] For purposes of clarity, and as will be apparent to one of skill in the art based on the subject matter exemplified herein, references herein to "affinity" include, for example, the dissociation constant (K D In general, references to the level of affinity of a binding protein described herein refer to the level of binding that can be quantified using the K DThus, as referred to herein, a K of at least 15 nM (15 nM or greater) having an affinity for EGFRvIII of at least 15 nM. D i.e., binding proteins with a dissociation constant value of 15 nM or less (e.g., 10 nM or 100 pM). In this context, reference to a higher affinity is to a binding protein with a lower numerical value K D is a reference to, and vice versa.

[0123] As used herein, the term "epitope" (synonym "antigenic determinant") is to be understood to mean a region of EGFRvIII to which a binding protein of the present disclosure binds. The term is not necessarily limited to the particular residues or structures that the binding protein contacts. For example, the term includes the region spanning the amino acids that the binding protein contacts and / or 5-10 or 2-5 or 1-3 amino acids outside of this region. In some embodiments, an epitope includes a series of non-contiguous amino acids that are located near each other when EGFRvIII is folded, i.e., a "conformational epitope." One of skill in the art will also recognize that the term "epitope" is not limited to peptides or polypeptides. For example, the term "epitope" includes chemically active surface groupings of molecules, such as sugar, phosphoryl, or sulfonyl side chains, and in certain instances may have specific three-dimensional structural characteristics and / or specific charge characteristics.

[0124] The term "competitively inhibit" should be understood to mean that the binding protein (or antigen-binding site thereof) of the present disclosure reduces or prevents the binding of the recited antibody or binding protein to EGFRvIII. This may be because the binding protein (or antigen-binding site) and the antibody bind to the same or overlapping epitope. It is clear from the above that the binding protein does not need to completely inhibit the binding of the antibody, but rather only needs to reduce the binding by a statistically significant amount, for example, at least about 10% or 20% or 30% or 40% or 50% or 60% or 70% or 80% or 90% or 95%. Preferably, the binding protein reduces the binding of the antibody by at least about 30%, more preferably at least about 50%, more preferably at least about 70%, even more preferably at least about 75%, even more preferably at least about 80% or 85%, even more preferably at least about 90%. Methods for determining competitive inhibition of binding are known in the art and / or described herein. For example, antibody is exposed to EGFRvIII in the presence or absence of binding protein.If less antibody binds in the presence of binding protein than in the absence of binding protein, it is considered that binding protein competitively inhibits the binding of antibody.In one embodiment, competitive inhibition is not due to steric hindrance.

[0125] "Derived from" as the term is used herein indicates a relationship between a first molecule and a second molecule. It generally refers to the structural similarity between the first molecule and the second molecule, and does not imply or include any process or source restrictions on the first molecule derived from the second molecule. For example, in the case of an intracellular signaling domain derived from a CD3 zeta molecule, the intracellular signaling domain retains sufficient CD3 zeta structure so that it has the required function, i.e., the ability to generate a signal under appropriate conditions. It does not imply or include any restrictions on the specific process of generating the intracellular signaling domain, for example, it does not mean that one must start from the CD3 zeta sequence and delete unnecessary sequences or impose mutations to arrive at the intracellular signaling domain to provide the intracellular signaling domain. The compositions and methods of the present invention encompass polypeptides and nucleic acids having the specified sequence or a sequence substantially identical or similar thereto, for example, a sequence that is at least 85%, 90%, or 95% identical or more identical to the specified sequence.

[0126] As used herein, the term "subject" may be any animal. In one embodiment, the animal is a vertebrate. For example, the animal may be a mammal, a bird, a chordate, an amphibian, or a reptile. Exemplary subjects include, but are not limited to, humans, primates, livestock (e.g., sheep, cows, chickens, horses, donkeys, pigs), companion animals (e.g., dogs, cats), laboratory test animals (e.g., mice, rabbits, rats, guinea pigs, hamsters), and captive wild animals (e.g., foxes, deer). In one embodiment, the mammal is a human.

[0127] As used herein, the term "treat" or "treatment" refers to both direct treatment of a subject by a medical professional (e.g., by administering a therapeutic agent to a subject) or indirect treatment by at least one party (e.g., a doctor, a nurse, a pharmacist, or a pharmaceutical salesperson) by giving any form of instruction, (i) instructing a subject to self-treat (e.g., self-administer a drug) according to the claimed method, or (ii) instructing a third party to treat the subject according to the claimed method.Prevention or reduction of the disease being treated, for example, by administering a therapeutic agent at a sufficiently early stage of the disease to prevent or slow its progression, is also included within the meaning of the term "treat" or "treatment".

[0128] As used herein, the terms "co-administration" or "administered in combination" and the like are meant to encompass the administration of selected therapeutic agents to a single subject, and are intended to include therapeutic regimens in which the agents are administered by the same or different routes of administration or at the same or different times.

[0129] EGFR and EGFRvIII The epidermal growth factor receptor (human EGFR; ErbB-1; HER1) is a transmembrane protein that is a receptor for members of the epidermal growth factor family (EGF family) of extracellular protein ligands. The amino acid sequence of wild-type human EGFR is provided in SEQ ID NO:1.

[0130] EGFR is a member of the ErbB family of receptors, a subfamily of four closely related receptor tyrosine kinases: EGFR (ErbB-1), HER2 / neu (ErbB-2), Her3 (ErbB-3), and Her4 (ErbB-4). Mutations, amplification, or misregulation that affect EGFR expression or activity result in many different types of cancer.

[0131] As used herein, the term "EGFRvIII" (also known as de2-7EGFR and ΔEGFR) refers to a mutant form of EGFR, termed an EGFR class III mutant, or a biologically active fragment thereof, that exhibits any properties specific to EGFRvIII as opposed to those properties shared with normally expressed EGFR. The amino acid sequence of human EGFRvIII is provided in SEQ ID NO:2. EGFRvIII lacks amino acid residues 6-273 of mature EGFR and contains a new glycine residue at position 6 between amino acid residues 5 and 274.

[0132] Abnormal EGFR (as opposed to EGFRvIII) expression is also associated with cancer, often due to gene amplification, and has been observed in many epithelial tumors. Without wishing to be bound by theory, it is understood that overexpressed EGFR, for example, produced by cancer cells, has exposed epitopes that are not present in the EGFR expressed by wild-type cells. Thus, despite being encoded by the same gene, unique conformational forms of EGFR protein can be found in tumorigenic, hyperproliferative, or abnormal cells, but are undetectable or transitional in normal or wild-type cells. Commercially available cell lines that overexpress EGFR include U87, A431, and U251 cells. Advantageously, in some embodiments, the binding proteins of the present invention bind to EGFRvIII and EGFR present on cancer cells but not on wild-type cells.

[0133] antibody In one aspect, the binding proteins described herein are antibodies or fragments thereof.

[0134] Those skilled in the art will recognize that an "antibody" generally comprises multiple polypeptide chains, e.g., V L and a polypeptide comprising V HIt will be appreciated that an antibody can be considered to be a protein comprising a variable region made up of a polypeptide comprising: H and V L interact to form an Fv that contains an antigen-binding site that can specifically bind to one or a few closely related antigens. Generally, mammalian light chains are either kappa or lambda light chains, and mammalian heavy chains are alpha, delta, epsilon, gamma, or mu. Antibodies may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. The term "antibody" also encompasses humanized antibodies, primatized antibodies, human antibodies, and chimeric antibodies.

[0135] The terms "full length antibody", "intact antibody" or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antigen-binding fragment of the antibody. In particular, a whole antibody includes an antibody having a heavy chain and a light chain, including an Fc region. The constant domain may be a wild-type sequence constant domain (e.g., a human wild-type sequence constant domain) or an amino acid sequence variant thereof.

[0136] As used herein, "variable region" refers to a portion of the light and / or heavy chain of an antibody as defined herein that is capable of specifically binding to an antigen and includes the amino acid sequences of the complementarity determining regions (CDRs); i.e., CDR1, CDR2, and CDR3, and framework regions (FRs). An exemplary variable region includes three or four FRs (e.g., FR1, FR2, FR3, and optionally FR4) along with three CDRs. In the case of a protein derived from an IgNAR, the protein may lack CDR2. H refers to the variable region of the heavy chain. L refers to the variable region of the light chain.

[0137] As used herein, the term "complementarity determining region" (synonyms CDR; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of an antibody variable region whose presence is necessary for antigen binding. Each variable region typically has three CDR regions, identified as CDR1, CDR2, and CDR3. The amino acid positions assigned to CDRs and FRs can be defined according to Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991, or other numbering systems in the practice of this disclosure, such as the standard numbering systems of Chothia and Lesk (Chothia et al., 1987) and / or Al-Lazikani (Al-Lazikani et al., 1997); the IMGT numbering system described in Lefranc et al. (2003); or the AHO numbering system described in Honnegher and Plukthun (2001). For example, according to the Kabat numbering system, V H The framework regions (FRs) and CDRs of are positioned as follows: residues 1-30 (FR1), 31-35 (CDR1), 36-49 (FR2), 50-65 (CDR2), 66-94 (FR3), 95-102 (CDR3), and 103-113 (FR4). According to the Kabat numbering system, V L The FRs and CDRs are positioned as follows: residues 1-23 (FR1), 24-34 (CDR1), 35-49 (FR2), 50-56 (CDR2), 57-88 (FR3), 89-97 (CDR3), and 98-107 (FR4). The present disclosure is not limited to FRs and CDRs defined by the Kabat numbering system, but includes all numbering systems, including those described above. In one aspect, references to CDRs (or FRs) herein refer to these regions according to the Kabat numbering system.

[0138] "Framework regions" (FR) are those variable region residues other than the CDR residues.

[0139] As used herein, the term "Fv" refers to a V polypeptide, whether composed of multiple polypeptides or a single polypeptide. L and V H The term "antigen-binding protein" should be taken to mean any binding protein that can bind to and form a complex with an antigen-binding site, i.e., that can specifically bind to an antigen. H and V L The V may be present in a single polypeptide chain or in different polypeptide chains. Furthermore, an Fv of the disclosure (and any binding protein of the disclosure) may have multiple antigen binding sites, which may or may not bind to the same antigen. It is to be understood that the term encompasses fragments derived directly from antibodies, and binding proteins corresponding to such fragments produced using recombinant means. In some embodiments, the V H is the heavy chain constant domain (C H ) 1 and / or V L is the light chain constant domain (C L Exemplary Fv-containing polypeptides or binding proteins include Fab fragments, Fab' fragments, F(ab') fragments, scFvs, diabodies, triabodies, tetrabodies, or higher order complexes, or constant regions or domains thereof, such as C H 2 or C H Any of the above linked to three domains, e.g., minibodies. A "Fab fragment" consists of a monovalent antigen-binding fragment of an immunoglobulin and can be produced by digesting whole antibody with the enzyme papain to yield a fragment consisting of an intact light chain and a portion of the heavy chain, or can be produced using recombinant means. An "Fab' fragment" of an antibody can be produced by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain. Hand a single constant domain. Two Fab' fragments are obtained per antibody thus treated. Fab' fragments can also be produced by recombinant means. An "F(ab')2 fragment" of an antibody consists of a dimer of two Fab' fragments linked by two disulfide bonds and can be obtained by treating a whole antibody molecule with the enzyme pepsin without subsequent reduction. "Fab2" fragments can be obtained by, for example, treating the whole antibody molecule with the leucine zipper or C H A "single-chain Fv" or "scFv" is a recombinant molecule that contains an antibody variable region fragment (Fv) in which the variable region of the light chain and the variable region of the heavy chain are covalently linked by a suitable flexible polypeptide linker.

[0140] As used herein, the term "antigen-binding domain" should be taken to mean an antibody region capable of specifically binding to an antigen, i.e., Fv, including VH or VL or both VH and VL. The antigen-binding domain does not have to be in the context of a complete antibody, for example, it can be separate (e.g., domain antibody) or in another form (e.g., scFv). Typically, as used herein, an antigen-binding domain can include an antigen-binding site capable of binding or specifically binding to an antigen.

[0141] As used herein, the term "antigen-binding site" should be interpreted as meaning a structure formed by a binding protein that can bind or specifically bind to an antigen.An antigen-binding site does not have to be a series of consecutive amino acids, nor does it have to be amino acids in a single polypeptide chain.For example, in an Fv generated from two different polypeptide chains, the antigen-binding site is a V domain that interacts with an antigen and is generally, but not necessarily, present in one or more of the CDRs in each variable region. L and V HIn some embodiments, the antigen-binding site comprises one or more CDRs. In some embodiments, the antigen-binding site comprises three CDRs. In some embodiments, the antigen-binding site comprises six CDRs. In some embodiments, the antigen-binding site comprises a V H or V L or Fv.

[0142] Methods for generating antibodies are known in the art and / or described in Harlow and Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988). Generally, in such methods, EGFRvIII or a region thereof (e.g., extracellular domain), or an immunogenic fragment or epitope thereof, or a cell expressing and presenting the same (i.e., immunogen) may be formulated with any suitable or desired carrier, adjuvant, or pharma- ceutically acceptable excipient, and administered to a non-human animal, such as a mouse, chicken, rat, rabbit, guinea pig, dog, horse, cow, goat, or pig. The immunogen can be administered intranasally, intramuscularly, subcutaneously, intravenously, intradermally, intraperitoneally, or by other known routes.

[0143] Polyclonal antibody production can be monitored by sampling blood from immunized animals at various times after immunization. One or more additional immunizations can be performed if necessary to achieve a desired antibody titer. The process of boosting and titering is repeated until a suitable titer is achieved. Once a desired level of immunogenicity is obtained, the immunized animal is bled and serum is isolated and stored, and / or the animal is used to produce monoclonal antibodies (mab).

[0144] Monoclonal antibody is one exemplary form of antibody contemplated by the present invention.The term "monoclonal antibody" or "mAb" refers to a homogeneous antibody population that can bind to the same antigen, for example, to the same epitope within the antigen.This term is not intended to be limited with respect to the source of antibody or the manner of making antibody.

[0145] For the production of mAbs, any one of a number of known techniques can be used, such as, for example, the procedures exemplified in US 4,196,265.

[0146] For example, suitable animals are immunized with immunogen under conditions sufficient to stimulate antibody-producing cells.Rodents such as rabbits, mice and rats are exemplary animals.Mice that are genetically engineered to express human antibodies and not express, for example, murine antibodies, can also be used to generate the antibodies of the present invention (e.g., as described in WO2002 / 066630).

[0147] Following immunization, somatic cells with the potential to produce antibodies, specifically B lymphocytes (B cells), are selected for use in mAb generation protocols. These cells can be obtained from biopsies of the spleen, tonsils, or lymph nodes, or from a peripheral blood sample. The B cells from the immunized animal are then fused with cells of an immortal myeloma cell, generally derived from the same species as the animal immunized with the immunogen.

[0148] The hybrids are amplified by culturing in selection medium containing agents that block de novo synthesis of nucleotides in tissue culture medium. Exemplary agents are aminopterin, methotrexate, and azaserine.

[0149] The amplified hybridomas are subjected to functional selection for antibody specificity and / or titer, for example, by flow cytometry and / or immunohistochemistry and / or immunoassay (e.g., radioimmunoassay, enzyme immunoassay, cytotoxicity assay, plaque assay, dot immunoassay, etc.). Alternatively, ABL-MYC technology (NeoClone, Madison WI 53713, USA) is used to generate cell lines secreting MAbs.

[0150] Antibodies can also be generated or isolated by screening display libraries, eg, phage display libraries, as described in, eg, US6300064 and / or US5885793.

[0151] In another example, a phage display library is screened or an animal is immunized with EGFRvIII or a fragment thereof and the identified binding proteins and / or antibodies are screened to identify those that are cross-reactive with EGFRvIII and / or its fragments.

[0152] In a further example, EGFRvIII or a fragment thereof is contacted with GCT01 or GCT02 (as described in Example 2). A phage display library is then contacted with EGFRvIII or a fragment thereof, and phage expressing proteins that compete with GCT01 or GCT02 for binding are selected.

[0153] In yet another example, a chimeric protein is made comprising, for example, mouse EGFRvIII, in which the epitope of interest from human EGFRvIII is replaced with the corresponding mouse sequence. This chimeric protein is then used to immunize mice (which are unlikely to induce an immune response against mouse proteins) and / or screen phage display libraries. The resulting antibodies / proteins are then screened to identify those that bind to human EGFRvIII (particularly at the epitope of interest) but do not bind to mouse EGFRvIII.

[0154] The antibody of the invention may be a synthetic antibody, for example, the antibody is a chimeric antibody, a humanized antibody, a human antibody, or a deimmunized antibody.

[0155] Chimeric Proteins In one embodiment, the binding protein described herein is a chimeric protein (e.g., a chimeric antibody). The term "chimeric protein" refers to a binding protein in which a portion of the protein (e.g., the heavy and / or light chains of an antibody) is identical or homologous to a corresponding sequence in a protein from a particular species (e.g., a murine animal such as a mouse) or belonging to a particular protein class or subclass, while the remainder of the protein is identical or homologous to a corresponding sequence from another species (e.g., a primate such as a human) or belonging to another protein class or subclass. For example, a chimeric antibody can utilize a non-human, e.g., rodent or rabbit, variable region and a human constant region to generate an antibody with a predominantly human domain. Methods for generating chimeric antibodies are described, for example, in US4816567 and US5807715.

[0156] Humanized and Human Proteins The binding proteins or antibodies of the invention may be humanized or human.

[0157] The term "humanized protein" or "humanized binding protein" should be understood to refer to a subclass of chimeric proteins that have an antigen-binding site or variable region derived from a protein of a non-human species and the remaining protein structure based on the structure and / or sequence of a human protein. For example, in a humanized antibody, the antigen-binding site generally comprises the complementarity determining regions (CDRs) from a non-human antibody grafted into the appropriate FRs in the variable region of the human antibody and the remaining regions from the human antibody. The antigen-binding site may be wild-type (i.e., identical to that of the non-human antibody) or modified by one or more amino acid substitutions. In some cases, the FR residues of the human antibody are replaced with the corresponding non-human residues.

[0158] Methods for humanizing non-human proteins or parts thereof (e.g., variable regions) are known in the art. Humanization can be carried out according to the methods of US5225539 or US5585089. Other methods for humanizing proteins are not excluded.

[0159] As used herein, the term "human protein" or "human binding protein" refers to a protein having an amino acid sequence that corresponds to a sequence found in humans, e.g., in human germline or somatic cells. A "human" protein can include amino acid residues that are not encoded in human sequences, e.g., mutations introduced by in vitro random or site-specific mutation (particularly mutations that include conservative substitutions, or mutations in a small number of residues of a protein, e.g., in 1, 2, 3, 4, 5, or 6 residues of a protein, e.g., in 1, 2, 3, 4, 5, or 6 residues that constitute one or more of the CDRs of an antibody). These "human proteins" do not necessarily have to be produced by humans, but rather can be produced using recombinant means and / or isolated from transgenic animals (e.g., mice) that contain nucleic acids encoding the human proteins. Human antibodies can be produced using a variety of techniques known in the art, including, for example, phage display libraries (as described in US5885793).

[0160] Human antibodies that recognize a selected epitope can also be generated using a technique called "guided selection," in which a selected non-human monoclonal antibody, e.g., a murine antibody, is used to guide the selection of a fully human antibody that recognizes the same epitope (e.g., as described in US5565332).

[0161] Exemplary human proteins are described herein and include GCT01 and GCT02. These human proteins offer the advantage of reduced immunogenicity in humans compared to non-human proteins.

[0162] Other antigen-binding site-containing binding proteins Single Domain Antibodies In some embodiments, the binding proteins of the present invention are or comprise single domain antibodies (used interchangeably with the terms "domain antibodies" or "dAbs"). Single domain antibodies are single polypeptide chains that contain all or part of the heavy chain variable region of an antibody. In certain instances, single domain antibodies are human single domain antibodies (see, e.g., US6248516).

[0163] Diabodies, triabodies, tetrabodies In some embodiments, the binding proteins of the invention are or include diabodies, triabodies, tetrabodies, or higher order protein complexes, such as those described in WO98 / 044001 and / or WO94 / 007921.

[0164] For example, a diabody is a protein that includes two linked polypeptide chains, each of which has the structure V L -XV H or V H -XV L wherein V L is the light chain variable region of an antibody, and V H is the heavy chain variable region of an antibody, and X is the V H and V L a linker that contains insufficient residues to allow the V of one polypeptide chain to bind (or form an Fv) or is absent H V of the other polypeptide chain L to form an antigen-binding site, i.e., to form an Fv molecule that can specifically bind to one or more antigens. L and V H may be the same in each polypeptide chain, or V L and V H may be different in each polypeptide chain, so as to form bispecific diabodies (i.e., containing two Fvs with different specificities).

[0165] Single chain Fv (scFv) Those skilled in the art will appreciate that scFvs are H and V L The V domains are then combined into a single polypeptide chain, and the scFv is combined for antigen binding (i.e., the V domains of the single polypeptide chain). H and V L A polypeptide linker is provided that enables V to form the desired structure (for linking together to form Fv). H and V L For example, the linker comprises more than 12 amino acid residues, and (Gly4Ser)3 is one of the more preferred linkers for scFvs.

[0166] The present invention also contemplates disulfide-stabilized Fvs (or diFvs or dsFvs), in which a single cysteine ​​residue is located at the V H FR and V L and cysteine ​​residues are linked by disulfide bonds to create a stable Fv.

[0167] Alternatively, or in addition, the invention encompasses dimeric scFvs, i.e., proteins comprising two scFv molecules linked by non-covalent or covalent bonds, e.g., by a leucine zipper domain (e.g., from Fos or Jun). Alternatively, the two scFvs are linked by a peptide linker of sufficient length to allow both scFvs to form and bind to an antigen, e.g., as described in US20060263367.

[0168] Heavy Chain Antibodies Heavy chain antibodies are structurally different from many other forms of antibodies insofar as they contain heavy chains but no light chains. Therefore, these antibodies are also called "heavy chain-only antibodies". Heavy chain antibodies are found, for example, in camelids and cartilaginous fish (also called IgNAR).

[0169] The variable regions present in naturally occurring heavy chain antibodies are the heavy chain variable regions present in conventional four-chain antibodies ("V HThe light chain variable region (referred to as the "V domain") present in conventional four-chain antibodies L In camelid antibodies, the term "V domain" is generally used to distinguish it from the "V domain" HH domain”, referred to as V-NAR in IgNAR.

[0170] Reviews of camelid-derived heavy chain antibodies and their variable regions and methods for their production and / or isolation and / or use can be found, inter alia, in the following references: WO94 / 04678, WO97 / 49805, and WO97 / 49805.

[0171] A review of cartilaginous fish-derived heavy chain antibodies and their variable regions and methods for their production and / or isolation and / or use can be found, inter alia, in WO2005 / 118629.

[0172] BiTE (Bispecific T cell engager) As known in the art, BiTEs generally refer to single polypeptide chain molecules that have two antigen-binding sites, one of which binds to an immune effector cell antigen (e.g., CD3) and the other of which binds to an antigen present on the surface of a target cell, e.g., EGFRvIII. BiTEs and methods for their production are described in WO05 / 061547, Baeuerle et al. (2008), and Bargou et al. (2008).

[0173] When both targets are bound, the BiTE molecule bridges the gap between cytotoxic T cells and tumor cells, allowing the T cells to recognize tumor cells and fight them by injecting toxic molecules. The tumor-binding arm of the molecule can be varied to create different BiTE antibody constructs that target different types of cancer. BiTEs are typically produced as recombinant glycosylated proteins secreted by higher eukaryotic cell lines. Thus, in another embodiment of the present invention, the protein of the present invention is a BiTE.

[0174] Other antigen-binding proteins The present invention relates to antigen-binding sites, such as: (i) "lock and key" bispecific proteins as described in US5731168; (ii) heteroconjugate proteins, e.g. as described in US4676980; (iii) heteroconjugate proteins produced using chemical cross-linkers, e.g., as described in US4676980; and (iv) Fab3 (e.g., as described in EP19930302894) Also included are other binding proteins, including

[0175] Deimmunized antibodies and proteins In some embodiments, the binding protein of the present invention is a deimmunized antibody or protein.Deimmunized antibodies and proteins have one or more epitopes, such as B cell epitopes or T cell epitopes, removed (i.e., mutated), thereby reducing the possibility that a mammal will mount an immune response against the antibody or protein.Methods for producing deimmunized antibodies and proteins are known in the art and are described, for example, in WO00 / 34317, WO2004 / 108158, and WO2004 / 064724.

[0176] Methods for introducing appropriate mutations and expressing and assaying the resulting proteins will be apparent to those of skill in the art based on the description herein.

[0177] Constant region The present invention also encompasses the binding proteins described herein that comprise an antibody constant region, including full length antibodies as well as antigen-binding fragments of antibodies fused to Fc.

[0178] The sequences of constant regions useful for generating the binding proteins of the present invention can be obtained from several different sources. In some embodiments, the constant region of the protein or a portion thereof is derived from a human antibody. The constant region or a portion thereof may be derived from any antibody class, including IgM, IgG, IgD, IgA, and IgE, and any antibody isotype, including IgG1, IgG2, IgG3, and IgG4. In one embodiment, the constant region is a human isotype IgG4 or a stabilized IgG4 constant region.

[0179] In one embodiment, the Fc region of the constant region has a reduced ability to induce effector function, for example, compared to native or wild-type human IgG1 or IgG3 Fc region. In one embodiment, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC). Methods for assessing the level of effector function of Fc region-containing proteins are known in the art and / or described herein.

[0180] In one embodiment, the Fc region is an IgG4 Fc region (i.e., derived from an IgG4 constant region), e.g., a human IgG4 Fc region. The sequences of suitable IgG4 Fc regions will be apparent to those of skill in the art and / or available from publicly available databases (e.g., available from the National Center for Biotechnology Information).

[0181] In one embodiment, the constant region is a stabilized IgG4 constant region. The term "stabilized IgG4 constant region" is understood to mean an IgG4 constant region that has been modified to reduce the tendency to undergo Fab-arm exchange or to form half-antibodies.

[0182] Mutations to proteins The present invention also encompasses mutant forms of the binding proteins described herein.For example, substitutions can be made in the CDRs of the binding proteins of the present invention and in the FRs of the variable region-containing proteins without inhibiting or significantly reducing their function in the context of the present invention.In this regard, amino acid substitutions can be introduced into the binding proteins of the present invention, and the products can be screened for desired activity, such as retaining / improving antigen binding, reducing immunogenicity, or improving ADCC or CDC.

[0183] For example, such mutant binding proteins contain one or more conservative amino acid substitutions compared to the sequences described herein. In some embodiments, the binding proteins contain 30 or fewer or 20 or fewer or 10 or fewer conservative amino acid substitutions, e.g., 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain and / or hydropathicity and / or hydrophilicity.

[0184] In one embodiment, the mutant binding protein has only one or two or three or four or five or six or fewer conservative amino acid changes compared to naturally occurring protein.Details of conservative amino acid changes are provided below.For example, as the skilled artisan will recognize from the present disclosure herein, such minor changes can be reasonably expected not to change the activity of the binding protein.

[0185] In some embodiments, the binding protein has no more than 3, 2, or 1 amino acid substitutions in CDR-L1, no more than 2 or 1 amino acid substitutions in CDR-L2, no more than 3, 2, or 1 amino acid substitutions in CDR-L3, no more than 2 or 1 amino acid substitutions in CDR-H1, no more than 4, no more than 3, 2, or 1 amino acid substitutions in CDR-H2, and / or no more than 3, 2, or 1 amino acid substitutions in CDR-H3 with respect to any one or more of the amino acid sequences of the CDRs provided herein. In some embodiments, each CDR comprises no more than 1, 2, 3, or 4 amino acid substitutions. In some embodiments, the amino acid substitutions are conservative substitutions. In some embodiments, the binding protein comprises amino acid substitutions in the framework regions. As one of skill in the art will appreciate, routine site-directed or random mutagenesis techniques can be performed to alter the amino acid sequence of any one of the CDR or framework sequences provided herein, for example, to alter binding affinity (e.g., affinity maturation), reduce susceptibility of the protein to degradation or oxidation, or to confer or modify other physicochemical or functional properties of the binding protein.

[0186] Examples of conservative amino acid substitutions are provided in Table 1 below.

[0187] Table 1. Exemplary conservative amino acid changes TIFF2024516748000001.tif132138

[0188] The present invention also contemplates non-conservative amino acid changes (e.g., substitutions) in the binding proteins of the invention, e.g., in the CDRs, e.g., CDR3. In one embodiment, the binding protein contains less than 6 or 5 or 4 or 3 or 2 or 1 non-conservative amino acid substitutions, e.g., in the CDR3, e.g., in the CDR3.

[0189] For example, amino acid substitutions can be introduced into the CDRs using routine techniques to improve binding affinity. Such substitutions can be made at CDR "hot spots," i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, 2008), and / or residues that contact EGFRvIII, resulting in mutant VIII. H and / or V L are tested for binding affinity. Affinity maturation, error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis are commonly used techniques.

[0190] In certain instances, substitutions, insertions, or deletions may occur within one or more CDRs, as long as such changes do not substantially reduce the ability of the binding protein to bind to EGFRvIII. In some embodiments, the binding protein that comprises amino acid substitutions binds to EGFRvIII with similar affinity as the binding protein that does not have the substitution. Such substitutions may, for example, be outside of the antigen contact residues in the CDR. In some embodiments, the binding protein that comprises amino acid substitutions binds to EGFRvIII with higher affinity than the binding protein that does not have the substitution. In some embodiments, the binding protein that comprises amino acid substitutions binds to EGFRvIII with lower affinity than the binding protein that does not have the substitution. In certain instances, each CDR is either unchanged or contains no more than one, two, three, or four amino acid substitutions. In some embodiments, the substitutions are conservative substitutions.

[0191] A useful method for identifying antibody residues or regions to be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham (1989). In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced with a neutral amino acid such as alanine to determine whether antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of an antigen-antibody complex can be used to identify contact points between the antibody and antigen. Such contact and adjacent residues can be targeted or eliminated as candidates for substitution. The mutants can be screened to determine whether they contain the desired properties.

[0192] The present invention also contemplates one or more insertions or deletions compared to the sequences described herein. In some embodiments, the binding protein contains 10 or fewer, e.g., 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 insertions and / or deletions.

[0193] Conjugates In some embodiments, the binding protein of the present invention is conjugated to another compound. The conjugate may be useful for extending half-life or imparting other biological activity. Methods for conjugating the binding protein are clear to those skilled in the art and / or described herein. All forms and methods of conjugation (i.e., binding) are contemplated by the present invention, including, for example, direct conjugation between the binding protein and another compound / moiety described herein, or indirect binding (e.g., by a linker between the binding protein and the other compound / moiety). In one embodiment, the conjugate is formed by chemical conjugation (e.g., by amine or disulfide bond) or by genetic fusion.

[0194] In one aspect, the present disclosure provides a fusion protein comprising a binding protein of the present invention and another compound. For example, the other compound can be located at the N-terminus of the protein, the C-terminus of the protein, or any combination thereof.

[0195] In one embodiment, the binding protein is conjugated to the other compound via a linker. For example, the linker can be a peptide linker.

[0196] In one embodiment, the linker is a flexible linker. A "flexible" linker is an amino acid sequence that does not have a fixed structure (secondary or tertiary structure) in solution. Thus, such a flexible linker is free to adopt various conformations. Flexible linkers suitable for use in the present invention are known in the art. An example of a flexible linker for use in the present invention is the linker sequence TIFF2024516748000002.tif3128 or (Gly4Ser)3. Flexible linkers are also disclosed in WO1999045132.

[0197] The linker can comprise any amino acid sequence that does not substantially interfere with the interaction of the binding domain with its target. Preferred amino acid residues for flexible linker sequences include, but are not limited to, glycine, alanine, serine, threonine, proline, lysine, arginine, glutamine, and glutamic acid.

[0198] The linker sequence between the binding regions preferably comprises 5 or more amino acid residues. Flexible linker sequences according to the invention consist of 5 or more residues, preferably 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or 25 or 30 or more residues. In one embodiment of the invention, the flexible linker sequence consists of 5, 7, 10, 13 or 16 or 30 residues.

[0199] Exemplary compounds that can be conjugated to the binding proteins of the invention can be selected from the group consisting of human serum albumin or a functional fragment thereof, an immunoglobulin Fc region or a functional fragment thereof, afamin, alpha-fetoprotein, vitamin D binding protein, an antibody fragment that binds to albumin, and a polymer. Other exemplary compounds include.

[0200] Cytotoxic agents In one embodiment, the binding protein is conjugated to a cytotoxic agent. A cytotoxic agent includes any agent that is detrimental to the growth, viability, or proliferation of a cell.Examples of suitable cytotoxic and chemotherapeutic agents that can be conjugated to the binding proteins according to this aspect of the invention include, for example, 1-(2-chloroethyl)-1,2-dimethanesulfonylhydrazide, 1,8-dihydroxy-bicyclo[7.3.1]trideca-4,9-diene-2,6-diyn-13-one, 1-dehydrotestosterone, 5-fluorouracil, 6-mercaptopurine, 6-thioguanine, 9-aminocamptothecin, actinomycin D, amanitin, aminopropanediol, and the like. Therin, anguidine, anthracycline, anthramycin (AMC), auristatin, bleomycin, busulfan, butyric acid, calicheamicin, camptothecin, carminomycin, carmustine, cemadotin, cisplatin, colchicine, combretastatin, cyclophosphamide, cytarabine, cytochalasin B, dactinomycin, daunorubicin, decarbazine, diacetoxypentyldoxorubicin, dibromomannitol, dihydroxy anthracin dione dione), disorazole, dolastatin, doxorubicin, duocarmycin, echinomycin, eleutherobin, emetine, epothilone, esperamicin, estramustine, ethidium bromide, etoposide, fluorouracil, geldanamycin, gramicidin D, glucocorticoids, irinotecan, leptomycin, leurosine, lidocaine, lomustine (CCNU), maytansinoids, mechlorethamine, melphalan, mercaptopurine, methopterin, methotrexate, mithramycin, mitomycin, mitoxantrone These include, for example, cyclosporine, N8-acetylspermidine, podophyllotoxin, procaine, propranolol, pteridine, puromycin, pyrrolobenzodiazepines (PDB), rhizoxin, streptozotocin, tallysomycins, taxol, tenoposide, tetracaine, thioepa, chlorambucil, tomaymycin, topotecan, tubulysin, vinblastine, vincristine, vindesine, vinorelbine, and derivatives of any of the foregoing.According to certain embodiments, the cytotoxic agent conjugated to the binding protein is a maytansinoid, such as DM1 or DM4, a tomaymycin derivative, or a dolastatin derivative. Other cytotoxic agents known in the art are also contemplated within the scope of the present invention, including, for example, protein toxins such as ricin, C. difficile toxin, pseudomonas exotoxin, ricin, diphtheria toxin, botulinum toxin, bryodin, saporin, pokeweed toxin (i.e., phytolaccatoxin and phytolacchigenin), and the like.

[0201] Production of binding proteins In one aspect, the binding proteins described herein, according to any of the examples, are produced using methods known in the art, for example, by culturing hybridomas under conditions sufficient to produce the binding protein.

[0202] Recombinant Expression In another example, the binding proteins described herein, according to any example, are recombinant.

[0203] In the case of recombinant binding proteins, the nucleic acid encoding the recombinant binding protein is cloned into an expression construct or vector, which is then transfected into a host cell, such as an Escherichia coli (E. coli) cell, a yeast cell, an insect cell, or a mammalian cell, such as a monkey COS cell, a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) cell, or a myeloma cell, which would not otherwise produce the binding protein. Exemplary cells for expressing binding proteins are CHO cells, myeloma cells, or HEK cells. Molecular cloning techniques to achieve these ends are known in the art and are described, for example, in Ausubel et al., (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date) or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989). A wide variety of cloning and in vitro amplification methods are suitable for constructing recombinant nucleic acids. Methods for producing recombinant antibodies are also known in the art, see, for example, US4816567 or US5530101.

[0204] Following isolation, the nucleic acid is inserted in operably linked to a promoter in an expression construct or expression vector for further cloning (amplification of the DNA) or for expression in a cell-free system or in a cell.

[0205] Means for introducing isolated nucleic acid or expression constructs containing it into cells for expression are known to those skilled in the art. The technique used for a given cell depends on known successful techniques. Means for introducing recombinant DNA into cells include, among others, microinjection, DEAE-dextran-mediated transfection, liposome-mediated transfection, for example by using lipofectamine (Gibco, MD, USA) and / or cellfectin (Gibco, MD, USA), PEG-mediated DNA uptake, electroporation, and microparticle bombardment, for example by using DNA-coated tungsten or gold particles (Agracetus Inc., WI, USA).

[0206] Protein isolation Methods for isolating binding proteins are known in the art and / or described herein. For example, if the binding protein is secreted into the culture medium, the supernatant from such an expression system can first be concentrated using a commercially available protein concentration filter, e.g., an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor, such as PMSF, can be included in any of the aforementioned steps to inhibit protein degradation, and an antibiotic can be included to prevent the growth of adventitious contaminating bacteria. Alternatively, or in addition, the supernatant can be filtered and / or separated from the cells expressing the binding protein, e.g., by continuous centrifugation.

[0207] For example, the prepared binding protein can be purified from cells using ion exchange, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, affinity chromatography (e.g., protein A affinity chromatography or protein G chromatography), or any combination of the above. These methods are known in the art. Those skilled in the art will also recognize that the binding protein can be modified to include a tag, for example, a polyhistidine tag, such as a hexahistidine tag, or an influenza virus hemagglutinin (HA) tag, or a simian virus 5 (V5) tag, or a FLAG tag, or a glutathione S-transferase (GST) tag, to facilitate purification or detection. The resulting binding protein is then purified using a method known in the art, for example, affinity purification.

[0208] Assay of binding protein activity Binding to EGFR and its mutations Methods for evaluating binding to binding proteins are known in the art, for example as described in Scopes (In: Protein purification: principles and practice, Third Edition, Springer Verlag, 1994). Such methods generally include labeling the binding protein and contacting it with immobilized antigen, or vice versa. After washing to remove non-specific binding proteins, the amount of label and, consequently, the amount of binding protein is detected. Of course, the binding protein may be immobilized and the antigen may be labeled. Panning type assays may also be used. Alternatively, or in addition, surface plasmon resonance assays may be used. Thus, in one embodiment, the affinity of the binding proteins described herein is determined using a biosensor.

[0209] Optionally, the dissociation constant (Kd) of the binding protein for EGFRvIII is determined. "Kd" or "K D " or "Kd value" is, in one embodiment, measured by a radiolabeled or fluorescently labeled EGFRvIII binding assay, which equilibrates the binding protein with a minimum concentration of labeled EGFRvIII in the presence of a series of increasing amounts of unlabeled EGFRvIII. After washing to remove unbound EGFRvIII, the amount of label is determined, which indicates the Kd of the binding protein.

[0210] According to another example, Kd is measured by using a surface plasmon resonance assay, for example, using BIAcore surface plasmon resonance (BIAcore, Inc., Piscataway, NJ) with immobilized proteins of the invention or vice versa. Thus, in one embodiment, the affinity of a binding protein is determined using a biosensor (e.g., by surface plasmon resonance) in an assay in which the binding protein is immobilized and EGFRvIII is contacted with the immobilized binding protein.

[0211] Epitope mapping In another example, the epitope to which the binding protein described herein binds is mapped. Methods for epitope mapping are apparent to those skilled in the art. For example, a series of overlapping peptides spanning the EGFRvIII sequence or a region thereof that contains the epitope of interest is generated, for example, peptides that contain 10-15 amino acids. A binding protein is then contacted with each peptide, and the peptide to which the binding protein binds is determined. This allows the peptide containing the epitope to which the binding protein binds to be determined. If the binding protein binds to multiple non-contiguous peptides, it may bind to a conformational epitope.

[0212] Alternatively, or in addition, as exemplified herein, amino acid residues within EGFRvIII are mutated, for example by alanine scanning mutagenesis, to determine mutations that reduce or prevent protein binding. Any mutation that reduces or prevents binding of the binding protein may be present within the epitope to which the binding protein binds.

[0213] A further method involves binding EGFRvIII or a region thereof to an immobilized binding protein of the invention and digesting the resulting complex with a protease. The peptides that remain bound to the immobilized protein are then isolated and analyzed, for example using mass spectrometry, to determine their sequence.

[0214] A further method involves converting hydrogens in EGFRvIII or regions thereof to deuterons and binding the resulting protein to an immobilized binding protein of the invention, then converting the deuterons back to hydrogen, isolating the EGFRvIII or regions thereof, digesting with an enzyme, and analyzing, for example, using mass spectrometry, to identify those regions containing deuterons that would have been protected from conversion to hydrogen by binding of the binding protein described herein.

[0215] Competitive binding determinations The assay for determining whether a protein competitively inhibits the binding of the binding protein described herein, for example, GCT01 or GCT02, is clear to those skilled in the art.For example, the binding protein can be conjugated to a detectable label, for example, a fluorescent label or a radioactive label.The labeled binding protein and the test protein are then mixed and contacted with EGFRvIII or its region or cells expressing them.Then, the level of the labeled binding protein is determined and compared with the level determined when the labeled binding protein is contacted with EGFRvIII, its region, or cells in the absence of the test protein.If the level of the labeled binding protein is reduced in the presence of the test protein compared to the absence of the test protein, the test protein is considered to competitively inhibit the binding of the labeled binding protein.

[0216] Optionally, the test protein is conjugated to a label different from the labeled binding protein, the alternative label allowing detection of the level of binding of the test protein to EGFRvIII or a region thereof or to a cell.

[0217] In another example, a binding protein is allowed to bind to EGFRvIII or a region thereof or a cell expressing the same before contacting the EGFRvIII, region, or cell with a test protein. A reduction in the amount of binding protein bound in the presence of the test protein compared to the absence of the test protein indicates that the test protein competitively inhibits binding to EGFRvIII. A reciprocal assay can also be performed using a labeled binding protein, with an unlabeled protein first binding to EGFRvIII. In this case, a reduction in the amount of labeled binding protein bound to EGFRvIII in the presence of the unlabeled protein compared to the absence of the unlabeled protein indicates that the labeled binding protein competitively inhibits binding of the unlabeled protein to EGFRvIII.

[0218] Determination of sequence identity Percent identity in two or more polypeptide or nucleotide sequences refers to the percentage of amino acids or nucleotides that are the same within a given region of polypeptide or nucleotide sequence.For example, two sequences can have, for example, 60% identity, optionally 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity across a region in the sequence, or across the entire sequence, if specified, when compared and aligned for maximum agreement across a comparison window or designated region, when measured by using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about 20 amino acids in length, or over a region that is 30, 40, 50, 75, 100, 125, 150, 175, 200 or more amino acids in length.

[0219] The method of aligning sequences for comparison is well known in the art.The optimal alignment of sequences for comparison can be carried out, for example, by Smith and Waterman (1970) local homology algorithm, by Needleman and Wunsch (1970) homology alignment algorithm, by Pearson and Lipman, (1988) similarity search method, by computer implementation of these algorithms (for example, GAP, BESTFIT, FASTA and TFASTA of Wisconsin Genetics Software Package, Genetics Computer Group), or by manual alignment and visual inspection (for example, see Brent et al., 2003).

[0220] Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977); and Altschul et al. (1990), respectively.

[0221] The percent identity between two amino acid or nucleotide sequences can also be determined using the Meyers and Miller (1988) algorithm incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent identity between two amino acid or nucleotide sequences can be determined using the Needleman and Wunsch (1970) algorithm incorporated into the GAP program in the GCG software package (available at www.gcg.com) using either a Blossom 62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0222] composition In some embodiments, the binding proteins described herein can be administered orally, parenterally, by inhalation spray, adsorption, absorption, topically, rectally, nasally, bucally, vaginally, intraventricularly, via an implanted reservoir in a dosage formulation containing a conventional non-toxic pharma- ceutically acceptable carrier, or by any other convenient dosage form. As used herein, the term "parenteral" includes subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal, and intracranial injection or infusion techniques.

[0223] Methods for preparing proteins in a form suitable for administration to a subject (e.g., a pharmaceutical composition) are known in the art and include, for example, those methods described in Remington's Pharmaceutical Sciences (18th ed., Mack Publishing Co., Easton, Pa., 1990) and the US Pharmacopeia: National Formulary (Mack Publishing Company, Easton, Pa., 1984).

[0224] The pharmaceutical compositions of the present disclosure are particularly useful for parenteral administration, e.g., intravenous administration, or administration into a body cavity or the interior of an organ or joint. A composition for administration will generally comprise a solution of the protein dissolved in a pharma- ceutically acceptable carrier, e.g., an aqueous carrier. A variety of aqueous carriers, e.g., buffered saline, and the like, can be used. The composition can include pharma- ceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, toxicity adjusting agents, and the like, as required to approximate physiological conditions, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like. The concentration of the protein of the present invention in these formulations can vary widely and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, according to the particular mode of administration selected and the needs of the patient. Exemplary carriers include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles, such as mixed oils and ethyl oleate, can also be used. Liposomes can also be used as carriers. The vehicle can include minor amounts of additives, e.g., buffers and preservatives, which enhance isotonicity and chemical stability.

[0225] Once formulated, the protein of the present invention will be administered in a manner compatible with the dosage formulation and in an amount that is therapeutically / prophylactically effective.The formulation is easily administered in a variety of dosage forms, such as the above-mentioned types of injectable solutions, but other pharmacologic acceptable forms are also contemplated, such as tablets, pills, capsules, or other solids for oral administration, suppositories, pessaries, nasal drops or nasal sprays, aerosols, inhalants, liposomal forms, and the like.Pharmaceutical "slow-release" capsules or compositions can also be used.Slow-release formulations are generally designed to provide a constant drug level over an extended period of time and can be used to deliver the compounds of the present invention.

[0226] WO2002 / 080967 describes compositions and methods for administering aerosolized compositions containing antibodies, for example for the treatment of asthma, which are also suitable for administration of proteins of the invention.

[0227] Chimeric antigen receptors (CARs) The term "chimeric antigen receptor" or alternatively "CAR" refers to a polypeptide or a series of polypeptides that, when present in an immune effector cell, provides the cell with specificity for a target cell, e.g., a cancer cell, and the generation of an intracellular signal. The CAR described herein comprises at least an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain (also referred to herein as a "cytoplasmic signaling domain" or "intracellular signaling domain") that comprises a binding protein of the invention.

[0228] In some embodiments, the CAR domains are in the same polypeptide chain (e.g., including chimeric fusion proteins). In some embodiments, the domains are adjacent to each other. In some embodiments, the domains are not adjacent to each other and are in different polypeptide chains, such as, for example, a split CAR. In some embodiments, the different polypeptide chains contain a dimerization switch that can couple the polypeptides to each other when a dimerization molecule is present, e.g., can couple an antigen binding domain to an intracellular signaling domain.

[0229] In some embodiments, the CAR further comprises a leader sequence. As used herein, the term "leader sequence" refers to an amino acid sequence that, when fused to the CAR sequence, helps localize the CAR to the cell membrane of the cell expressing the CAR. In some embodiments, the leader sequence is an N-terminal leader sequence. In some embodiments, the leader sequence comprises an amino acid sequence that is at least 70% or at least 80% or at least 90% or at least 95% identical to SEQ ID NO:39. In some embodiments, the leader sequence, when expressed in a cell, is cleaved from the CAR during intracellular processing and localization of the CAR to the cell membrane.

[0230] Antigen-binding domain As described herein, the CAR of the present invention comprises a target-specific binding element, otherwise referred to as an "antigen-binding domain". The antigen-binding domain can be any domain that binds to EGFRvIII, including, but not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, human antibodies, and functional fragments thereof, such as, but not limited to, single-domain antibodies, such as the heavy chain variable domain (VH), light chain variable domain (VL), and variable domain (VHH) of camelid-derived nanobodies, as well as alternative scaffolds known in the art to function as antigen-binding domains, such as recombinant fibronectin domains, T cell receptors (TCRs), or fragments thereof, such as single-chain TCRs. In some cases, it is advantageous for the antigen-binding domain to be derived from the same species as the CAR will ultimately be used. For example, for use in humans, it may be advantageous for the antigen-binding domain of the CAR to comprise human or humanized residues for the antigen-binding domain of an antibody or antibody fragment.

[0231] In some embodiments, the antigen binding domain comprises a single domain antigen binding (SDAB) molecule, which includes molecules in which the complementarity determining region is part of a single domain polypeptide. Examples include, but are not limited to, heavy chain variable domains, binding molecules that are naturally devoid of light chains, single domains derived from traditional four-chain antibodies, engineered domains, and single domain scaffolds other than those derived from antibodies. SDAB molecules can be any in the art or any future single domain molecule. SDAB molecules can be derived from any species, including, but not limited to, mouse, human, camel, llama, lamprey, fish, shark, goat, rabbit, and cow. The term also includes naturally occurring single domain antibody molecules from species other than Camelidae and shark. In one embodiment, SDAB molecules can be derived from the variable region of immunoglobulins found in fish, such as those derived from the immunoglobulin isotype known as the novel antigen receptor (NAR) found in the serum of sharks. Methods for generating single domain molecules derived from the variable regions of NARs ("IgNARs") are described in WO03 / 014161 and Streltsov (2005).

[0232] In one embodiment, SDAB molecules are naturally occurring single domain antigen binding molecules, known as heavy chains without light chains. Such single domain molecules are disclosed, for example, in WO9404678 and Hamers-Casterman et al. (1993). For clarity, this variable domain derived from heavy chain molecules without light chains in nature is known herein as VHH or nanobody, to distinguish it from the conventional VH of four-chain immunoglobulins. Such VHH molecules can be derived from Camelidae species, such as camel, llama, dromedary, alpaca, and guanaco. Other species outside Camelidae may also produce heavy chain molecules without light chains in nature; such VHHs are within the scope of the present invention. SDAB molecules may be recombinant, CDR-grafted, humanized, camelized, deimmunized, and / or in vitro generated (e.g., selected by phage display).

[0233] In one embodiment, the antigen-binding domain is characterized by a particular functional feature or property of an antibody or antibody fragment. For example, in one embodiment, the portion of the CAR composition of the present invention that comprises the antigen-binding domain specifically binds to a tumor antigen as described herein.

[0234] In one embodiment, the antigen-binding domain is an antibody fragment, e.g., a single-chain variable fragment (scFv). In one embodiment, the antigen-binding domain is an Fv, Fab, (Fab')2, or a bifunctional (e.g., bispecific) hybrid antibody (e.g., Lanzavecchia et al., 1987). In one embodiment, the antibodies and fragments of the present invention bind to their targets with wild-type affinity or enhanced affinity. In some cases, scFvs can be prepared according to methods known in the art (see, e.g., Bird et al., 1988 and Huston et al., 1988). ScFv molecules can be generated by linking VH and VL regions together using a flexible polypeptide linker. The scFv molecule includes a linker (e.g., a Ser-Gly linker) that is optimized for length and / or amino acid composition. The length of the linker can greatly affect how the variable regions of the scFv fold and interact. In fact, when short polypeptide linkers (e.g., 5-10 amino acids) are used, intrachain folding is prevented. Interchain folding is also required to bring the two variable regions together to form a functional epitope binding site. For examples of linker orientations and sizes, see Hollinger et al. 1993, US2005 / 0100543, US2005 / 0175606, US2007 / 0014794, WO2006 / 020258 and WO2007 / 024715.

[0235] An scFv can comprise a linker of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 or more amino acid residues between its VL and VH regions. The linker sequence can comprise any naturally occurring amino acid. In some embodiments, the linker sequence comprises the amino acids glycine and serine. In another embodiment, the linker sequence comprises a series of glycine and serine repeats, such as (Gly4Ser)n, where n is a positive integer equal to or greater than 1. In one embodiment, the linker can be (Gly4Ser)4 or (Gly4Ser)3. Variation in the length of the linker can retain or enhance activity, resulting in better efficacy in activity studies.

[0236] In another embodiment, the antigen-binding domain is a T cell receptor ("TCR") or a fragment thereof, such as a single-chain TCR (scTCR). Methods for producing such TCRs are known in the art (see, for example, Willemsen et al., 2000; Zhang et al., 2004; Aggen et al., 2012). For example, a scTCR can be engineered that includes Va and νβ genes from a T cell clone linked by a linker (e.g., a flexible peptide). This approach is very useful for cancer-related targets that are themselves intracellular, but where fragments (peptides) of such antigens are presented on the surface of cancer cells by MHC.

[0237] Other types of suitable antigen-binding domains are discussed herein in the context of the binding proteins of the invention.

[0238] Transmembrane domain With regard to the transmembrane domain, in various embodiments, the CAR can be designed to include a transmembrane domain that is linked to the extracellular domain of the CAR to position the CAR in the cell membrane. The transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, such as one or more amino acids that are linked to the extracellular region of the protein from which the transmembrane is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 to 15 amino acids of the extracellular region) and / or one or more additional amino acids that are linked to the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 to 15 amino acids of the intracellular region). In one embodiment, the transmembrane domain is linked to one of the other domains of the CAR, for example, in one embodiment, the transmembrane domain can be from the same protein from which the primary signaling domain, the costimulatory domain, or the hinge region is derived. In another embodiment, the transmembrane domain is not from the same protein from which any other domain of the CAR is derived. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid such domain binding to the transmembrane domain of the same or different surface membrane protein, for example, to minimize interaction with other members of the receptor complex. In one embodiment, the transmembrane domain can homodimerize with another CAR on the cell surface of a CAR-expressing cell. In a different embodiment, the amino acid sequence of the transmembrane domain can be modified or substituted to minimize interaction with the binding domain of a native binding partner present in the same CAR-expressing cell.

[0239] The transmembrane domain can be derived from either natural or recombinant sources.When the source is natural, the domain can be derived from any membrane-bound or transmembrane protein.In one embodiment, the transmembrane domain can transmit a signal to the intracellular domain whenever the CAR binds to its target.The transmembrane domain of particular use in the present invention can include at least the transmembrane regions of, for example, the alpha, beta or zeta chain of T cell receptor, TNFR2, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In some embodiments, the transmembrane domain may be, for example, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, C At least some examples of such proteins include D11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKG2D, and the transmembrane domain of NKG2C, or functional mutants thereof.

[0240] In some cases, the transmembrane domain can be connected to the extracellular region of the CAR, e.g., the antigen-binding domain of the CAR, via a hinge, e.g., a hinge derived from a human protein. As used herein, the term "hinge region" refers to any suitable amino acid sequence located between the transmembrane domain and the antigen-binding domain. Suitable hinge sequences are known in the art. For example, in one embodiment, the hinge can be a human Ig (immunoglobulin) hinge (e.g., an IgG4 hinge, an IgD hinge), a GS linker (e.g., a GS linker described herein), a KIR2DS2 hinge, or a CD8 alpha hinge. In one embodiment, the hinge or spacer comprises an IgG4 hinge. In one embodiment, the hinge region comprises an IgD hinge. In another embodiment, the hinge comprises a CD8 alpha hinge region.

[0241] In one aspect, the transmembrane domain may be recombinant, in which case it will comprise primarily hydrophobic residues such as leucine and valine, hi one aspect, triplets of phenylalanine, tryptophan, and valine can be found at each end of the recombinant transmembrane domain.

[0242] Optionally, a short oligo- or polypeptide linker between 2 and 10 amino acids in length can form a link between the transmembrane domain and the cytoplasmic region of the CAR. A glycine-serine doublet provides a particularly suitable linker. For example, in one embodiment, the linker comprises the amino acid sequence GGGGSGGGGS. In one embodiment, the hinge or spacer comprises the hinge of KIR2DS2.

[0243] Intracellular domain The intracellular domain of a CAR is generally involved in the activation of at least one of the normal effector functions of an immune cell into which the CAR is introduced. The term "effector function" refers to a specialized function of a cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity, such as secretion of cytokines. Thus, the term "intracellular domain" refers to the portion of a CAR that transmits an effector function signal and instructs the cell to perform a specialized function. Usually, the entire intracellular domain can be used, but in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular domain is used, such a truncated portion can be used instead of the intact chain, so long as it transmits an effector function signal. Thus, the term intracellular domain is meant to include any truncated portion of the intracellular domain that is sufficient to transmit an effector function signal.

[0244] Examples of intracellular domains for use in the CARs of the present invention include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor binding, as well as any derivatives or variants of these sequences, and any recombinant sequences that have the same functional capabilities.

[0245] It is known that signals generated by TCR alone are insufficient for full activation of T cells, and that secondary and / or costimulatory signals are also required. Thus, T cell activation can be said to be mediated by two separate classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through TCR (herein referred to as "primary signaling domains") and those that act antigen-independently to provide secondary or costimulatory signals (herein referred to as "costimulatory domains").

[0246] As used herein, the phrase "primary signaling domain" refers to a domain present in the intracellular domain of a CAR that can induce or inhibit antigen-dependent primary activation signaling in cells expressing the CAR. For example, primary activation signaling, such as that initiated by binding of the TCR / CD3 complex to a peptide-loaded MHC molecule, leads to mediation of T cell responses, such as proliferation, activation, differentiation, etc. In this context, the primary signaling domain regulates the primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary signaling domains that act in a stimulatory manner can include signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs.

[0247] Examples of ITAM-containing primary signaling domains that are particularly useful in the present invention include those of CD3 zeta, common FcR gamma (FCERIG), Fc gamma RIIa, FcR beta (Fc epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12, or functional variants thereof. In one embodiment, the CAR of the present invention comprises an intracellular signaling domain, such as a primary signaling domain of CD3 zeta, or a functional variant thereof. In one embodiment, the CAR of the present invention comprises a primary signaling domain of CD3 zeta (e.g., SEQ ID NO:35).

[0248] In one embodiment, the primary signaling domain comprises a modified ITAM domain, e.g., a mutated ITAM domain that has altered (e.g., increased or decreased) activity compared to the native ITAM domain. In one embodiment, the primary signaling domain comprises a primary signaling domain that comprises a modified ITAM, e.g., a primary signaling domain that comprises an optimized and / or truncated ITAM. In one embodiment, the primary signaling domain comprises one, two, three, four or more ITAM motifs.

[0249] The intracellular domain of the CAR can comprise the primary signaling domain alone or in combination with any other desired intracellular signaling domain useful in the CAR of the present invention. For example, in some embodiments, the intracellular domain of the CAR comprises a primary signaling domain and a costimulatory domain.

[0250] "Costimulatory domain" refers to a portion of a CAR that is derived from the intracellular domain of a costimulatory molecule and can provide a costimulatory signal in a cell expressing the CAR upon antigen binding. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is required for an efficient response of lymphocytes to antigens. Examples of such molecules include TNFR2, CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83. For example, CD27 costimulation has been shown to enhance the expansion, effector function, and survival of human CAR-T cells in vitro, as well as to increase the persistence and antitumor activity of human T cells in vivo (Song et al., 2012). Further examples of such costimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, These include ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), NKG2D, CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, and CD19a.

[0251] The primary signaling domain and the costimulatory domain of the cytoplasmic part of the CAR of the present invention can be linked together in a random or specific order.Optionally, a short oligo- or polypeptide linker, for example, 2 to 10 amino acids (for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in length, can form a link between the intracellular signaling sequences.In one embodiment, a glycine-serine doublet can be used as a suitable linker.In one embodiment, a single amino acid, for example, alanine, glycine, can be used as a suitable linker.

[0252] In one embodiment, the intracellular domain is designed to include two or more, for example, two, three, four, five or more, costimulatory domains. In one embodiment, the two or more, for example, two, three, four, five or more, costimulatory domains are separated by a linker molecule, for example, a linker molecule described herein. In one embodiment, the intracellular domain includes two costimulatory domains. In some embodiments, the linker molecule is a glycine residue. In some embodiments, the linker is an alanine residue.

[0253] In one embodiment, the intracellular domain is designed to include the signaling domain of CD3 zeta and the signaling domain of CD28. In one embodiment, the intracellular domain is designed to include the signaling domain of CD3 zeta, the signaling domain of CD28, and the signaling domain of 4-1BB, or a functional variant thereof. In one embodiment, the intracellular domain is designed to include the signaling domain of CD3 zeta, the signaling domain of CD28, and the signaling domain of CD27.

[0254] Multispecific CAR In some embodiments, the CAR is a multispecific (e.g., bispecific or trispecific) CAR. Protocols for making bispecific or heterodimeric binding molecules are known in the art, including, for example, but not limited to, those described in US5731168; WO09 / 089004; WO06 / 106905 and WO2010 / 129304; WO07 / 110205; WO08 / 119353, WO2011 / 131746, and WO2013 / 060867; US5273743; US5534254; US5582996; US5591828; US5635602; US5637481; US5837242; US5837821; US5844094; US5864019 and US5869620.

[0255] Within each antibody or antibody fragment (e.g., scFv) of a bispecific antibody molecule, the VH may be upstream or downstream of the VL. In some embodiments, the upstream antibody or antibody fragment (e.g., scFv) has its VH (VHi) positioned upstream of its VL (VLi) and the downstream antibody or antibody fragment (e.g., scFv) has its VL (VL2) positioned upstream of its VH (V3 / 4), such that the overall bispecific antibody molecule has the configuration VH1-VL1-VL2-VH2. In other embodiments, the upstream antibody or antibody fragment (e.g., scFv) has its VL (VLi) positioned upstream of its VH (VHi) and the downstream antibody or antibody fragment (e.g., scFv) has its VH (VH2) positioned upstream of its VL (VL2), such that the overall bispecific antibody molecule has the configuration VL1-VH1-VH2-VL2. Optionally, a linker is placed between two antibodies or antibody fragments (e.g., scFvs), for example, between VL1 and VL2 when the construct is arranged as VH1-VL1-VL2-VH2, or between VH2 and VH2 when the construct is arranged as VL1-VH1-VH2-VL2. The linker can be a linker as described herein. In general, the linker between two scFvs should be long enough to avoid mispairing between the domains of the two scFvs. Optionally, a linker is placed between the VL and VH of the first scFv. Optionally, a linker is placed between the VL and VH of the second scFv. In constructs with multiple linkers, any two or more linkers may be the same or different. Thus, in some embodiments, the bispecific CAR comprises a VL, a VH, and optionally, one or more linkers, in an arrangement as described herein.

[0256] Adjustable Car Regulatable CAR (RCAR) is a CAR whose activity can be controlled, which is desirable for optimizing the safety and efficacy of CAR therapy.There are many ways that CAR activity can be regulated.For example, inducible apoptosis using caspases fused to dimerization domains (see, for example, Di et al., 2011) can be used as a safety switch in the CAR therapy of the present invention.

[0257] In embodiments, an RCAR can comprise a "multi-switch." A multi-switch can comprise a heterodimerization switch domain or a homodimerization switch domain.

[0258] One embodiment provides an RCAR in which the antigen binding member is not tethered to the surface of the CAR cell, allowing a cell bearing an intracellular signaling member to conveniently pair with one or more antigen binding domains without transforming the cell with sequences encoding the antigen binding member.

[0259] One embodiment provides an RCAR with a configuration that allows for switching of proliferation. In this embodiment, the RCAR comprises: 1) an intracellular signaling member comprising: optionally a transmembrane domain or membrane tethering domain; one or more costimulatory signaling domains, e.g., selected from 41BB, CD28, CD27, ICOS, and OX40, and a switch domain; and 2) an antigen binding member comprising: an antigen binding domain, a transmembrane domain, and a primary signaling domain, e.g., a CD3 zeta domain, where the antigen binding member does not comprise a switch domain or does not comprise a switch domain that dimerizes with a switch domain on the intracellular signaling member. In one embodiment, the antigen binding member does not comprise a costimulatory signaling domain. In one embodiment, the intracellular signaling member comprises a switch domain from a homodimerization switch. In one embodiment, the intracellular signaling member comprises a first switch domain of a heterodimerization switch, and the RCAR comprises a second switch domain of a heterodimerization switch that comprises a second intracellular signaling member. In such an embodiment, the second intracellular signaling member comprises the same intracellular signaling domain as the intracellular signaling member. In one aspect, the dimerization switch is intracellular. In one aspect, the dimerization switch is extracellular.

[0260] In any of the RCAR configurations described herein, the first and second switch domains comprise an FKBP-FRB-based switch as described herein.

[0261] Split CAR In some embodiments, the CAR of the invention is a split CAR. The split CAR approach is described in more detail in publications WO2014 / 055442 and WO2014 / 055657.

[0262] Determining the validity of CAR Once the CAR described herein is constructed, various assays can be used to evaluate the activity of the molecule in suitable in vitro and animal models, including but not limited to, the ability to expand T cells after antigen stimulation, the ability to sustain T cell expansion in the absence of re-stimulation, and anti-cancer activity. The assays for evaluating the effect of the CAR of the present invention are described in more detail below.

[0263] Western blot analysis of CAR expression in primary T cells can be used to detect the presence of monomers and dimers (see, e.g., Milone et al., 2009). Very briefly, T cells expressing CAR (1:1 mixture of CD4+ and CD8+ T cells) are expanded in vitro for more than 10 days, followed by lysis and SDS-PAGE under reducing conditions. CARs containing the full-length TCR-ζ cytoplasmic domain and endogenous TCR-ζ chain are detected by Western blotting using an antibody against the TCR-ζ chain. The same T cell subsets are used for SDS-PAGE analysis under non-reducing conditions to allow assessment of covalent dimer formation.

[0264] In vitro expansion of CAR-T cells after antigen stimulation can be measured by flow cytometry. For example, a mixture of CD4+ and CD8+ T cells is stimulated with aCD3 / aCD28 aAPCs and subsequently transduced with a lentiviral vector expressing GFP under the control of the promoter to be analyzed. Exemplary promoters include the promoters of the CMV IE gene, EF-la, ubiquitin C, or phosphoglycerokinase (PGK). GFP fluorescence is assessed by flow cytometry on day 6 of culture of subsets of CD4+ and / or CD8+ T cells (see, for example, Milone et al., 2009). Alternatively, on day 0, a mixture of CD4+ and CD8+ T cells is stimulated with aCD3 / aCD28 coated magnetic beads, and on day 1, the CAR is transduced using a bicistronic lentiviral vector expressing the CAR together with eGFP using a 2A ribosomal skipping sequence. After washing, the culture is restimulated with cells expressing the antigen, for example, K562 cells (K562 expressing antigen), wild type K562 cells (K562 wild type), or K562 cells expressing hCD32 and 4-1BBL in the presence of anti-CD3 and anti-CD28 antibodies (K562-BBL-3 / 28). Exogenous IL-2 is added to the culture at 100 IU / ml every other day. GFP-expressing T cells are counted by flow cytometry using bead-based counting. Sustained CAR-T cell expansion in the absence of restimulation can also be measured (see, for example, Milone et al., 2009). Briefly, after stimulation with aCD3 / aCD28 coated magnetic beads on day 0 and transduction with the indicated CAR on day 1, mean T cell volume (fl) is measured on day 8 of culture using a Coulter Multisizer III particle counter, Nexcelom Cellometer Vision, or Millipore Scepter.

[0265] Animal models can also be used to measure the activity of CARs. For example, xenograft models. Specific CAR-T cells can be used to treat primary human pre-B ALL in immunodeficient mice (see, for example, Milone et al., 2009). Very briefly, after the establishment of ALL, mice are randomized for treatment groups. Different numbers of CAR-engineered T cells are co-injected into B-ALL-bearing NOD-SCID mice at a 1:1 ratio. At various times after injection of T cells, the copy number of the specific CAR vector in the spleen DNA of the mice is evaluated. Animals are evaluated at weekly intervals for leukemia. Peripheral blood blast cell counts are measured in mice injected with antigen peptides against CAR-T cells or mock-transduced T cells. Survival curves for these groups are compared using the log-rank test. In addition, the absolute number of peripheral blood CD4+ and CD8+ T cells 4 weeks after T cell injection in NOD-SCID mice can also be analyzed. Mice are injected with leukemia cells and 3 weeks later, injected with T cells engineered to express the CAR by a bicistronic lentiviral vector encoding the CAR linked to eGFP. T cells are normalized to 45-50% input GFP+ T cells by mixing with mock-transduced cells prior to injection and confirmed by flow cytometry. Animals are evaluated at weekly intervals for leukemia. Survival curves for CAR-T cell groups are compared using the log-rank test.

[0266] Assessment of cell proliferation and cytokine production has been described previously (e.g., Milone et al., 2009). Briefly, assessment of CAR-mediated proliferation is performed by mixing washed T cells with K562 cells expressing antigen (e.g., K19) or CD32 and CD137 (KT32-BBL) in microtiter plates to a final T cell:K562 ratio of 2:1. Prior to use, K562 cells are gamma irradiated. Anti-CD3 (clone OKT3) and anti-CD28 (clone 9.3) monoclonal antibodies are added to cultures containing KT32-BBL cells to serve as positive controls for stimulating T cell proliferation, as these signals support long-term CD8+ T cell expansion ex vivo. CountBright 1000 ng / mL T cells were used as described by the manufacturer. (商標) T cells are counted in culture using fluorescent beads (Invitrogen, Carlsbad, CA) and flow cytometry. CAR-T cells are identified by GFP expression using T cells engineered with a CAR-expressing lentiviral vector linked to eGFP-2A. For CAR+T cells that do not express GFP, CAR+T cells are detected with biotinylated protein and a secondary avidin-PE conjugate. CD4+ and CD8+ expression on T cells is also detected simultaneously with specific monoclonal antibodies (BD Biosciences). Cytokine measurements (e.g., IFN-gamma) are performed on supernatants collected 24 hours after restimulation using a Human TH1 / TH2 Cytokine Cytometry Bead Array Kit (BD Biosciences, San Diego, CA) according to the manufacturer's instructions. Fluorescence is assessed using a FACScalibur flow cytometer and data are analyzed according to the manufacturer's instructions.

[0267] Imaging techniques can be used to assess the specific trafficking and proliferation of CARs in tumor-bearing animal models. Such assays are described, for example, in Barrett et al., (2011).

[0268] Alternatively, the therapeutic efficacy and specificity of a single injection of CAR-T cells in the Nalm-6 xenograft model can be measured as follows: NSG mice are injected with Nalm-6 transduced to stably express firefly luciferase, followed 7 days later by a single tail vein injection of T cells electroporated with the CAR of the present invention. Animals are imaged at various time points after injection. For example, photon density heat maps of firefly luciferase positive leukemia in a representative mouse at day 5 (2 days before treatment) and day 8 (24 hours after CAR+PBL) can be generated.

[0269] Other assays can also be used to evaluate the CARs described herein, including those described in the Examples section herein and those known in the art.

[0270] Nucleic acids and vectors The present invention also provides a vector into which the nucleic acid construct encoding the binding protein or CAR of the present invention is inserted. The expression of the natural or synthetic nucleic acid encoding the binding protein or CAR is typically achieved by functionally linking the nucleic acid encoding the binding protein or CAR polypeptide or a part thereof to a promoter and incorporating the construct into an expression vector. The vector can be suitable for replication and integration in eukaryotes. A typical cloning vector includes transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence. The expression construct of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods for gene delivery are known in the art. See, for example, US5399346, US5580859, and US5589466. In another aspect, the present invention provides a gene therapy vector.

[0271] The nucleic acid can be cloned into several types of vectors, including but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids.

[0272] In one embodiment, the vector comprising the nucleic acid encoding the desired protein or CAR of the present invention is an adenoviral vector (A5 / 35). In another embodiment, expression of the nucleic acid encoding the protein or CAR can be achieved using transposons, such as sleeping beauty, crisper, CAS9, and zinc finger nucleases (see, e.g., June et al., 2009).

[0273] Additionally, the expression vector can be provided to cells in the form of a viral vector. Viral vector technology is well known in the art and described, for example, in Sambrook Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, and other virology and molecular biology manuals. Using techniques known in the art, a selected gene can be inserted into a vector and packaged into a retroviral particle. The recombinant virus can then be isolated and delivered to cells of a subject either in vivo or ex vivo. Several retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Several adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used. Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although some promoters have also been shown to contain functional elements downstream of the start site. The spacing between promoter elements is often flexible, so that promoter function is preserved when elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be extended to as far as 50 bp apart before activity begins to decline.

[0274] Methods for producing cells expressing binding proteins or CARs Also provided herein is a method for producing a cell that expresses a binding protein or CAR by introducing a vector or nucleic acid that codes for the protein or CAR into the cell.The method of introducing and expressing a gene in a cell is known in the art.Regarding expression vector, the vector can be easily introduced into host cell, for example, mammalian cell, bacterial cell, yeast cell or insect cell, by any method in the art.For example, the expression vector can be transferred into host cell by physical, chemical or biological means.

[0275] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art (see, for example, Sambrook Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0276] Biological methods for introducing polynucleotide of interest into host cell include the use of DNA vector and RNA vector.Viral vector, especially retroviral vector, has become the most widely used method for inserting genes into mammalian, for example human cell.Other viral vectors can be derived from lentivirus, poxvirus, herpes simplex virus I, adenovirus, adeno-associated virus, etc.See, for example, US5350674 and US5585362.

[0277] Chemical means for introducing polynucleotide into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, such as oil-in-water emulsions, micelles, mixed micelles, and liposomes.Exemplary colloidal systems for use as delivery vehicles in vitro and in vivo are liposomes (e.g., artificial membrane vesicles).Other methods of advanced targeted delivery of nucleic acids are available, such as the delivery of polynucleotides using targeted nanoparticles or other suitable submicron-sized delivery systems.

[0278] An exemplary non-viral delivery vehicle is liposome. For the introduction of nucleic acid into host cells (in vitro, ex vivo or in vivo), lipid formulations are contemplated. In another embodiment, nucleic acid can be bound to lipid. Nucleic acid bound to lipid can be encapsulated in the aqueous interior of liposome, dispersed in the lipid bilayer of liposome, bound to liposome via a linking molecule that binds to both liposome and oligonucleotide, enclosed in liposome, complexed with liposome, dispersed in a solution that contains lipid, mixed with lipid, combined with lipid, contained as a suspension in lipid, contained with or complexed with micelles, or otherwise bound to lipid.

[0279] Regardless of the method used to introduce exogenous nucleic acid into host cells or otherwise expose cells to the inhibitors of the present invention, various assays can be carried out to confirm the presence of recombinant DNA sequences in host cells.Such assays include, for example, "molecular biological" assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as detecting the presence or absence of specific peptides, for example, by immunological means (ELISA and Western blot) or by the assays described herein to identify agents that fall within the scope of the present invention.

[0280] Generally, immune effector cells, such as T cells, can be activated and expanded using well known methods.

[0281] In general, a population of immune effector cells, for example, regulatory T cell depleted cells, can be expanded by contacting with a surface to which an agent that stimulates CD3 / TCR complex-associated signals and a ligand that stimulates costimulatory molecules on the surface of T cells are bound. In particular, as described herein, a T cell population can be stimulated by contacting with, for example, an anti-CD3 antibody or its antigen-binding fragment or an anti-CD2 antibody immobilized on a surface, or by contacting with a protein kinase C activator (e.g., bryostatin) together with a calcium ionophore. For costimulation of accessory molecules on the surface of T cells, a ligand that binds to the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions that are suitable for stimulating the proliferation of T cells. An anti-CD3 antibody and an anti-CD28 antibody can be used to stimulate the proliferation of either CD4+ T cells or CD8+ T cells. Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France), which can be used as well as other methods commonly known in the art (Berg et al., 1998; Haanen et al., 1999; Garland et al., 1999).

[0282] Conditions suitable for culturing immune effector cells include an appropriate medium (e.g., Minimum Essential Medium or RPMI Medium 1640 or X-vivo 15 (Lonza)), which may contain factors necessary for growth and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF, and TNF-a, or any other additives for the growth of cells known to one of skill in the art.

[0283] Cells containing CAR or binding protein In another aspect, the present invention provides a cell comprising the binding protein, CAR, nucleic acid, or vector of the present invention. Suitable cell types are described herein. In some embodiments, the cell is an immune effector cell. As used herein, the phrase "immune effector cell" refers to an immune cell that can affect or induce an immune response upon recognition of an antigen. In some embodiments, the immune effector cell is a T cell or a NK cell. In some embodiments, the immune effector cell is a CD8+ T cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.

[0284] In one embodiment, the cells described herein can further comprise a second (or more) CAR, e.g., a second CAR comprising a different antigen binding domain, e.g., to the same target (e.g., a target described above) or to a different target. In one embodiment, the second CAR comprises an antigen binding domain that binds to a target expressed in the same cancer cell type as the target of the first CAR.

[0285] In another embodiment, the cells comprising the binding protein or CAR described herein can further express another agent, e.g., an agent that enhances the activity of the CAR-expressing cell. For example, in one embodiment, the agent can be an agent that inhibits an inhibitory molecule. In some embodiments, an inhibitory molecule, e.g., PD1, can reduce the ability of a CAR-expressing cell to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGF beta. In one embodiment, the agent that inhibits an inhibitory molecule is, e.g., a molecule described herein, e.g., an agent that includes a first polypeptide that binds to a second polypeptide that provides a positive signal to the cell, e.g., an inhibitory molecule, as described herein. In one embodiment, the agent comprises a first polypeptide, e.g., an inhibitory molecule, e.g., PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, or TGFbeta, or a fragment of any of these (e.g., at least a portion of the extracellular domain of any of these), and a second polypeptide which is an intracellular signaling domain as described herein (e.g., a costimulatory domain (e.g., including 41BB, CD27, or CD28, e.g., as described herein, or a functional variant thereof), and / or a primary signaling domain (e.g., a CD3 zeta signaling domain or a functional variant thereof as described herein).In one embodiment, the agent comprises a first polypeptide of PD1 or a fragment thereof (e.g., at least a portion of the extracellular domain of PD1 or a functional variant thereof) and a second polypeptide of an intracellular signaling domain described herein (e.g., a CD28 signaling domain described herein and / or a CD3 zeta signaling domain described herein or a functional variant thereof). PD1 is an inhibitory member of the CD28 family of receptors, which also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated B cells, T cells, and myeloid cells (Agata et al., 1996). Two ligands for PDl, namely PD-L1 and PD-L2, have been shown to downregulate T cell activation upon binding to PDl (Freeman et al., 2000; Latchman et al., 2001; Carter et al., 2002). PD-L1 is abundant in human cancers (Dong et al., 2003;Blank et al., 2005;Konishi et al., 2004). Immune suppression can be reversed by inhibiting the local interaction of PDl with PD-L1.

[0286] In one embodiment, the agent comprises an inhibitory molecule, such as the extracellular domain (ECD) of programmed death 1 (PD1), fused to a transmembrane domain and an intracellular signaling domain, such as 41BB and CD3 zeta (also referred to herein as a PD1 CAR). In one embodiment, the PD1 CAR improves T cell persistence when used in combination with an XCAR described herein. In one embodiment, the CAR is a PD1 CAR that comprises the extracellular domain of PD1.

[0287] In another embodiment, the present invention provides a population of CAR-expressing cells, e.g., CAR-T cells. In some embodiments, the population of CAR-expressing cells comprises a mixture of cells expressing different CARs. For example, in one embodiment, the population of CAR-T cells can comprise a first cell expressing a CAR with an antigen binding domain and a second cell expressing a CAR with a different antigen binding domain, e.g., an antigen binding domain for a different target, e.g., an antigen binding domain for a target different from the target to which the antigen binding domain of the CAR expressed by the first cell binds. As another example, the population of CAR-expressing cells can comprise a first cell expressing a CAR that comprises an antigen binding domain for a first target and a second cell expressing a CAR that comprises an antigen binding domain for a second target. In one embodiment, the population of CAR-expressing cells can comprise, for example, a first cell expressing a CAR that comprises a primary signaling domain and a second cell expressing a CAR that comprises a secondary signaling domain.

[0288] In the embodiments described herein, the immune effector cell can be an allogeneic immune effector cell, such as a T cell or a NK cell. For example, the cell can be an allogeneic T cell, such as an allogeneic T cell that lacks a functional T cell receptor (TCR) and / or human leukocyte antigen (HLA), such as HLA class I and / or HLA class II expression.

[0289] A T cell lacking a functional TCR can, for example, be engineered not to express any functional TCR on its surface, can be engineered not to express one or more subunits that comprise a functional TCR, or can be engineered to produce very little functional TCR on its surface. Alternatively, the T cell can express a substantially impaired TCR, for example, by expression of a mutated or truncated form of one or more of the subunits of the TCR. The term "substantially impaired TCR" means that the TCR will not elicit a deleterious immune response in the host.

[0290] The T cells described herein can be engineered, for example, to not express functional HLA on its surface.For example, the T cells described herein can be engineered to downregulate cell surface expression HLA, for example, HLA class 1 and / or HLA class II.In some embodiments, the T cells can lack functional TCR and functional HLA, for example, HLA class I and / or HLA class II.

[0291] Modified T cells lacking expression of a functional TCR and / or HLA can be obtained by any suitable means, including knocking out or knocking down one or more subunits of the TCR or HLA. For example, the T cells can include knockdown of the TCR and / or HLA using siRNA, shRNA, clustered regularly interspaced short palindromic repeats (CRISPR), transcription activator-like effector nucleases (TALENs), or zinc finger endonucleases (ZFNs).

[0292] Treatment Method Condition being treated The binding proteins, compositions, and CARs of the present invention are particularly useful for the treatment, prevention, and / or amelioration of any disease or disorder that is associated with or mediated by EGFRvIII expression or activity, or that can be treated by blocking the interaction between EGFRvIII and EGFR ligands, or otherwise inhibiting EGFRvIII activity and / or signaling, and / or promoting receptor internalization, and / or reducing the number of cell surface receptors. For example, the binding proteins, compositions, and CARs of the present invention are useful for treating tumors that express EGFRvIII and / or respond to ligand-mediated signaling. The binding proteins, compositions, and CARs of the present invention can also be used to treat primary and / or metastatic tumors that occur in the brain and meninges, oropharynx, lungs and bronchial tree, gastrointestinal tract, male and female reproductive systems, muscles, bones, skin and appendages, connective tissue, spleen, immune system, hematopoietic cells and bone marrow, liver and urinary tract, and special sensory organs such as the eye. In certain embodiments, the binding proteins, compositions, and CARs of the invention are used to treat one or more of the following cancers: renal cell carcinoma, pancreatic cancer, head and neck cancer, prostate cancer, malignant glioma, osteosarcoma, colorectal cancer, gastric cancer (e.g., gastric cancer with MET amplification), malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, breast cancer, or melanoma.

[0293] In the methods of treatment described herein, the binding protein, composition, or CAR can be administered as a monotherapy (i.e., the therapeutic agent alone) or in combination with one or more additional therapeutic agents (examples of which are described elsewhere herein).

[0294] In one embodiment, the binding proteins of the invention are also useful for treating cancers associated with overexpressed EGFR, preferably in cancers where EGFR is misfolded, in which the binding proteins are useful for treating glioma, pancreatic cancer, colorectal cancer, lung cancer, breast cancer, gastric cancer, renal cancer, cervical cancer, ovarian cancer, adenocarcinoma, bladder cancer, or head and neck cancer.

[0295] Dosage and timing Appropriate dosages of the binding proteins, compositions, and cells of the invention will vary depending on the condition being treated and / or the subject being treated, and are within the ability of a skilled physician to determine appropriate dosages.

[0296] In some aspects, the methods of the invention comprise administering a prophylactically or therapeutically effective amount of a binding protein, composition, or cell described herein.

[0297] The term "therapeutically effective amount" is an amount that, when administered to a subject in need of treatment, improves the subject's prognosis and / or condition and / or reduces or inhibits one or more symptoms of the clinical conditions described herein to a level below that observed and tolerated as a clinical diagnosis or clinical characteristic of the condition.

[0298] As used herein, the term "prophylactically effective amount" should be taken to mean an amount of binding protein, composition, or cell sufficient to prevent or inhibit or delay the onset of one or more detectable symptoms of a clinical condition. Those skilled in the art will recognize that such amounts will vary, for example, depending on the specific binding protein administered, and / or the particular subject, and / or the type or severity or level of the condition, and / or predisposition to the condition (genetic or otherwise).

[0299] Administration of binding proteins, compositions, or cells according to the methods of the invention can be continuous or intermittent, depending, for example, on the physiological condition of the recipient, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to those of skill in the art. Administration can be essentially continuous over a preselected period of time, or can be a series of spaced doses, for example, either during or after the onset of a condition.

[0300] As will be clear to those skilled in the art, the "reduction" of cancer symptoms in a subject will be compared with another subject that also suffers from cancer but is not treated by the methods described herein.This does not necessarily require a side-by-side comparison of two subjects.Instead, population data can be relied upon.For example, a population of subjects with cancer that are not treated by the methods described herein (optionally a population of subjects that are similar to the subjects that are treated, for example, in terms of age, weight, race) can be evaluated, and the average value can be compared with the results of a subject or a population of subjects that are treated by the methods described herein.

[0301] CAR-expressing cells The present invention includes certain cell therapies in which immune effector cells (e.g., T cells, NK cells) are genetically modified to express the CAR described herein, and the CAR-expressing cells are infused into a recipient in need thereof. The infused cells can kill diseased cells in the recipient that express the target of the CAR. Unlike antibody therapies, CAR-modified immune effector cells (e.g., T cells, NK cells) can replicate in vivo and provide long-term persistence that can lead to sustained tumor control. In various embodiments, the immune effector cells (e.g., T cells, NK cells) administered to a patient or their progeny persist in the patient for at least 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years, or 5 years after administration of the T cells or NK cells to the patient.

[0302] The present invention also includes certain cell therapies in which immune effector cells (e.g., T cells, NK cells) are modified to transiently express chimeric antigen receptors (CARs), for example, by in vitro transcribed RNA, and the CAR T cells or NK cells are infused into recipients in need thereof. The infused cells can kill tumor cells in the recipient. Thus, in various embodiments, the immune effector cells (e.g., T cells, NK cells) administered to a patient are present for less than one month, for example, 3 weeks, 2 weeks, 1 week, after administration of the T cells or NK cells to the patient. Without wishing to be bound by any particular theory, the anti-tumor immune response elicited by the CAR-modified immune effector cells (e.g., T cells, NK cells) can be an active or passive immune response, or alternatively, can be a direct immune response versus an indirect immune response. In one embodiment, CAR-transduced immune effector cells (e.g., T cells, NK cells) exhibit specific proinflammatory cytokine secretion and potent cytolytic activity in response to human cancer cells expressing EGFRvIII.

[0303] Ex vivo procedures are well known in the art. Briefly, cells are isolated from a mammal (e.g., human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing a CAR as described herein. The CAR-expressing cells can be administered to a mammalian recipient to provide therapeutic benefits. The mammalian recipient can be a human, and the CAR-expressing cells can be autologous to the recipient. Alternatively, the cells can be allogeneic, syngeneic, or xenogeneic to the recipient.

[0304] The procedure for ex vivo expansion of hematopoietic stem cells and hematopoietic progenitor cells is described in US5,199,942 and can be applied to the cells of the present invention. Other suitable methods are known in the art, and therefore the present invention is not limited to any particular method of ex vivo expansion of cells. In brief, the ex vivo culture and expansion of immune effector cells (e.g., T cells, NK cells) includes the following steps: (1) collecting mammalian CD34+ hematopoietic stem cells and hematopoietic progenitor cells from peripheral blood collection or bone marrow explant; and (2) ex vivo expansion of such cells. In addition to the cell growth factors described in US5,199,942, other factors such as flt3-L, IL-1, IL-3, and c-kit ligand can be used to culture and expand cells.

[0305] The CAR-expressing immune effector cells (e.g., T cells, NK cells) of the present invention can be administered either alone or as a pharmaceutical composition in combination with diluents and / or other components, such as IL-2 or other cytokines, or cell populations, as described herein. The immune effector cells can be administered either alone or as a pharmaceutical composition in combination with diluents and / or other components, such as IL-2, IL-15, or other cytokines, or cell populations. Briefly, a pharmaceutical composition can include the immune effector cells described herein in combination with one or more pharma- ceutical or physiologically acceptable carriers, diluents, or excipients. Such compositions can include buffers, such as neutral buffered saline, phosphate buffered saline, and the like; carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. In some embodiments, the compositions for use in the disclosed methods are formulated for intravenous administration.

[0306] Pharmaceutical compositions comprising the cells described herein may be used in any combination of 10 to 20 mg of 10 ... 4 ~10 9 Cells / kg body weight, e.g., 10 5 ~10 6 The cells can be administered in a dosage of cells / kg body weight. The cell composition can also be administered multiple times at these dosages. The cells can be administered by injection techniques commonly known in immunotherapy (see, for example, Rosenberg et al., 1988). The optimal dosage and treatment regime for a particular patient can be easily determined by those skilled in the medical arts by monitoring the patient for symptoms of disease and adjusting the treatment accordingly.

[0307] Combination therapy The binding protein or CAR of the invention can be co-formulated with and / or administered in combination with one or more additional therapeutically active ingredients of one or more chemotherapeutic agents.

[0308] In vitro and diagnostic uses The binding proteins of the invention can also be used to detect and / or measure EGFRvIII or EGFRvIII expressing cells in a sample, e.g., for diagnostic purposes. Alternatively, the binding proteins of the invention can also be used to detect and / or measure EGFR or EGFR expressing cells in a sample, e.g., for diagnostic purposes, preferably when EGFR is misfolded. For example, the binding proteins of the invention can be used to diagnose a condition or disease characterized by expression of EGFRvIII. An exemplary diagnostic assay for EGFRvIII or EGFR can include, for example, contacting a sample obtained from a patient with a binding protein of the invention, where the binding protein is labeled with a detectable label or reporter molecule. Alternatively, the unlabeled binding proteins of the invention can be used in diagnostic applications in combination with a secondary protein that is itself detectably labeled. The detectable label or reporter molecule can be a radioisotope, e.g., 3 H,14 C. 32 S, or 125 I; fluorescent or chemiluminescent moiety, such as fluorescein isothiocyanate or rhodamine; or enzyme, such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure EGFRvIII in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

[0309] The sample that can be used in the EGFRvIII or EGFR diagnostic assay according to the present invention includes any tissue or body fluid sample that can be obtained from a patient and contains a detectable amount of EGFRvIII or EGFR protein or their fragments under normal or pathological conditions.Generally, the level of EGFRvIII or EGFR in a particular sample obtained from a healthy patient (e.g., a patient not suffering from a disease or condition related to the expression of EGFRvIII) is measured to first establish a baseline or reference level of EGFRvIII or EGFR.This baseline level of EGFRvIII or EGFR can then be compared with the level of EGFRvIII or EGFR measured in a sample obtained from an individual suspected of having a disease or condition related to EGFRvIII or EGFR. EXAMPLES

[0310] Example 1 - Materials and Methods Generation of anti-EGFRvIII scFv Recombinant soluble EGFRvIII protein (catalog no. EGI-H52H4), consisting of the extracellular domain and no transmembrane or endodomain, and wild-type EGFR (catalog no. EGR-H5222) were purchased from ACRO Biosystems. Recombinant EGFRvIII was labeled with the chemical fluorophores DyLight 405 NHS ester (ThermoFisher, catalog no. 46400) and ATTO 488 NHS ester (ATTO-TEC, catalog no. AD 488-31) according to the manufacturer's instructions. Recombinant EGFR was labeled with DyLight 405 NHS ester.

[0311] Affinity Biosciences' Retained Display (ReD) protein display platform (WO2011075761A1 and WO2013000023A1) and the RUBY library (WO2013023251A1) were used to screen for scFv antibodies with human germline scaffolds that would selectively target the EGFRvIII form compared to the wild-type EGFR protein. Following two rounds of panning of the RUBY bacteriophage library using EGFRvIII protein bound to Dynabeads (ThermoFisher, Cat. No. 65002), the panning output was switched to the ReD cell display modality and processed as described in patent WO2013000023A1. Detergent-permeabilized cells displaying the capsid-bound scFv were labeled with EGFRvIII-ATTO488, and target-bound clones were selected by FACS. The FACS output was lysed, infected with bacteriophage, and clones were recovered on LB agar plates. The second and third rounds of FACS were performed in a similar manner, using EGFRvIII labeled with Dy405 for the second round of FACS and ATTO488 for the third round. Two rounds of counterscreening FACS were then performed, first using Dy405-labeled EGFRvIII and ATTO488-labeled EGFR to gate clones that selectively bound to Dy405-labeled EGFRvIII. The second FACS counterscreen reversed the dye label and selected clones that bound to the ATTO488-labeled target. The output clones of the second counterscreen were sequenced and unique scFvs were cloned into expression vectors that produced scFv proteins as His6 and AviTag fusions to aid in purification and characterization.

[0312] Sequencing of the output of the EGFRvIII screen yielded 13 clones that preferentially bound to EGFRvIII compared to the wild-type EGFR extracellular domain. Selectivity was demonstrated by assaying biotinylated scFvs by first binding to streptavidin Dynabeads, which were then washed and blocked with free biotin before binding to soluble EGFRvIII or EGFR labeled with ATTO488. An irrelevant biotinylated scFv was used as a control to establish baseline binding and fluorescence. Figure 1 shows the relative binding of scFv clones to either EGFRvIII or EGFR. Two clones, 70 and 205, newly named GCT01 (VH / VL SEQ ID NO:3 / 4) and GCT02 (VH / VL SEQ ID NO:5 / 6), showed negligible binding to EGFR wild-type but maintained strong binding to the EGFRvIII isoform. GCT01 was notable because its VH CDR3 was related to 10 other unique clones, suggesting a conserved binding epitope in EGFRvIII. Although all 10 clones in this clade used the same VL germline scaffold (IGLV3-1), their VL CDR3s showed only weak sequence conservation (a central Gly was a feature of the library structure using the IGLV3-1 scaffold), suggesting that the VH CDR3 creates a crucial binding determinant. However, subtle differences in the GCT01 sequence compared to other clones in this clade gave this clone a higher discrimination of EGFRvIII against EGFR.

[0313] Generation, purification, and activation of mouse T cells Lymph nodes from 6-8 week old C57BL / 6 mice were harvested and filtered through a 0.7 μm nylon filter. Cells were washed with PBS and centrifuged (1500 RPM, 5 min). Cells were diluted to 1 × 10 8Cells / ml were resuspended and CD4 or CD8 T cells were positively selected using the EasySep™ Positive Selection Kit according to the manufacturer's instructions. Primary T cells were activated with mouse CD3z / CD28 Dynabeads® and incubated overnight at 37° C., 5% CO2.

[0314] Detection of EGFRvIII expression on target cells Target cells were labeled with GCT01, GCT02 scFv for 30 minutes on ice. Cells were labeled with streptavidin-PE for 30 minutes. Samples were analyzed by flow cytometry and cells were gated for PE positive cells.

[0315] Surface Plasmon Resonance (SPR) SPR experiments were performed using a ProteOn XPR36 instrument (Bio-Rad) at 25 °C in PBS containing 0.05% Tween (PBS-T). Streptavidin was diluted in 10 mM sodium acetate (pH 4.0) and 300 response units (RU) were immobilized by amine coupling onto three flow cells of a GLC sensor chip. Biotinylated GCT01, GCT02, and GFP scFv chains (200–400 RU) were captured on separate flow cells of the ProteOn GLC streptavidin-coupled sensor chip. A separate flow cell containing immobilized streptavidin with no scFv bound served as a control channel.

[0316] Recombinant EGFR Catalog Number (Cat) EGR-H5222 Lot: R102-616S1-A3 and EGFRvIII (Protein Catalog Number EGI-H52H4 Lot: 2144-65RS1-BG) were obtained from Acrobiosystems and reconstituted in PBS to 1ug / ul, then diluted in PBS-T and injected over the test and control surfaces at the indicated concentrations (flow rate 30μl / min). GFP protein was injected over the chip to serve as a negative control for non-specific scFv binding. Injected protein was cleared from the chip between injections using 10mM glycine HCl buffer (pH 3). Results from at least two independent injections were analyzed. Interactions were analyzed with ProteOn Manager software (version 2.1) after subtracting data from control cells. KD values ​​were obtained from kinetic fit analysis using a 1:1 Langmuir binding model.

[0317] Generation of EGFRvIII CAR-expressing cells To confirm cell surface expression, a MYC tag was inserted into the scFv region. Gene blocks were ordered, subcloned into the pMSCV plasmid backbone using Gibson cloning, and fully sequence verified.

[0318] CAR plasmids were combined with retroviral coat and packaging plasmids and transfected into HEK293T cells using FuGENE-6 (Promega) according to the manufacturer's instructions. Retronectin (32ug / mL) was coated onto 12-well plates overnight at 4°C, then washed and transduced. HEK293T viral supernatants were filtered and centrifuged with primary activated T cells and incubated for >4 hours, then centrifuged at 1000G for 1 hour and incubated overnight at 37°C, 5% CO2. Lymph nodes from 6-8 week old C57BL / 6 mice were harvested and CD4 + and CD8 +T cell populations were positively selected and then activated with CD3ζ / CD28 Dynabeads. Retrovirus in the supernatant was collected at 24 h and used to transduce activated T cells. 24 h after transduction, cells were analyzed by flow cytometry to assess transduction efficiency by expression of mCherry and cell surface Myc tagging.

[0319] Generation of non-signaling EGFRvIII-expressing cell lines Lentivirus containing non-signaling EGFRvIII (SEQ ID NO:40) was generated in HEK293T cells and transduced into tumor cell lines, which were stained using an in-house anti-EGFRvIII binder, and EGFRvIII-positive cells were FACS sorted, expanded, and used in the assay.

[0320] Chromium release assay 1×10 6 Incubate target tumor cells for 1 h (37 °C) in chromium ( 51 Cr), then washed and co-cultured with CAR-T cells at an effector to target cell ratio of 10:1. Minimum killing was measured in target cells alone and maximum killing was measured in target cells with 10% Triton X-100. Target and effector cells were co-incubated in 96-well plates (in triplicate) at 37°C and 5% CO2 for 24 hours. Lysis was measured in viral supernatants using a Genesys GeneII scintillation counter.

[0321] In vivo testing of CAR-T cells in intracranial tumor-bearing mice One hour before surgery, mice were given 10 mg / kg of prophylactic Baytril (enrofloxacin) antibiotics. The general anesthesia (GA) Ketamine to Xylazine (20,000 mg / Kg (i.e., 0.4 g (400 μl) per 20 g mouse combination) was administered i.p., and a subcutaneous injection of 5 mg / kg Carprofen analgesic was administered. Once the mouse was secured in the stereotaxic frame, the injection site was identified as 2 mm lateral to the bregma at the coronal suture to a depth of 3 mm. On the determined target, the scalp was incised sagittally with a sterile blade. A small burr hole of 3 mm was made in the right frontal region using a motorized burr. A 26-gauge needle attached to a Hamilton 10 μl microliter syringe via a thin plastic tube driven by World Precision Instruments™ was used to inject 10 μl of carprofen into the right frontal region. 5 Tumor cells were injected into the brain at a depth of 3 mm under stereotactic guidance. Tumor cells were dispensed at 5 μl over 3 minutes using an Aladdin™ AL-1000 electronic programmable syringe pump. The injection site was closed with bone wax and the scalp was closed with Vicryl dissolving braided sutures using an interrupted suture technique. Mice were administered a subcutaneous injection of 10 mg / kg Baytril for 3 days after surgery.

[0322] Tumor growth was monitored using an IVIS system (IVIS® Lumina III series hardware (Perkin Elmer) with Living Image® software). Briefly, mice were injected intraperitoneally with 200 μL of Promega D-luciferin (in PBS) using a 27G insulin syringe. After approximately 5-10 minutes to allow the luciferin to circulate throughout the body, mice were anesthetized using isoflurane inhalant (levels between 4% and maintained at 2%) and transferred to the IVIS imaging system for imaging.

[0323] Following positive IVIS imaging of tumor engraftment, performed 5-8 days after tumor inoculation, mice were randomly assigned to groups and administered between 2e6-10e6 CD4 and CD8 CAR T cells intravenously. Mice were then imaged weekly using IVIS® Lumina to monitor tumor cell growth.

[0324] Example 2 - High affinity anti-EGFRvIII scFv Two high affinity anti-EGFRvIII scFvs that specifically bind EGFRvIII compared to wild type EGFR were generated using a phage display screen at Myrio Therapeutics, Victoria, as described in Example 1. The two high affinity anti-EGFRvIII scFvs were named "GCT01" and "GCT02". Their amino acid sequences are shown below. SEQ ID NO:3-GCT01 VH amino acid sequence (CDRs are underlined) TIFF2024516748000003.tif15138SEQ ID NO:4-GCT01 VL amino acid sequence (CDRs are underlined) TIFF2024516748000004.tif10137SEQ ID NO:5-GCT02 VH amino acid sequence (CDRs are underlined) TIFF2024516748000005.tif16138SEQ ID NO:6-GCT02 VL amino acid sequence (CDRs are underlined) TIFF2024516748000006.tif15138

[0325] Example 3 - Binding to EGFRvIII To determine the binding characteristics of GCT01 and GCT02 to EGFRvIII, surface plasmon resonance (SPR) experiments were performed using a ProteOn XPR36 instrument (Bio-Rad). Biotinylated GCT01, GCT02, and GFP scFv chains (200–400 RU) were captured on separate streptavidin-conjugated sensor chips. A separate flow cell containing immobilized streptavidin with no scFv bound served as a control channel.

[0326] Figure 2 shows that GCT01 scFv and GCT02 scFv are more specific for EGFRvIII protein than EGFR wild type protein. Both GCT01 and GCT02 scFv clones bound specifically to recombinant EGFRvIII protein compared to EGFR (wild type) recombinant protein as determined by surface plasmon resonance (SPR). GFP protein was used as a negative control. Recombinant EGFR and EGFRvIII (extracellular domain only) proteins were reconstituted in PBS to 1ug / ul, then diluted in PBS-T and injected over the test and control surfaces at concentrations between 0.1nM and 10nM. As shown in Figure 2, both GCT01 and GCT02 bound to EGFRvIII, and only very limited binding was detected for wild type EGFR. Dissociation constant (KD) values ​​were obtained from kinetic fit analysis using a 1:1 Langmuir binding model and were calculated to be 4.57×10-11 M for GCT01 and 3.27×10-10 M for GCT02. The affinity of GCT01 and GCT02 for EGFRvIII was several orders of magnitude greater than that previously reported for a humanized scFv called "2173" or "N2173" (1.01×10-7 M) and a murine scFv called "3C10" (2.58×10-8 M) in Johnson et al. (2015).

[0327] Flow cytometry was also performed to determine the ability of GCT01 and GCT02 to bind to EGFR expressed on the surface of cells. Importantly, both GCT01 and GCT02 scFv clones bound specifically to U87 EGFRvIII cells, while only GCT01 showed binding to U87 wild-type cells. This confirmed that GCT01 and GCT02 bound specifically to both recombinant soluble EGFRvIII protein (Figure 2) and EGFRvIII expressed on the target cell membrane (Figure 3).

[0328] Interestingly, Figure 3 also shows that GCT01 exhibits potential binding not only to EGFRvIII but also to misfolded EGFR, as detected in HCC827 (lung adenocarcinoma cells), U251 (human glioblastoma cells), and low levels of binding to HCT116 (human colorectal cancer cells).

[0329] Example 4 - Activity of EGFRvIII CAR The genes for GCT01 scFv and GCT02 scFv were codon-optimized for expression in human cells and cloned into a retroviral vector for CAR T cell generation. The codon-optimized nucleotide sequences for both scFvs are provided in SEQ ID NOs:29-34.

[0330] Each CAR construct contained GCT01 scFv or GCT02 scFv, a CD8 hinge, a CD28 transmembrane domain, and an intracellular domain containing the CD28 costimulatory domain and the CD3 zeta primary signaling domain. The CARs were also expressed with an N-terminal leader sequence for T cell membrane localization and a Myc tag to confirm CAR expression. The amino acid sequences of the complete CARs are provided in SEQ ID NOs:25 and 26.

[0331] Retroviral vectors were transduced into primary T cells as described in Example 1, and transduction efficiency was measured by flow cytometry. Figure 4 shows that GCT01 and GCT02 chimeric antigen receptors are expressed on the surface of T cells. Cell surface CAR expression determined by flow cytometry using the Myc tag located under the scFv. GCT01 (left) shows more consistent expression of the intracellular fluorophore mCherry (used as a surrogate measure of transduction efficiency) than GCT02 (right). Both constructs show very high mCherry expression. These data are representative of 5 experiments. Figure 4 shows that more than 70% of primary T cells in any given case show positive CAR expression for both GCT01 and GCT02.

[0332] To determine whether GCT01 and GCT02 CAR-expressing T cells have cytotoxic activity against cells expressing EGFR, a chromium release assay was used. The chromium release assay measures the amount of radioactive chromium ( 51 Target cell death is measured by quantification of Cr).

[0333] The cytotoxicity of EGFRvIII CAR-T cells against various cell lines was tested and the results are shown in Figure 5. mCherry empty vector T cells were used as a negative control. The effector to target ratio was 10:1. Cytotoxicity was measured by chromium release measured after 24 hours of co-incubation (for cell lines marked with "**" in Figure 5, cytotoxicity was measured after 4 hours of co-incubation). The background level of non-specific target cell death was about 10%. As expected, there was no specific target cell killing by any of the CAR T cells of HEK293T, MC57 WT, or E0771 parental cell lines, which do not express EGFRvIII. As a positive control for cytotoxicity, MC57, U87, and E0771 target cells were transduced with non-signaling EGFRvIII constructs to achieve high EGFRvIII cell surface expression. In these cell lines, GCT01 and GCT02 CAR T cells induced EGFRvIII-specific target cell death.

[0334] GCT01 CD8 + There was also cytotoxicity of U87 WT, U251 and HCT116 cells induced by CART cells, which correlated with the binding of GCT01 scFv clones to these cell lines. Genomic amplification of EGFR is observed in 40-70% of glioblastomas and can lead to overexpression and misfolding at the cell surface. EGFR has been shown to be expressed in U87 cells, suggesting that GCT01 binds to misfolded EGFR on cancer cells, and not just EGFRvIII.

[0335] Example 5 - GCT01 and GCT02 CAR-T cells induce tumor regression in intracranial glioma tumors Firefly luciferase-labeled U87-EGFRvIII tumor cells were grown intracranially in immunocompromised NSG mice for 1 week. A 1:1 mixture of CD4 and CD8 T cells (5 × 10) expressing either the empty mCherry-labeled CAR, GCT01-CAR, or GCT02-CAR was then injected into the mice. 6 Cells) were administered as a single iv injection.

[0336] Figure 7 shows that GCT01 and GCT02 CAR T cells induce rapid and complete elimination of glioblastoma brain tumors in vivo. The data show that at 3 weeks, little or no luciferase activity was detected in GCT01-CAR and GCT02-CAR treated mice, indicating that cells expressing these receptors are able to induce tumor regression in this model of glioma.

[0337] Example 6 - GCT01 CAR-T cells induce tumor regression in a model of lung adenocarcinoma To further characterize the efficacy of GCT01 CAR T cells in vivo, a subcutaneous model of lung adenocarcinoma was used. Briefly, female NSG mice were subcutaneously injected with 2e6 HCC827 cells, and 5–8 days later, 2–10e6 CD4 and CD8 GCT01 CAR T cells were intravenously adoptively transferred into the mice. Tumor growth was measured three times a week using calipers. All tumor-bearing NSG mice treated with GCT01 CAR T cells had tumor regression within 14 days after adoptive transfer (Figure 6).

[0338] Those skilled in the art will recognize that numerous variations and / or modifications can be made to the invention as illustrated in the specific embodiments without departing from the spirit or scope of the invention as broadly described, which embodiments are therefore to be considered in all respects as illustrative and not restrictive.

[0339] All publications cited herein are incorporated herein by reference in their entirety. When referring to a URL or other such identifier or address, it is understood that such identifiers may change and particular information on the Internet may shift, but equivalent information may be found by searching the Internet. Reference thereto evidences the availability and public dissemination of such information.

[0340] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention and is not to be construed as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention by reason of their existence prior to the priority date of each claim of this application.

[0341] References TIFF2024516748000007.tif202101TIFF2024516748000008.tif33128

Claims

1. A human EGFRvIII binding protein comprising an antigen-binding domain that binds to EGFRvIII, wherein the antigen-binding domain competitively inhibits binding of EGFRvIII to an antibody comprising: A heavy chain variable region (V) comprising CDR1, CDR2, and CDR3 comprising the sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively. H ); and A light chain variable region (V) comprising CDR1, CDR2, and CDR3 comprising the sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively. L );or A heavy chain variable region (V) comprising CDR1, CDR2, and CDR3 comprising the sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively. H ); and A light chain variable region (V) comprising CDR1, CDR2, and CDR3 comprising the sequences set forth in SEQ ID NOs:22, 23, and 24, respectively. L ).

2. 2. The EGFRvIII binding protein of claim 1, comprising: (a) a heavy chain variable region (V) comprising CDR1, CDR2, and CDR3 comprising the sequences set forth in SEQ ID NOs:7, 8, and 9, respectively; H ); and / or A light chain variable region (V) comprising CDR1, CDR2, and CDR3 comprising the sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively. L );or (b) a heavy chain variable region (V H or a heavy chain variable region (V) comprising a CDR1 comprising an amino acid sequence having no more than two amino acid substitutions relative to SEQ ID NO:13, a CDR2 comprising an amino acid sequence set forth in SEQ ID NO:14 or having no more than four amino acid substitutions relative to SEQ ID NO:14, and a CDR3 comprising an amino acid sequence set forth in SEQ ID NO:15 or having no more than three amino acid substitutions relative to SEQ ID NO:

15. H ), and / or a light chain variable region (V) comprising CDR1 comprising the sequence set forth in SEQ ID NO:16 or an amino acid sequence having no more than three amino acid substitutions relative to SEQ ID NO:16, CDR2 comprising the sequence set forth in SEQ ID NO:17 or an amino acid sequence having no more than two amino acid substitutions relative to SEQ ID NO:17, and CDR3 comprising the sequence set forth in SEQ ID NO:18 or an amino acid sequence having no more than three amino acid substitutions relative to SEQ ID NO:18; L );or (c) a CDR1 comprising a sequence set forth in SEQ ID NO:19 or an amino acid sequence having no more than two amino acid substitutions relative to SEQ ID NO:19, a CDR2 comprising a sequence set forth in SEQ ID NO:20 or an amino acid sequence having no more than four amino acid substitutions relative to SEQ ID NO:20, and a CDR3 comprising a sequence set forth in SEQ ID NO:21 or an amino acid sequence having no more than three amino acid substitutions relative to SEQ ID NO:

21. H and / or CDR1 comprising the sequence set forth in SEQ ID NO:22 or an amino acid sequence having no more than three amino acid substitutions relative to SEQ ID NO:22, CDR2 comprising the sequence set forth in SEQ ID NO:23 or an amino acid sequence having no more than two amino acid substitutions relative to SEQ ID NO:23, and CDR3 comprising the sequence set forth in SEQ ID NO:24 or an amino acid sequence having no more than three amino acid substitutions relative to SEQ ID NO:

24. L .

3. EGFRvIII binding protein according to claim 1 or 2, comprising: (a) a heavy chain variable region (V) comprising an amino acid sequence set forth in SEQ ID NO:3 or at least about 70% identical, at least about 80% identical, at least about 90% identical, or at least about 95% identical to the sequence set forth in SEQ ID NO:3; H ), and / or A light chain variable region (V) comprising a sequence set forth in SEQ ID NO:4 or an amino acid sequence at least about 70% identical, at least about 80% identical, at least about 90% identical, or at least about 95% identical to the sequence set forth in SEQ ID NO:

4. L );or (b) a heavy chain variable region (V) comprising an amino acid sequence set forth in SEQ ID NO:5 or at least about 70% identical, at least about 80% identical, at least about 90% identical, or at least about 95% identical to the sequence set forth in SEQ ID NO:5; H ), and / or A light chain variable region (V) comprising a sequence set forth in SEQ ID NO:6 or an amino acid sequence at least about 70% identical, at least about 80% identical, at least about 90% identical, or at least about 95% identical to the sequence set forth in SEQ ID NO:

6. L ).

4. 2. The EGFRvIII binding protein of claim 1, comprising: (i) single chain Fv fragment (scFv); (ii) dimeric scFv (di-scFv); (iii) diabody; (iv) triabody; (v) tetrabody; (vi) Fab; (vii)F(ab') 2 ; (viii) Fv; or (ix) at least one of (i)-(viii) linked to an antibody constant region, a fragment crystallizable (Fc) region, or a heavy chain constant domain CH2 and / or CH3.

5. 2. The EGFRvIII binding protein of claim 1, which is a bispecific T cell engager (BiTE).

6. The EGFRvIII binding protein of claim 1 , which comprises an Fc region and is optionally an antibody.

7. A chimeric antigen receptor (CAR) comprising the binding protein of claim 1, a transmembrane domain, and an intracellular domain.

8. The CAR of claim 7, wherein the intracellular domain comprises a primary signaling domain and a costimulatory domain.

9. (a) the primary signaling domain comprises a primary signaling domain of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, common FcR gamma (FCER1G), FcR beta (Fc epsilon Rib), CD79a, CD79b, Fc gamma RIIa, DAP10, or DAP12; Preferably, the primary signaling domain is the primary signaling domain of CD3 zeta. (b) the costimulatory domain specifically binds to CD28, 4-1BB (CD137), OX40, CD27, CD30, CD40, CD134, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFl), CD160, CD19, CD4, CD8alpha, CD8beta, IL2Rbeta, IL2Rgamma, IL7Ralpha, ITGA4, VLAl, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA -1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, or the costimulatory domain of TNFR2. Preferably, the costimulatory domain is the costimulatory domain of CD28, and / or (c) the transmembrane domain is selected from the group consisting of CD28, CD3 epsilon, CD3 zeta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGBl, CD29, ITGB2, CD18, LFA-1, ITGB7, DNAMl (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT including the transmembrane domains of AM, Ly9 (CD229), CD160 (BY55), PSGLl, CDIOO (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, NKG2C, or TNFR2, or the alpha, beta, or zeta chains of the T cell receptor; Preferably, the transmembrane domain is the transmembrane domain of CD28.

9. The CAR of claim 8.

10. the binding protein is linked to the transmembrane domain by a hinge region; 8. The CAR of claim 7, optionally wherein the hinge region comprises a CD8 hinge, an IgG hinge, an IgD hinge, a CD28 hinge, a KIR2DS2 hinge, or a glycine-serine linker.

11. The CAR of claim 7, comprising an amino acid sequence that is at least about 70%, at least about 80%, at least about 90%, at least about 95% identical, or 100% identical to the sequence set forth in SEQ ID NO:25 or SEQ ID NO:

26.

12. A nucleic acid encoding a binding protein of claim 1 or 5 or a CAR of claim 7.

13. 13. The nucleic acid of claim 12, comprising a nucleotide sequence that is at least about 70%, at least about 80%, at least about 90%, at least about 95% identical, or 100% identical to any one or more of the sequences provided in SEQ ID NOs: 29, 30, 31, 32, 33, or 34.

14. A vector comprising the nucleic acid of claim 12, Optionally, the vector is a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenovirus or adeno-associated virus vector, or a retroviral vector.

15. 10. A cell comprising the binding protein of claim 1 or the CAR of claim 7, Optionally, the cell is an immune effector cell. Further optionally, the immune effector cells are T cells. The cells.

16. A composition comprising the protein of claim 1 and a pharma- ceutically acceptable carrier.

17. A composition comprising the CAR of claim 7 and a pharma- ceutically acceptable carrier.

18. A composition comprising the vector of claim 14 and a pharma- ceutically acceptable carrier.

19. 13. A method of producing a CAR-expressing cell, comprising introducing into a cell the nucleic acid of claim 12 under conditions such that the CAR is expressed.

20. 17. The EGFRvIII binding protein of claim 1 or the composition of claim 16 for use in the treatment of cancer associated with overexpression of EGFRvIII.

21. 18. The composition of claim 17 for use in the treatment of cancer associated with overexpression of EGFRvIII.

22. 19. The composition of claim 18 for use in the treatment of cancer associated with overexpression of EGFRvIII.

23. 21. The protein or composition for use according to claim 20, wherein the cancer associated with overexpression of EGFR is glioma, pancreatic cancer, colorectal cancer, lung cancer, breast cancer, gastric cancer, renal cancer, cervical cancer, ovarian cancer, adenocarcinoma, bladder cancer, or head and neck cancer.

24. 22. The composition of claim 21, wherein the cancer associated with overexpression of EGFR is glioma, pancreatic cancer, colorectal cancer, lung cancer, breast cancer, gastric cancer, renal cancer, cervical cancer, ovarian cancer, adenocarcinoma, bladder cancer, or head and neck cancer.

25. 23. The composition of claim 22, wherein the cancer associated with overexpression of EGFR is glioma, pancreatic cancer, colorectal cancer, lung cancer, breast cancer, gastric cancer, renal cancer, cervical cancer, ovarian cancer, adenocarcinoma, bladder cancer, or head and neck cancer.

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