Monoclonal antibodies that bind to EGFRvIII and uses thereof

Abstract

Disclosed are monoclonal antibodies and antigen-binding fragments thereof, conjugates thereof, and chimeric antigen receptors that specifically bind to epidermal growth factor receptor (EGFR) variant (v) III. Nucleic acid molecules encoding antibody heavy and light chain domains and chimeric antigen receptors (CARs) are also disclosed, as are host cells expressing the nucleic acid molecules. Furthermore, disclosed are the use of these monoclonal antibodies, antigen-binding fragments, conjugates, and T cells expressing CARs, for example, for the treatment of tumors expressing EGFRvIII. Also disclosed are methods for detecting tumors expressing EGFRvIII.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 869,956, filed July 2, 2019, which is incorporated by reference herein in its entirety.

[0002] Government support statement This invention was made with government support under Project No. Z01#:Z1A BC 008757 awarded by the National Cancer Institute of the National Institutes of Health. The U.S. Government has certain rights in this invention.

[0003] Areas of disclosure The present disclosure relates to the area of ​​cancer biology, specifically to monoclonal antibodies and antigen-binding fragments thereof that specifically bind to human epidermal growth factor receptor (EGFR) variant III (vIII). [Background technology]

[0004] background EGFR is frequently implicated in the oncogenic progression of human cancers. Various alterations in expression, including gene amplification and activating mutations, contribute to carcinogenesis. This large receptor has a 621-amino acid ectodomain (ECD), a 23-amino acid single transmembrane domain (TM), and a 542-amino acid enzymatically active intracellular domain (ICD). EGFR is a member of the receptor tyrosine kinase family and was the first receptor shown to be positively associated with human cancer. Ligand binding leads to receptor dimerization and activation of the kinase domain, which transduces signals into one of several pathways that can promote mammalian cell growth, survival, and distribution. Activating mutations can occur in either the ECD or ICD; gene amplification and large deletions, exemplified by the loss of exons 2–7, which produces EGFR variant (v)III, or the loss of exon 19, which generates a constitutively active enzyme mutant, also exist. Expression of EGFRvIII or the loss of exon 19 has only been reported in cancer cells. There remains a need for antibodies that can bind to EGFRvIII and not to wild-type EGFR. Summary of the Invention

[0005] Disclosure Overview Disclosed are isolated monoclonal antibodies and antigen-binding fragments thereof that specifically bind to EGFRvIII. In some embodiments, the monoclonal antibody or antigen-binding fragment is (a) Heavy chain variable region (V) shown as SEQ ID NOs: 1 and 2, respectively H ) and the light chain variable region (V L ) including heavy chain complementarity determining regions (HCDR) 1, HCDR2, and HCDR3, and light chain complementarity determining regions (LCDR) 1, LCDR2, and LCDR3, H and V L ; (b) V, shown as SEQ ID NOs: 11 and 12, respectively H and V LV, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L ; (c) V, shown as SEQ ID NOs: 13 and 14, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L ;or (d) V, shown as SEQ ID NOs: 15 and 16, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L Includes:

[0006] In some embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof of claim 1 comprises: (a) V, shown as SEQ ID NOs: 17 and 12, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L ; (b) V, shown as SEQ ID NOs: 26 and 12, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L ; (c) V, shown as SEQ ID NOs: 29 and 30, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L ; (d) V, shown as SEQ ID NOs: 39 and 40, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L ; (e) V, shown as SEQ ID NOs: 43 and 44, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L ;or (f) V, shown as SEQ ID NOs: 53 and 54, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L Includes:

[0007] In other embodiments, conjugates of these antibodies and antigen-binding fragments are disclosed. In yet other embodiments, chimeric antigen receptors comprising these antibodies or antigen-binding fragments are disclosed. In further embodiments, T cells expressing these chimeric antigen receptors are disclosed.

[0008] In some embodiments, the V of these monoclonal antibodies H and / or V L Disclosed are nucleic acid molecules encoding the same, vectors containing these nucleic acids, and host cells transformed with these nucleic acid molecules and / or vectors.

[0009] In a further embodiment, the use of these monoclonal antibodies for inhibiting tumors expressing EGFRvIII in a subject is disclosed. In another embodiment, the use of these monoclonal antibodies for detecting EGFRvIII is disclosed.

[0010] [The present invention 1001] (a) Heavy chain variable region (V) shown as SEQ ID NOs: 1 and 2, respectively H ) and the light chain variable region (V L ) including heavy chain complementarity determining regions (HCDR) 1, HCDR2, and HCDR3, and light chain complementarity determining regions (LCDR) 1, LCDR2, and LCDR3, H and V L ; (b) V, shown as SEQ ID NOs: 11 and 12, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L ; (c) V, shown as SEQ ID NOs: 13 and 14, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L ;or (d) V, shown as SEQ ID NOs: 15 and 16, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L 1. An isolated monoclonal antibody or antigen-binding fragment thereof comprising: An isolated monoclonal antibody or antigen-binding fragment thereof, wherein the monoclonal antibody specifically binds to epidermal growth factor receptor (EGFR) variant III (vIII). [The present invention 1002] (a) V, shown as SEQ ID NOs: 17 and 12, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L ; (b) V, shown as SEQ ID NOs: 26 and 12, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L ; (c) V, shown as SEQ ID NOs: 29 and 30, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L ; (d) V, shown as SEQ ID NOs: 39 and 40, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L ; (e) V, shown as SEQ ID NOs: 43 and 44, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L ;or (f) V, shown as SEQ ID NOs: 53 and 54, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L 1001. An isolated monoclonal antibody or antigen-binding fragment thereof of the present invention, comprising: [The present invention 1003] (a) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth as SEQ ID NOs:5, 6, 7, 8, 9, and 10, respectively; (b) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth as SEQ ID NOs:20, 21, 22, 23, 24, and 25, respectively; (c) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth as SEQ ID NOs: 20, 28, 22, 23, 24, and 25, respectively; (d) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth as SEQ ID NOs: 33, 34, 35, 36, 37, and 38, respectively; (e) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth as SEQ ID NOs: 47, 48, 49, 50, 51, and 52, respectively; or (f) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth as SEQ ID NOs: 47, 57, 49, 58, 51, and 52, respectively; The antibody or antigen-binding fragment thereof of the present invention 1001 or 1002. [The present invention 1004] (a)V H and V L comprises an amino acid sequence that is at least 90% identical to the amino acid sequences set forth as SEQ ID NOs: 1 and 2, respectively; (b)V H and V L comprises an amino acid sequence that is at least 90% identical to the amino acid sequences set forth as SEQ ID NOs: 17 and 12, respectively; (c)V H and V L comprises an amino acid sequence that is at least 90% identical to the amino acid sequences set forth as SEQ ID NOs: 26 and 12, respectively; (d)V H and V L comprises an amino acid sequence that is at least 90% identical to the amino acid sequences set forth as SEQ ID NOs: 29 and 30, respectively; (e)V H and V L comprises an amino acid sequence that is at least 90% identical to the amino acid sequences set forth as SEQ ID NOs: 39 and 40, respectively; (f)V H and V L comprises an amino acid sequence that is at least 90% identical to the amino acid sequences set forth as SEQ ID NOs: 43 and 44, respectively; or (g)V H and V L comprising an amino acid sequence that is at least 90% identical to the amino acid sequences set forth as SEQ ID NOs: 53 and 54, respectively; Any of the antibodies or antigen-binding fragments of the present invention. [The present invention 1005] Any of the preceding antibodies or antigen-binding fragments of the invention, comprising human framework regions. [The present invention 1006] (a)V H and V L comprises the amino acid sequences shown as SEQ ID NOs: 1 and 2, respectively; (b)V H and V L comprises the amino acid sequences shown as SEQ ID NOs: 17 and 12, respectively; (c)V H and V L comprises the amino acid sequences shown as SEQ ID NOs: 26 and 12, respectively; (d)V H and V L comprises the amino acid sequences shown as SEQ ID NOs:29 and 30, respectively; (e)V H and V L comprises the amino acid sequences shown as SEQ ID NOs: 39 and 40, respectively; (f)V H and V L comprises the amino acid sequences set forth as SEQ ID NOs: 43 and 44, respectively; or (g)V H and V L comprising the amino acid sequences set forth as SEQ ID NOs: 53 and 54, respectively; The antibody or antigen-binding fragment of any one of 1001 to 1004 of the present invention. [The present invention 1007] Any of the preceding antibodies of the invention, which comprise a human constant domain. [The present invention 1008] Any of the antibodies of the present invention, which is an IgG. [The present invention 1009] Any of the preceding antibodies of the invention comprising a recombinant constant domain comprising a modification that increases the half-life of the antibody. [The present invention 1010] Any of the preceding antibodies or antigen-binding fragments of the invention conjugated to a toxin or chemotherapeutic agent. [The present invention 1011] The antibody or antigen-binding fragment thereof of the present invention 1010, wherein the toxin is Pseudomonas exotoxin (PE), ricin, abrin, diphtheria toxin, ribotoxin, ribonuclease, saporin, calicheamicin, or botulinum toxin. [The present invention 1012] 1011. The antibody or antigen-binding fragment of the invention, wherein the toxin is PE, and PE is PE25, PE38, or PE40. [The present invention 1013] The antibody or antigen-binding fragment of the present invention 1010, wherein the chemotherapeutic agent is monomethyl auristatin E or a maytansinoid. [The present invention 1014] An antigen-binding fragment of any one of 1001 to 1006 or 1010 to 1013 of the present invention. [The present invention 1015] Fv fragment, dsFv fragment, ds-scvFv fragment, Fab fragment, F(ab') 2 fragment, scFV fragment, or scFV 2 An antigen-binding fragment of the present invention 1014, which is a fragment thereof. [The present invention 1016] Any of the preceding antibodies or antigen-binding fragments of the invention conjugated to a detectable marker. [The present invention 1017] A chimeric antigen T cell receptor comprising any of the antigen-binding fragments of the present invention. [The present invention 1018] A bispecific antibody comprising any one of the antibodies or antigen-binding fragments of the present inventions 1001 to 1016. [The present invention 1019] Any of the antibodies or antigen-binding fragments of the present invention 1001 to 1016, or V of the antibody or antigen-binding fragment H Or V L or the chimeric antigen T cell receptor of the present invention 1017 An isolated nucleic acid molecule encoding [The present invention 1020] (a) V, shown as SEQ ID NOs: 3 and 4, respectively H and / or V L nucleotide sequence of; (b) V, shown as SEQ ID NOs: 18 and 19, respectively H and / or V L nucleotide sequence of; (c) V, shown as SEQ ID NOs: 27 and 19, respectively H and / or V L nucleotide sequence of; (d) V, shown as SEQ ID NOs: 31 and 32, respectively H and / or V L nucleotide sequence of; (e) V, shown as SEQ ID NOs: 41 and 42, respectively H and / or V L nucleotide sequence of; (f) V, shown as SEQ ID NOs: 45 and 46, respectively H and / or V L or the nucleotide sequence of (g) V, shown as SEQ ID NOs: 55 and 56, respectively H and / or V L The nucleotide sequence of 1019. A nucleic acid molecule of the present invention comprising: [The present invention 1021] The nucleic acid molecule of invention 1019 or invention 1020, which is a cDNA sequence. [The present invention 1022] 1022. The nucleic acid molecule of any one of 1019 to 1021, operably linked to a promoter. [The present invention 1023] A vector comprising any one of the nucleic acid molecules of the present inventions 1019 to 1022. [The present invention 1024] An isolated host cell comprising any one of the nucleic acid molecules or vectors of the present inventions 1019 to 1023. [The present invention 1025] An isolated T cell expressing the chimeric antigen T cell receptor of the present invention. [The present invention 1026] an effective amount of any of the antibodies, antigen-binding fragments, nucleic acid molecules, or vectors of the present invention; a pharmaceutically acceptable carrier; 10. A pharmaceutical composition for use in treating an EGFRvIII-expressing cancer, comprising: [The present invention 1027] 1. A method for producing an antibody or antigen-binding fragment that specifically binds to EGFRvIII, or a bispecific antibody comprising the monoclonal antibody or antigen-binding fragment, comprising: expressing in a host cell one or more nucleic acid molecules encoding the antibody, antigen-binding fragment, or bispecific antibody of any of claims 1001 to 1016; and Purifying the antibody, antigen-binding fragment, or bispecific antibody A method comprising: [The present invention 1028] 1. A method for detecting the presence of EGFRvIII in a biological sample derived from a human subject, comprising: contacting the biological sample with an effective amount of the antibody or antigen-binding fragment of any of 1001-1016 of the present invention under conditions sufficient to form an immune complex; and detecting the presence of immune complexes in the biological sample, wherein the presence of immune complexes in the biological sample indicates the presence of EGFRvIII in the sample. A method comprising: [The present invention 1029] The method of claim 1028, wherein the subject has glioma, head and neck cancer, breast cancer, or bladder cancer. [The present invention 1030] 1029. The method of claim 1029, wherein the biological sample is a biopsy sample derived from glioma, head and neck cancer, breast cancer, or bladder cancer, respectively. [The present invention 1031] administering to a subject an effective amount of the antibody, antigen-binding fragment, nucleic acid molecule, vector, T cell, or pharmaceutical composition of any one of claims 1001 to 1026 of the present invention. 1. A method of inhibiting an EGFRvIII-expressing tumor in a subject, comprising: The method, wherein the subject has a tumor that expresses EGFRvIII. [The present invention 1032] The method of claim 1031, wherein the tumor is a glioma, head and neck cancer, breast cancer, or bladder cancer. [The present invention 1033] The method of claim 1031 or 1032, wherein the subject is a human. [The present invention 1034] The method of any of claims 1031 to 1033, wherein inhibiting tumors comprises reducing tumor growth, size, or metastasis. [This invention 1035] Administering an effective amount of the antibody, antigen-binding fragment, nucleic acid molecule, vector, T cell, or pharmaceutical composition of any one of claims 1001 to 1026 to a subject having a tumor that overexpresses EGFR. 1. A method of inhibiting an EGFR-overexpressing tumor in a subject, comprising: The antibody or antigen-binding fragment comprises a V comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NO:1. H and V comprising LCDR1, LCDR2, and LCDR3 of SEQ ID NO:2. H thereby inhibiting tumors in a subject. [The present invention 1036] The method of claim 1035, wherein the tumor is a glioma, head and neck cancer, breast cancer, or bladder cancer. [This invention 1037] The method of any one of claims 1035 to 1036, wherein the subject is a human. [The present invention 1038] The method of any of claims 1035 to 1037, wherein inhibiting tumors comprises reducing tumor growth, size, or metastasis. [This invention 1039] Use of any of the antibodies, antigen-binding fragments, nucleic acid molecules, vectors, chimeric antigen receptors, chimeric antigen receptor-expressing T cells, or pharmaceutical compositions of the present inventions 1001 to 1026 for inhibiting EGFRvIII-expressing tumors in a subject or for detecting the presence of EGFRvIII in a biological sample. These and other features and advantages of the present invention will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying drawings. [Brief explanation of the drawings]

[0011] [Figure 1] Schematic diagram of EGFRvIII peptide antigen (aa 286-303) conjugated with (1A) EGFR and (1B) KLH. The loop locations and approximate binding site of ma528 on domain III of EGFR are also shown. In both Figures 1A and 1B, SEQ ID NO:69 is shown. [Figure 2] Bar graph of the results of individual hybridoma supernatants by enzyme-linked immunosorbent assay (ELISA) reactivity to wtEGFR-His and EGFRvIII-His, including 528 as a positive control. [Figure 3] Bar graph of the reactivity of hybridoma supernatants with the following cells: MDA-MB-468 cells, A431 cells, F98EGFR cells, and F98npEGFRvIII cells. The results show that the antibodies bind specifically. ma528 is a control. [Figure 4] SDS-PAGE gel showing the purity of seven monoclonal antibodies eluted from the A / G column. [Figure 5] Reactivity of purified monoclonal antibodies with wtEGFR-His and EGFRvIII-His in an ELISA format. [Figure 6A] Reactivity of purified monoclonal antibodies in intact cells using flow cytometry. A. MDA-MB-468; A431 B. F98EGFR; F98npEGFRvIII; C. WI-38. [Figure 6B] See legend to Figure 6A. [Figure 6C] See legend to Figure 6A. [Figure 7] Reactivity of individual antibodies with wild-type (wt) and selected mutants of the 287-302-His peptide. [Figure 8A]Alignment of antibody sequences deduced from variable cDNA gene sequences. The VL for each antibody is listed in Figure 8A with "1-light" after the antibody name (SEQ ID NOs: 2, 12, 30, 40, 44, and 54), and the VH for each antibody is listed in Figure 8B with "1-heavy" after the antibody name (SEQ ID NOs: 1, 17, 26, 29, 39, 43, and 53). The following sequences are shown: 40H3: VH and VL are SEQ ID NOs: 1 and 2; 3D10: VH and VL are SEQ ID NOs: 17 and 12; 9G11: VH and VL are SEQ ID NOs: 26 and 12; 1D9: VH and VL are SEQ ID NOs: 29 and 30; 4A4: VH and VL are SEQ ID NOs: 39 and 40; 11E11: VH and VL are SEQ ID NOs: 43 and 44; 11G3: VH and VL are SEQ ID NOs: 53 and 54. [Figure 8B] See legend to Figure 8A. [Figure 9] Cytotoxicity of antibodies and immunotoxin proteins against cancer cell lines. [Figure 10A] Dose-response curves of cell viability after addition of 40H3-PE38 (immunotoxin) or PE64 (native toxin) to WI-38 cells, F98EGFR cells, or F98npEGFRvIII cells. WI-38 is a normal lung fibroblast cell line, "F98EGFR" is a rat glioma cell line transfected with full-length EGFR, and F98npEGFRvIII is a rat glioma cell line transfected with non-phosphorylated EGFRvIII. [Figure 10B] See legend to Figure 10A. [Figure 10C] See legend to Figure 10A. [Figure 11A] Binding data (using an Octet Biosensor) for 40H3 (full-length antibody) to immobilized EGFR loops, EGFRvIII extracellular domain (ECD), or wtEGFR ECD. [Figure 11B]See legend to Figure 11A. [Figure 11C] See legend to Figure 11A. [Figure 12A] Octet Biosensor data for the interaction of 40H3-MMAE (antibody drug conjugate ADC), 40H3-DM1 (ADC), or 40H3-PE38 immunotoxin to immobilized EGFR loops. The ADC is constructed by chemical attachment of a cytotoxic drug to the full-length 40H3 antibody. The 40H3-PE38 immunotoxin is a single-chain Fv (scFv) in which a protein toxin is fused to the scFv. The data confirm that there is no loss of binding activity for any of the drug conjugates. The immunotoxin, being monovalent, has a lower binding affinity due to a faster dissociation rate. [Figure 12B] See legend to Figure 12A. [Figure 12C] See legend to Figure 12A. [Figure 13A] Flow cytometry data for 40H3 and 528 antibodies on the PDX cell line GBM39. 40H3 is a full-length antibody. 528 antibody reacts with all species of EGFR. GBM39 is a human glioblastoma PDX sample. [Figure 13B] See legend to Figure 13A. [Figure 13C] See legend to Figure 13A. [Figure 14] Dose-response curve of cell viability after addition of 40H3-PE38 (immunotoxin) to GBM39 cells. [Figure 15A] Dose-response curves for cell viability following addition of 40H3-MMAE to MDA-MB-468, A431, or F98npEGFRvIII cells. [Figure 15B] See legend to Figure 15A. [Figure 16A] Dose-response curves for cell viability following addition of 40H3-DM1 to MDA-MB-468, A431, or F98npEGFRvIII cells. [Figure 16B] See legend to Figure 16A. [Figure 17] Flow cytometry data for 40H3, 528, or 40H3-PE38 against DKMG EGFRvIII. The DKMG cell line is a permanent cell line established from cells derived from glioblastoma. The cells were stably transduced with a viral vector encoding EGFRvIII, which underwent genomic integration. [Figure 18] Dose-response curves for cell viability following the addition of 40H3-MMAE, 40H3-DM1, or 40H3-PE38 to DKMG EGFRvIII. [Figure 19] scFv with a C-terminal cysteine ​​for drug conjugation. DETAILED DESCRIPTION OF THE INVENTION

[0012] Sequence Listing The listed nucleic acid and amino acid sequences are shown using standard abbreviations for nucleotide bases and amino acids as defined in 37 CFR 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood by referring to the displayed strand. In the heavy and light chain variable domain sequences, CDR sequences are underlined. The sequence listing is submitted as an ASCII text file [Sequence_Listing, July 1, 2020, size 41,999 bytes], which is incorporated herein by reference. In the attached sequence listing,

[0013] SEQ ID NO:1 is the amino acid sequence of the 40H3 heavy chain variable domain. TIFF0007815099000001.tif11158

[0014] SEQ ID NO:2 is the amino acid sequence of the 40H3 light chain variable domain. TIFF0007815099000002.tif11158

[0015] SEQ ID NO:3 is an exemplary nucleic acid sequence encoding the 40H3 heavy chain variable domain. TIFF0007815099000003.tif37159

[0016] SEQ ID NO:4 is an exemplary nucleic acid sequence encoding the 40H3 light chain variable domain. TIFF0007815099000004.tif36159

[0017] SEQ ID NOs:5 to 10 are the amino acid sequences of the 40H3 CDRs. TIFF0007815099000005.tif36128

[0018] SEQ ID NO:11 is the amino acid sequence of the consensus heavy chain variable domain for 3D10 / 9G11. TIFF0007815099000006.tif11160X1 is I or T, X2 is S or G.

[0019] SEQ ID NO:12 is the amino acid sequence of the light chain variable domain for 3D10 and 9G11. TIFF0007815099000007.tif11160

[0020] SEQ ID NO:13 is the amino acid sequence of the consensus heavy chain variable domain for 1D9 / 4A4. TIFF0007815099000008.tif11159X1=Q or L X2=T or I X3=D or E

[0021] SEQ ID NO:14 is the amino acid sequence of the consensus light chain variable domain for 1D9 / 4A4 (SEQ ID NO:14). TIFF0007815099000009.tif11160X is V or A.

[0022] SEQ ID NO:15 is the amino acid sequence of the consensus heavy chain variable domain for 11E11 / 11G3 (SEQ ID NO:15). TIFF0007815099000010.tif11157X1=I or V X2=K or M X3=R or K X4=T or A X5=A or T

[0023] SEQ ID NO:16 is the amino acid sequence of the consensus light chain variable domain for 11E11 / 11G3. TIFF0007815099000011.tif11159X1=A or P X2=Q or R

[0024] SEQ ID NO:17 is the amino acid sequence of the 3D10 heavy chain variable domain. TIFF0007815099000012.tif11157

[0025] SEQ ID NO:18 is an exemplary nucleic acid encoding the 3D10 heavy chain variable domain. TIFF0007815099000013.tif36160

[0026] SEQ ID NO:19 is an exemplary nucleic acid encoding the light chain variable domain of 3D10 and 9G11. TIFF0007815099000014.tif36160Due to the degeneracy of the genetic code, position 97 can be C or A and position 291 can be C or T.

[0027] SEQ ID NOs:20 to 25 are the amino acid sequences of the 3D10 CDRs. TIFF0007815099000015.tif36128

[0028] SEQ ID NO:26 is the amino acid sequence of the 9G11 heavy chain variable domain. TIFF0007815099000016.tif11159

[0029] SEQ ID NO:27 is an exemplary nucleic acid sequence encoding the 9G11 heavy chain variable domain. TIFF0007815099000017.tif36160

[0030] SEQ ID NOs:20, 28, 22, 23, 24, and 25 are the amino acid sequences of the 9G11 CDRs, five of which are identical to the CDRs of 3D10. TIFF0007815099000018.tif36128

[0031] SEQ ID NO:29 is the amino acid sequence of the 1D9 heavy chain variable domain. TIFF0007815099000019.tif11159

[0032] SEQ ID NO:30 is the amino acid sequence of the 1D9 light chain variable domain. TIFF0007815099000020.tif11160

[0033] SEQ ID NO:31 is an exemplary nucleic acid sequence encoding the 1D9-1 heavy chain variable domain. TIFF0007815099000021.tif36160

[0034] SEQ ID NO:32 is an exemplary nucleic acid sequence encoding the 1D9 light chain variable domain. TIFF0007815099000022.tif36160

[0035] SEQ ID NOs: 33 to 38 are the amino acid sequences of the CDRs of 1D9. TIFF0007815099000023.tif36128

[0036] SEQ ID NO:39 is the amino acid sequence of the 4A4 heavy chain variable domain. TIFF0007815099000024.tif11159

[0037] SEQ ID NO:40 is the amino acid sequence of the 4A4 light chain variable domain. TIFF0007815099000025.tif11160

[0038] SEQ ID NO:41 is an exemplary nucleic acid sequence encoding the 4A4 heavy chain variable domain. TIFF0007815099000026.tif36160

[0039] SEQ ID NO:42 is an exemplary nucleic acid sequence encoding the 4A4 light chain variable domain. TIFF0007815099000027.tif36160

[0040] SEQ ID NOs: 33-35 and 36-38 are the amino acid sequences of the CDRs for 4A4. Note that these are identical to the amino acid sequences of the CDRs of 1D9. TIFF0007815099000028.tif37128

[0041] SEQ ID NO:43 is the amino acid sequence of the 11E11 heavy chain variable domain. TIFF0007815099000029.tif11158

[0042] SEQ ID NO:44 is the amino acid sequence of the 11E11 light chain variable domain. TIFF0007815099000030.tif11159

[0043] SEQ ID NO:45 is an exemplary nucleic acid encoding the 11E11 heavy chain variable domain. TIFF0007815099000031.tif36160

[0044] SEQ ID NO:46 is an exemplary nucleic acid encoding the 11E11 light chain variable domain. TIFF0007815099000032.tif36160

[0045] SEQ ID NOs: 47-52 are the amino acid sequences of the 11E11 CDRs. TIFF0007815099000033.tif37128

[0046] SEQ ID NO:53 is the amino acid sequence of the 11G3 heavy chain variable domain. TIFF0007815099000034.tif11158

[0047] SEQ ID NO:54 is the amino acid sequence of the 11G3 light chain variable domain. TIFF0007815099000035.tif11158

[0048] SEQ ID NO:55 is an exemplary nucleic acid sequence encoding the 11G3 heavy chain variable domain. TIFF0007815099000036.tif36160

[0049] SEQ ID NO:56 is an exemplary nucleic acid sequence encoding the 11G3 light chain variable domain. TIFF0007815099000037.tif36160

[0050] SEQ ID NOs:47, 57, 49, 58, 51, and 52 are the amino acid sequences of the CDRs of 11G3. Note that some of the CDR sequences are identical to the CDRs of 11E11. TIFF0007815099000038.tif37128

[0051] SEQ ID NO:59 is the amino acid sequence of the linker. SEQ ID NO:61 is the amino acid sequence of the signal peptide. SEQ ID NO:61 is the amino acid sequence of an immunoglobulin domain. SEQ ID NOs: 62 and 63 are the amino acid sequences of the transmembrane domains. SEQ ID NOs:64 to 68 are the amino acid sequences of the intracellular domain. SEQ ID NO:69 is amino acids 287-302 of EGFR.

[0052] DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS Disclosed herein are monoclonal antibodies and antigen-binding fragments thereof that specifically bind to EGFRvIII and do not bind to wild-type EGFR expressed in normal cells. Also disclosed are methods of using these antibodies, for example, to detect EGFRvIII, inhibit tumor growth and tumor metastasis, and / or reduce tumor volume. Disclosed are chimeric antigen receptors and conjugates comprising these monoclonal antibodies and antigen-binding fragments.

[0053] Antibodies and antigen-binding fragments containing CDRs derived from monoclonal antibody 40H3 bind to EGFRvIII and also bind to mutant and overexpressed EGFR expressed in tumor cells, but not to wild-type (non-cancer) EGFR. 287-302 Antibodies and antigen-binding fragments comprising CDRs from additional antibodies disclosed herein also specifically bind to EGFRvIII and do not bind to wild-type EGFR expressed in wild-type (non-cancer) cells. These antibodies and antigen-binding fragments also do not bind to other forms of EGFR expressed in tumor cells.

[0054] Activation of wild-type EGFR involves ligand binding and dimerization, which requires a monomer-to-dimer transition, accompanied by a change in receptor conformation. Several structures have been reported for the extracellular domain of EGFR, both in the monomeric and dimeric conformations. Analysis of these structures indicates the presence of residues that are not exposed in the wild-type receptor. However, under oncogenic conditions, when the receptor is highly expressed and may not fold correctly, or when mutant versions of the receptor are expressed, cryptic structures can become exposed. One structural element that is not sterically accessible under normal conditions is the disulfide-restricted loop at positions 287-302 (mature receptor numbering or 301-326 in the full-length receptor). This loop is exposed in EGFRvIII and can become exposed when receptor expression is extremely high or when ECD mutations alter the wild-type structure. In some embodiments, methods are provided for inhibiting tumors that have this loop exposed on cells.

[0055] In some embodiments, the antibody comprises the heavy and light chain CDRs of antibody 40H3 and binds to tumor cells that overexpress EGFR. Thus, methods are provided for inhibiting EGFR-overexpressing tumors in a subject.

[0056] In some embodiments, D290 and E293 are selected from the group consisting of EGFR 287-302 In other embodiments, E293 is required for binding to the EGFR loop. Exemplary antibodies having this binding characteristic (such as 1D9, 3D10, 4A4, 9G11, and 11E3) are provided. In other embodiments, E293 is required for binding to the EGFR loop. 287-302 In a further embodiment, R300 is required for binding to the EGFR loop. Exemplary antibodies (such as 11E11) having this binding characteristic are provided. 287-302 The present invention relates to a method for binding to the 40H3 loop (SEQ ID NO:69). Exemplary antibodies (such as 40H3) that possess this binding characteristic are provided.

[0057] I. Terminology Overview Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes X, published by Jones & Bartlett Publishers, 2009; and Meyers et al. (eds.), Encyclopedia of Cell Biology and Molecular Medicine, published by Wiley-VCH in 16 volumes, 2008; and other similar references.

[0058] As used herein, the singular forms "a," "an," and "the" refer to both the singular and the plural unless the context clearly indicates otherwise. For example, the term "an antigen" includes a single antigen or multiple antigens and can be considered equivalent to the phrase "at least one antigen." As used herein, the term "comprises" means "includes." Furthermore, all given base or amino acid sizes and all molecular weight or molecular mass values ​​for nucleic acids or polypeptides are approximate unless otherwise indicated and are provided for descriptive purposes. Although many methods and materials similar or equivalent to those described herein can be used, specific suitable methods and materials are described herein. In the case of conflict, the present specification, including explanations of terms, will control. Furthermore, the materials, methods, and examples are illustrative only and not limiting. To facilitate review of the various embodiments, the following explanations of terms are provided:

[0059] About: Unless the context indicates otherwise, "about" refers to plus or minus 5% of the referenced value. For example, "about" 100 refers to 95 to 105.

[0060] Administration: The introduction of a composition into a subject by a selected route. Administration can be local or systemic. For example, if the selected route is intravenous, a composition (such as a composition comprising a disclosed antibody or antigen-binding fragment, etc.) is administered by introducing the composition into a subject's vein. Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), sublingual, rectal, transdermal (e.g., topical), intranasal, vaginal, and inhalation routes.

[0061] Drug: A substance or combination of substances useful for achieving a certain purpose or result; for example, a substance or combination of substances useful for inhibiting tumor growth or metastasis in a subject. Drugs include proteins, nucleic acid molecules, compounds, small molecules, organic compounds, inorganic compounds, or other molecules of interest. Drugs can include therapeutic agents (such as chemotherapeutic agents), diagnostic agents, or pharmaceutical agents. In some embodiments, the drug is an antibody that specifically binds to EGFRvIII, its antigen-binding fragment, a conjugate thereof, or a chimeric antigen receptor (CAR) comprising the antibody or antigen-binding fragment. Those skilled in the art will understand that a particular drug may be useful for achieving multiple results.

[0062] Amino acid substitution: The replacement of an amino acid in a polypeptide with a different amino acid or no amino acid at all (i.e., deletion). In some instances, an amino acid in a polypeptide is substituted with an amino acid from a homologous polypeptide, for example, an amino acid in an antibody or antigen-binding fragment thereof that specifically binds to EGFRvIII can be substituted with the corresponding amino acid from another antibody or antigen-binding fragment thereof that specifically binds to EGFRvIII.

[0063] Antibodies and antigen-binding fragments: Immunoglobulins, antigen-binding fragments, or derivatives thereof that specifically bind to and recognize an analyte (antigen), such as EGFRvIII. The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antigen-binding fragments, so long as they exhibit the desired antigen-binding activity.

[0064] Non-limiting examples of antibodies include, for example, intact immunoglobulins and variants and antigen-binding fragments thereof that retain binding affinity to an antigen. Examples of antigen-binding fragments include, but are not limited to, Fv, Fab, dsFv, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv and ds-scFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen-binding fragments produced by modification of intact antibodies or those synthesized de novo using recombinant DNA methodologies (see, e.g., Kontermann and Dubel (Eds.), Antibody Engineering, Vols. 1-2, 2014). nd ed., Springer-Verlag, 2010).

[0065] Antibodies also include genetically engineered forms such as chimeric antibodies (such as humanized murine antibodies) and heteroconjugate antibodies (such as bispecific antibodies).

[0066] An antibody can have one or more binding sites. When multiple binding sites are present, the binding sites may be identical to each other or different. For example, naturally occurring immunoglobulins have two identical binding sites, single-chain antibodies or Fab fragments have one binding site, and bispecific or bifunctional antibodies have two different binding sites.

[0067] Typically, naturally occurring immunoglobulins have heavy (H) chains and light (L) chains interconnected by disulfide bonds. Immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable domain genes. There are two types of light chains, lambda (λ) and kappa (κ). There are five major heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE.

[0068] The heavy and light chains each contain a constant region (or constant domain) and a variable region (or variable domain). The combination of the heavy and light chain variable regions specifically binds to an antigen.

[0069] "V H References to "V" or "VH" refer to the variable region of an antibody heavy chain, including that of an antigen-binding fragment such as an Fv, scFv, dsFv, or Fab. L References to "VL" or "VL" refer to the variable domain of an antibody light chain, including that of an Fv, scFv, ds-scFv or Fab.

[0070] V H and V L contains a "framework" region interspersed with three hypervariable regions, also called "complementarity-determining regions" or "CDRs" (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5 th(See, e.g., NIH Publication No. 91-3242, Public Health Service, National Institutes of Health, USDapartment of Health and Human Services, 1991). The sequences of framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, which is the combination of the framework regions of the constituent light and heavy chains, functions to position and align the CDRs in three-dimensional space.

[0071] CDRs are primarily responsible for binding to an epitope of an antigen. The amino acid sequence boundaries of a particular CDR are defined by Kabat et al. (Sequences of Proteins of Immunological Interest, 5 th ed., NIH Publication No. 91-3242, Public Health Service, National Institute of Health, USDapartment of Health and Human Services, 1991; "Kabat" numbering scheme), Al-Lazikani et al. ("Standard conformations for canonical structures of immunoglobulins," J. Mol. Bio., 273(4):927-948, 1997; "Chothia" numbering scheme), and Lefranc et al. ("IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev. Comp. Immunol., 27(1):55-77, 2003; "IMGT" numbering scheme). The CDRs of each chain are typically referred to (from N-terminus to C-terminus) as CDR1, CDR2, and CDR3, and are typically identified by the chain in which the particular CDR is located.H CDR3 is the V of the antibody in which it is found H CDR3 derived from V L CDR1 is the V of the antibody in which it is found L The CDRs of the light chain are sometimes referred to as LCDR1, LCDR2, and LCDR3. The CDRs of the heavy chain are sometimes referred to as HCDR1, HCDR2, and HCDR3.

[0072] In some embodiments, the disclosed antibodies comprise a heterologous constant domain, e.g., the antibody comprises a constant domain that differs from the native constant domain, such as a constant domain that includes one or more modifications (such as an "LS" mutation) to increase half-life.

[0073] A "monoclonal antibody" is an antibody obtained from a population of substantially homogeneous antibodies; i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variant antibodies containing, for example, naturally occurring mutations or mutations that arose during the generation of the monoclonal antibody preparation (such variants are generally present in minor amounts). In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci; such methods and other exemplary methods for producing monoclonal antibodies are described herein. In some instances, monoclonal antibodies are isolated from a subject. Monoclonal antibodies may have conservative amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions (see, e.g., Greenfield (Ed.), Antibodies: A Laboratory Manual, 2002). nd ed. New York: Cold Spring Harbor Laboratory Press, 2014).

[0074] A "humanized" antibody or antigen-binding fragment comprises a human framework region and one or more CDRs derived from a non-human antibody or antigen-binding fragment (such as a mouse, rat, or synthetic antibody). The non-human antibody or antigen-binding fragment providing the CDRs is referred to as the "donor," and the human antibody or antigen-binding fragment providing the framework is referred to as the "acceptor." In one embodiment, in a humanized immunoglobulin, all CDRs are derived from the donor immunoglobulin. Constant regions may be absent, but if present, can be substantially identical to human immunoglobulin constant regions, e.g., at least about 85-90%, e.g., about 95% or more identical. Thus, all parts of a humanized antibody or antigen-binding fragment, except possibly the CDRs, are substantially identical to the corresponding parts of a natural human antibody sequence.

[0075] A "chimeric antibody" is an antibody that contains sequences from two different antibodies, typically from different species. In some instances, a chimeric antibody contains one or more CDRs and / or framework regions from one human antibody and CDRs and / or framework regions from another human antibody.

[0076] A "fully human antibody" or "human antibody" is an antibody that contains sequences derived from the human genome and no sequences derived from another species. In some embodiments, a human antibody contains CDRs, framework regions, and (if present) an Fc region derived from the human genome. Human antibodies can be identified and isolated using techniques to make antibodies based on sequences derived from the human genome, for example, by phage display or using transgenic animals (see, e.g., Barbas et al., Phage display: Laboratory Manuel. 1999). stEd. New York: Cold Spring Harbor Laboratory Press, 2004. Print.; Lonberg, Nat. Biotech., 23:1117-1125, 2005; Lonenberg, Curr. Opin. Immunol., 20:450-459, 2008).

[0077] Biological sample: A sample obtained from a subject. Biological samples include all clinical samples useful for detecting disease or tumors (e.g., head and neck cancer, breast cancer, or bladder cancer) in a subject, including, but not limited to, cells, tissues, and bodily fluids, such as blood, blood derivatives and fractions (e.g., serum), cerebrospinal fluid; and biopsied or surgically removed tissues, such as unfixed tissues, frozen tissues, or formalin or paraffin-fixed tissues. In a specific example, the biological sample is obtained from a subject who has or is suspected to have a tumor, such as, but not limited to, head and neck cancer, breast cancer, or bladder cancer.

[0078] Bispecific antibodies: Recombinant molecules composed of two different antigen-binding domains and therefore capable of binding to two different antigenic epitopes. Bispecific antibodies include molecules in which two antigen-binding domains are chemically or genetically linked. The antigen-binding domains may be linked using a linker. The antigen-binding domains may be monoclonal antibodies, antigen-binding fragments (e.g., Fab, scFv, ds-scFv), or combinations thereof. Bispecific antibodies may, but do not necessarily, contain one or more constant domains.

[0079] Cancer: A malignant tumor containing transformed epithelial cells. Non-limiting examples of cancer include adenocarcinoma, squamous cell carcinoma, undifferentiated carcinoma, large cell carcinoma, and small cell carcinoma. In some examples, the cancer is breast cancer, head and neck cancer, or bladder cancer.

[0080] Chemotherapeutic agent: A chemical agent that has therapeutic utility in the treatment of diseases characterized by abnormal cell growth. For example, chemotherapeutic agents are useful for the treatment of cancer, e.g., head and neck cancer, breast cancer, and bladder cancer. In one embodiment, the chemotherapeutic agent is an agent useful in the treatment of cancer. Specific examples of additional therapeutic agents that may be used include microtubule binding agents, DNA intercalators or cross-linking agents, DNA synthesis inhibitors, inhibitors of DNA and RNA transcription, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors. In one embodiment, the chemotherapeutic agent is a radioactive compound. Other examples include antineoplastic agents, 5-fluorouracil (5-FU) and IRT. Those of skill in the art can readily identify useful chemotherapeutic agents (see, e.g., Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2 nded., (Copyright) 2000 Churchill Livingstone, Inc.; Baltzer, L., Berkery, R. (eds): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer, DS, Knobf, MF, Durivage, HJ (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993; Chabner and Longo, Cancer Chemotherapy and Biotherapy: Principles and Practice (4th ed.). Philadelphia: Lippincott Williams & Wilkins, 2005; Skeel, Handbook of Cancer Chemotherapy (6th ed.). Lippincott Williams & Wilkins, 2003. Combination chemotherapy is the administration of multiple drugs to treat cancer.

[0081] Chimeric antibody: An antibody that contains sequences from two different antibodies, e.g., from different species. In some instances, a chimeric antibody contains one or more CDRs and / or framework regions from one human antibody and CDRs and / or framework regions from another human antibody.

[0082] Chimeric Antigen Receptor (CAR): An engineered T cell receptor that has an extracellular antibody-derived targeting domain (such as an scFv) joined to one or more intracellular signaling domains of the T cell receptor. A "chimeric antigen receptor T cell" is a T cell that expresses a CAR and has antigen specificity determined by the antibody-derived targeting domain of the CAR. Methods for generating CARs (e.g., for the treatment of cancer) are available (see, e.g., Park et al., Trends Biotechnol., 29:550-557, 2011; Grupp et al., N Engl J Med., 368:1509-1518, 2013; Han et al., J. Hematol Oncol., 6:47, 2013; PCT Publications WO2012 / 079000, WO2013 / 059593; and U.S. Publication 2012 / 0213783, each of which is incorporated by reference in its entirety).

[0083] Conditions sufficient for immune complex formation: Conditions that allow an antibody or antigen-binding fragment thereof to bind to its cognate epitope to an extent that is detectably greater than binding to substantially all other epitopes and / or to an extent that binding to substantially all other epitopes is substantially excluded. Conditions sufficient for immune complex formation depend on the format of the binding reaction and are typically those utilized in immunoassay protocols or conditions found in vivo. For immunoassay formats and conditions, see Harlow & Lane, Antibodies, A Laboratory Manual, 2002. nded. Cold Spring Harbor Publications, New York (2013). The conditions utilized in the methods are "physiological conditions," which includes reference to conditions (e.g., temperature, osmolality, pH) typical in living mammals or mammalian cells. While it is recognized that some organs are subject to extreme conditions, the environment within organisms and cells is typically approximately pH 7 (e.g., pH 6.0 to pH 8.0, more typically pH 6.5 to 7.5), contains water as the primary solvent, and exists at a temperature above 0°C and below 50°C. Osmolality is within a range that supports cell survival and growth.

[0084] Conjugate: A complex of two molecules linked together, e.g., covalently linked. In one embodiment, an antibody is linked to an effector molecule; for example, an antibody that specifically binds to EGFRvIII is linked to an effector molecule. Linkage can be by chemical or recombinant means. In one embodiment, linkage is chemical, where reaction between the antibody moiety and the effector molecule generates a covalent bond formed between the two molecules, forming a single molecule. A peptide linker (a short peptide sequence) can optionally be included between the antibody and the effector molecule. Because conjugates can be prepared from two molecules with separate functionalities, such as an antibody and an effector molecule, they are sometimes called "chimeric molecules."

[0085] Conservative variant: A "conservative" amino acid substitution is a substitution that does not substantially affect or reduce the function of a protein, such as the ability of a protein to interact with a target protein. For example, an antibody specific for EGFRvIII can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 conservative substitutions compared to a reference antibody sequence and still retain its specific binding activity to EGFRvIII. The term "conservative variation" also includes the use of a substituted amino acid instead of a non-substituted parent amino acid.

[0086] Individual substitutions, deletions, or additions that alter, add, or delete a single amino acid or a small percentage of amino acids (e.g., less than 5%, in some embodiments, less than 1%) of the encoded sequence are conservative variations in which the alteration results in the substitution of chemically similar amino acids.

[0087] The following six groups are examples of amino acids that are considered to be conservative substitutions for one another: (1) alanine (A), serine (S), threonine (T); (2) aspartic acid (D), glutamic acid (E); (3) asparagine (N), glutamine (Q); (4) arginine (R), lysine (K); (5) isoleucine (I), leucine (L), methionine (M), valine (V); and (6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0088] Non-conservative substitutions are those that reduce the activity or function of EGFRvIII-specific antibodies, such as the ability to specifically bind to EGFRvIII or bind to cancer cells that express EGFRvIII.For example, if an amino acid residue is essential for the function of a protein, even if it is otherwise conservative, its substitution may interfere with activity.Therefore, conservative substitutions do not change the basic function of the protein of interest.

[0089] Contact: A direct physical association arrangement; includes both solid and liquid, and can occur either in vivo or in vitro. Contact includes contact of one molecule with another molecule, for example, amino acids on the surface of a polypeptide, such as a peptide, in contact with another polypeptide. Contact can also include contact of cells, for example, by placing a polypeptide in direct physical association with a cell.

[0090] Control: Reference standard. In some embodiments, the control is a negative control sample obtained from a healthy patient. In other embodiments, the control is a positive control sample obtained from a patient diagnosed with a tumor that expresses EGFRvIII, or recombinantly produced purified EGFRvIII. In yet other embodiments, the control is a historical control or a standard reference value or range of values ​​(e.g., a previously tested control sample, e.g., a group of patients with a known prognosis or outcome, or a group of samples representing baseline or normal values).

[0091] The difference between the test sample and the control may be an increase or, conversely, a decrease. The difference may be a qualitative difference or a quantitative difference, for example, a statistically significant difference. In some examples, the difference may be at least about 5%, for example, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, or more than 500% increase or decrease compared to the control. In some embodiments, tumor growth, volume, and / or metastasis are reduced.

[0092] Reduce or reduce: To reduce the quality, quantity, or intensity of something; for example, a reduction in tumor burden. In one example, a treatment reduces a tumor or one or more symptoms associated with a tumor (such as tumor size, number of tumors, tumor metastasis, or a combination thereof), for example, compared to the response in the absence of treatment. In a specific example, a treatment reduces tumor size, number of tumors, tumor metastasis, or a combination thereof after treatment, for example, the reduction is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. Such reduction can be measured using the methods disclosed herein.

[0093] Degenerate variant: In the context of this disclosure, a "degenerate variant" refers to a polynucleotide encoding a polypeptide (such as an antibody) that contains a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by multiple codons. Thus, all degenerate nucleotide sequences that encode a peptide are included, as long as the amino acid sequence of the peptide encoded by the nucleotide sequence is unchanged.

[0094] Detectable marker: A detectable molecule (also known as a label) that is directly or indirectly conjugated to a second molecule, such as an antibody, to facilitate detection of the second molecule. For example, a detectable marker may be detectable by ELISA, spectrophotometry, flow cytometry, microscopy, or imaging techniques (such as CT scans, MRI, ultrasound, fiberoptic examination, and laparoscopy). Specific, non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzyme linkages, radioactive isotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). Methods for using detectable markers and guidance on selecting appropriate detectable markers for various purposes can be found, for example, in Green and Sambrook (Molecular Cloning: A Laboratory Manual, 4 th ed., New York: Cold Spring Harbor Laboratory Press, 2012) and Ausubel et al. (Eds.) (Current Protocols in Molecular Biology, New York: John Wiley and Sons, including supplements, 2017).

[0095] Detect: To identify the existence, presence, or fact of something.

[0096] Effective amount: The amount of a particular substance sufficient to achieve a desired effect in a subject to which the substance is administered, such as a therapeutically effective amount for treatment. For example, this may be the amount of an antibody required to inhibit tumor growth and / or metastasis, or to measurably alter the outward symptoms of a tumor.

[0097] In some embodiments, administration of an effective amount of the disclosed antibodies or antigen-binding fragments that bind EGFRvIII can reduce or inhibit tumor growth, tumor metastasis, or tumor volume by a desired amount, e.g., at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination of the tumor), compared to a suitable control.

[0098] The effective amount of an antibody or antigen-binding fragment that specifically binds to EGFRvIII to be administered to a subject will vary depending on numerous factors related to the subject, such as the subject's overall health and / or weight. An effective amount can be determined by varying the dosage and measuring the resulting response, such as a reduction in tumor burden. An effective amount can also be determined through various in vitro, in vivo, or in situ immunoassays.

[0099] Effective amount includes divided doses that contribute to achieving effective response by combining with previous or subsequent administration.For example, effective amount of drug can be administered once, or can be administered several times, for example, daily, in the course of treatment that lasts for several days or weeks.However, effective amount can depend on the subject to be treated, the severity and type of the condition to be treated, and the mode of administration.The unit dosage form of drug can be packaged in a certain amount or multiples of effective amount, for example, in a vial (for example, with a pierceable cap) or a syringe with sterile components.

[0100] Effector molecule: A molecule that has or is intended to have a desired effect on, for example, a cell to which the effector molecule is targeted. Effector molecules include molecules such as chemical compounds, polypeptides, radioisotopes, and small molecules. Non-limiting examples of effector molecules include toxins, chemotherapeutic agents, and anti-angiogenic agents. Those skilled in the art will understand that some effector molecules can have or exert multiple desired effects. In one example, the effector molecule is a portion of a chimeric molecule, for example, a chimeric molecule comprising a disclosed antibody or fragment thereof, that is intended to have a desired effect on a cell to which the chimeric molecule is targeted.

[0101] Epidermal Growth Factor Receptor (EGFR): EGFR (also known as HER1 or ERBB1) is a receptor belonging to the ERBB family of receptor thymidine kinases (RTKs). In vivo, ligand binding by EGF leads to activation of the RTK / RAS / PI(3)K pathway via receptor phosphorylation, resulting in increased cell proliferation, angiogenesis, and local tissue invasion, as well as resistance to apoptosis. The nucleic acid sequence for human EGFR can be found in GENBANK® Accession No. NM_005228.5 (June 18, 2019) and GENBANK® Accession No. NC_000007.14 (EGFR within the chromosome) (June 14, 2019), both of which are incorporated herein by reference. EGFR has a 621-amino acid ectodomain (ECD), a 23-amino acid single transmembrane domain (TM), and a 542-amino acid enzymatically active intracellular domain (ICD). Ligand binding leads to receptor dimerization and activation of the kinase domain, which transmits signals to one of several pathways that can promote mammalian cell growth, survival, and distribution. In tumors, loss of exons 2-7 produces the constitutively active EGFR variant III (EGFRvIII). The cDNA sequence for EGFRvIII can be found in GENBANK® Accession No. NM_001346941 (June 18, 2019), incorporated herein by reference, and the amino acid sequence can be found in NP_001333870.1 (June 14, 2019), incorporated herein by reference. Activation of wild-type EGFR involves ligand binding and dimerization, which requires a change in receptor conformation and a transition from a monomer to a dimer. Activating mutations can occur in either the ECD or ICD. Loss of exon 19 generates a constitutively active enzyme variant. EGFR can be overexpressed either by gene amplification or loss of transcriptional regulation. High-level expression (such as greater than about 50,000 receptors per cell) leads to either misfolding of the receptor or mutations in one or more of the gene copies.Overexpression can result in a two-fold or greater increase in EGFR present in cells compared to wild-type controls. One structural element that is sterically inaccessible under normal conditions is the disulfide-restricted loop at positions 287-302 (numbering in the mature receptor or 301-326 in the full-length receptor). This loop is exposed in EGFRvIII and can become exposed when receptor expression is extremely high or when ECD mutations alter the wild-type structure. See also Figures 1A and 1B and SEQ ID NO:69.

[0102] Epitope: An antigenic determinant. These are specific chemical groups or peptide sequences on an antigenic molecule that induce a specific immune response. For example, an epitope is a region of an antigen to which B cells and / or T cells respond. An antibody can bind to a specific antigenic epitope, such as an epitope on EGFRvIII.

[0103] Expression: The transcription or translation of a nucleic acid sequence. For example, a gene is expressed when its DNA is transcribed into RNA or RNA fragments, which, in some instances, are processed into mRNA. A gene can also be expressed when its mRNA is translated into an amino acid sequence, such as a protein or protein fragment. In a specific example, a heterologous gene is expressed when it is transcribed into RNA. In another example, a heterologous gene is expressed when its RNA is translated into an amino acid sequence. The term "expression" is used herein to mean either transcription or translation. Control of expression can include regulation of transcription, translation, RNA transport and processing, degradation of intermediate molecules such as mRNA, or the activation, inactivation, compartmentalization, or degradation of a specific protein molecule after it is produced.

[0104] Expression control sequence: A nucleic acid sequence that controls the expression of an operably linked heterologous nucleic acid sequence. An expression control sequence is operably linked to a nucleic acid sequence when it controls and regulates the transcription and, optionally, the translation of the sequence nucleic acid. Thus, an expression control sequence may include an appropriate promoter, enhancer, transcription terminator, a start codon (ATG) in front of a protein-encoding gene, splicing signals for introns, maintenance of the correct reading frame of the gene to allow proper translation of mRNA, and a stop codon. The term "regulatory sequence" includes at least components whose presence affects expression, and also includes additional components whose presence is advantageous, such as leader sequences and fusion partner sequences. An expression control sequence may include a promoter.

[0105] A promoter is a minimal sequence sufficient to direct transcription. Also included are promoter elements sufficient to render promoter-dependent gene expression regulatable, e.g., cell-type specific, tissue-specific, or inducible by external signals or agents; such elements can be located in the 5' or 3' region of the gene. Both constitutive and inducible promoters are included (see, e.g., Bitter et al., Methods in Enzymology 153:516-544, 1987). For example, when cloning in a bacterial system, inducible promoters such as pL, plac, ptrp, and ptac (ptrp-lac hybrid promoter) of bacteriophage lambda can be used. In one embodiment, when cloning in a mammalian cell system, promoters derived from the genome of mammalian cells (such as the metallothionein promoter) or mammalian viruses (such as the retroviral long terminal repeat; the adenovirus late promoter; or the vaccinia virus 7.5K promoter) can be used. Promoters produced by recombinant DNA or synthetic techniques can also be used to provide transcription of nucleic acid sequences.

[0106] The polynucleotide may be inserted into an expression vector containing a promoter sequence that facilitates efficient transcription of the inserted gene sequence in the host. Expression vectors typically contain an origin of replication, a promoter, and also contain specific nucleic acid sequences that allow for phenotypic selection of transformed cells.

[0107] Expression vector: A vector containing a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression may be supplied by the host cell or an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) into which the recombinant polynucleotide is incorporated.

[0108] Fc region: The constant region of an antibody excluding the first heavy chain constant domain. The Fc region generally refers to the last two heavy chain constant domains of IgA, IgD, and IgG, and the last three heavy chain constant domains of IgE and IgM. The Fc region may also include part or all of the flexible hinge N-terminal to these domains. For IgA and IgM, the Fc region may or may not include the tailpiece, and may or may not have an attached J chain. For IgG, the Fc region is typically understood to include immunoglobulin domains Cγ2 and Cγ3, and optionally the portion below the hinge between Cγ1 and Cγ2. Although the boundaries of the Fc region can vary, the human IgG heavy chain Fc region is generally defined as comprising residues from C226 or P230 to the Fc carboxyl terminus (numbering according to Kabat). For IgA, the Fc region comprises immunoglobulin domains Cα2 and Cα3, and optionally the lower portion of the hinge between Cα1 and Cα2.

[0109] Framework region: The amino acid sequences found between the CDRs in the variable region of an antibody's heavy or light chain. Includes the framework regions of the variable light chain and variable heavy chain. Framework regions serve to maintain the CDRs in the proper orientation.

[0110] Heterologous: originating from a different genetic origin. A nucleic acid molecule that is heterologous to a cell is one that originates from a genetic origin other than the cell in which it is expressed. In one specific, non-limiting example, a heterologous nucleic acid molecule encoding a protein such as an scFv is expressed in a cell such as a mammalian cell. Methods for introducing heterologous nucleic acid molecules into cells or organisms, such as nucleic acid transformation, for example, electroporation, lipofection, particle gun acceleration, and homologous recombination, are well known in the art.

[0111] Host cell: A cell in which a vector can be propagated and its DNA expressed. The cell can be prokaryotic or eukaryotic. The term includes progeny of the original host cell. Not all progeny are identical to the parent cell because of possible mutations that occurred during replication. However, when the term "host cell" is used, such progeny are included.

[0112] IgG: A polypeptide belonging to a class or isotype of antibody substantially encoded by the recognized immunoglobulin gamma genes. In humans, this class includes IgG1, IgG2, IgG3, and IgG4.

[0113] Immune complex: An antibody or an antigen-binding fragment (such as an scFv) binds to a soluble antigen to form an immune complex. The formation of an immune complex can be detected by conventional methods, such as immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, ELISA, immunoblotting (e.g., Western blot), magnetic resonance imaging, CT scan, X-ray examination, and affinity chromatography.

[0114] Immune response: A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In one embodiment, the response is specific for a particular antigen (an "antigen-specific response"). In one embodiment, the immune response is a T cell response, such as a CD4+ or CD8+ response. In another embodiment, the response is a B cell response, resulting in the production of specific antibodies.

[0115] Immunogen: A compound, composition, or substance capable of stimulating the production of antibodies or a T-cell response in an animal, for example, a composition that is injected or absorbed into an animal, for example, EGFRvIII coupled to a carrier. Immunogens can be used to produce antibodies such as those disclosed herein.

[0116] Tumor inhibition or treatment: A therapeutic intervention that reduces the signs or symptoms of a tumor (e.g., administering a therapeutically effective amount of an antibody that specifically binds to EGFRvIII or a conjugate thereof). Treatment can also induce alleviation, such as a reduction in tumor size. In a specific example, treatment includes inhibiting metastasis.

[0117] The term "reducing" is a relative term, and therefore, if a disease or condition is quantitatively reduced after administration of the drug, or if it is reduced compared to a reference drug after administration of the drug, the drug reduces the disease or condition. Reduction of signs or symptoms refers to an observable beneficial effect of treatment. Reduction of tumor-related signs or symptoms can be evidenced, for example, by delaying the onset of clinical symptoms of the disease in susceptible subjects (such as subjects with tumors that have not yet metastasized), reducing the severity of some or all clinical symptoms of the disease, slowing the progression of the disease (e.g., extending the lifespan of tumor-bearing subjects), reducing the number of tumors or the time between tumor removal and tumor recurrence, improving the overall health or well-being of the subject, or other parameters known in the art specific to a particular tumor. A "prophylactic" treatment is a treatment administered to subjects who do not show signs of tumors but have a genetic predisposition to tumors, or to subjects who only show early symptoms such as precancerous lesions, with the aim of reducing the risk of tumor development. The term "prevent" does not necessarily mean that an agent completely eliminates a disease or condition, so long as at least one characteristic of the disease or condition is eliminated. Thus, a composition that reduces or prevents tumors may prevent the risk of tumor development, but not necessarily prevent it completely.

[0118] Isolated: A biological component (such as a nucleic acid, peptide, protein, or protein complex, e.g., an antibody) that has been substantially separated, prepared separately, or purified from other biological components of the cells of the organism in which it naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA and proteins. Thus, isolated nucleic acids, peptides, and proteins include nucleic acids and proteins purified by standard purification methods. The term includes nucleic acids, peptides, and proteins prepared by recombinant expression in a host cell, and also includes chemically synthesized nucleic acids. An isolated nucleic acid, peptide, or protein, e.g., an antibody, can be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure.

[0119] Kabat location: Kabat et al. (Sequences of Proteins of Immunological Interest, 5 th The position of a residue within an amino acid sequence according to the numbering convention established by the National Institutes of Health (NIH Publication No. 91-3242, 1991).

[0120] Linker: A bifunctional molecule that can be used to link two molecules into one contiguous molecule, for example, to link an effector molecule to an antibody or to link a detectable marker to an antibody. Non-limiting examples of peptide linkers include glycine-serine linkers.

[0121] The terms "conjugate," "join," "couple," or "link" can refer to bringing two molecules into one continuous molecule; for example, linking two polypeptides into one continuous polypeptide, or covalently attaching an effector molecule or detectable marker radionuclide or other molecule to a polypeptide such as an scFv. Linking can be by either chemical or recombinant means. "Chemical means" refers to a reaction between an antibody moiety and an effector molecule such that a covalent bond is formed between the two molecules, forming one molecule.

[0122] Neoplasm, Cancer, or Tumor: A neoplasm is an abnormal growth of tissue or cells resulting from excessive cell division. Neoplastic growth can give rise to a tumor. The amount of tumor in an individual is the "tumor burden," which can be measured as the number, volume, or mass of tumors. Tumors that do not metastasize are called "benign." Tumors that can invade surrounding tissues or metastasize (or both) are called "malignant."

[0123] Tumors of the same histological type are primary tumors that originate in a particular organ (such as the head and neck, breast, or bladder). Tumors of the same histological type can be classified into different subtypes. For example, lung cancer can be classified into adenocarcinoma, small cell, squamous, or non-small cell tumors.

[0124] Examples of solid tumors such as sarcomas (connective tissue cancers) and carcinomas (epithelial cell cancers) include fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colorectal cancer, lymphoid malignancies, pancreatic cancer, breast cancer, head and neck cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, brown cell carcinoma, and ovarian cancer. Included are pheochromocytoma, sebaceous carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, and CNS tumors (such as glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma).

[0125] Nucleic acid: A polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and non-naturally occurring synthetic analogs thereof) linked via phosphodiester bonds, related naturally occurring structural variants, and non-naturally occurring synthetic analogs thereof. Thus, the term includes nucleotide polymers in which the nucleotides and the linkages between them contain non-naturally occurring synthetic analogs, such as, but not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methylribonucleotides, peptide nucleic acids (PNAs), and the like. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "oligonucleotide" typically refers to short polynucleotides, generally less than about 50 nucleotides. When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), it will be understood that this also includes RNA sequences in which "T" is replaced by "U" (i.e., A, U, G, C).

[0126] "Nucleotide" includes, but is not limited to, a monomer containing a base linked to a sugar, such as a pyrimidine, purine, or synthetic analogue thereof, or a base linked to an amino acid, such as peptide nucleic acid (PNA). A nucleotide is one monomer within a polynucleotide. A nucleotide sequence refers to the sequence of bases within a polynucleotide.

[0127] Conventional notation for describing nucleotide sequences is used herein: the left-hand end of a single-stranded nucleotide sequence is the 5'-end; the left-hand direction of a double-stranded nucleotide sequence is referred to as the 5'-direction. The 5' to 3' direction in which nucleotides are added to a nascent RNA transcript is referred to as the transcription direction. The DNA strand with the same sequence as the mRNA is referred to as the "coding strand"; the sequence on the DNA strand with the same sequence as the mRNA transcribed from that DNA and located 5' to the 5'-end of the RNA transcript is referred to as the "upstream sequence"; the sequence on the DNA strand with the same sequence as the RNA and located 3' to the 3'-end of the coding RNA transcript is referred to as the "downstream sequence."

[0128] "cDNA" refers to a DNA that is complementary to or identical to an mRNA, in either single- or double-stranded form.

[0129] "Encoding" refers to the inherent property of a particular nucleotide sequence of a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, having either a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence, and the biological properties attributed thereto. Thus, if transcription and translation of the mRNA produced by a gene produces a protein in a cell or other biological system, the gene encodes that protein. Both the coding sequence of a gene or cDNA, whose nucleotide sequence is identical to the mRNA sequence and generally provided in a sequence listing, and the non-coding strand, used as a transcription template, can be referred to as encoding the protein or other product of the gene or cDNA. Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. Protein- and RNA-encoding nucleotide sequences can contain introns.

[0130] A polynucleotide having a first sequence is "antisense" to a polynucleotide having a second sequence if the first sequence specifically hybridizes to the polynucleotide having the second sequence.

[0131] Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when it is placed into a functional relationship with the second nucleic acid sequence. For example, a promoter, such as a CMV promoter, is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

[0132] Pharmaceutically acceptable carriers: Useful pharmaceutically acceptable carriers are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 19th Edition, 1995, describes suitable compositions and formulations for pharmaceutical delivery of the disclosed antibodies and antigen-binding fragments thereof.

[0133] Generally, the nature of the carrier will depend on the particular mode of administration being employed. For example, parenteral formulations generally comprise injectable fluids containing pharmaceutically and physiologically acceptable liquids as a vehicle, such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, or the like. Conventional non-toxic solid carriers for solid compositions (e.g., in powder, pill, tablet, or capsule forms) can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical composition to be administered may contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, for example, sodium acetate or sorbitan monolaurate. In specific embodiments, carriers suitable for administration to a subject may be sterile and / or may be suspended or otherwise contained in a unit dosage form containing one or more measured doses of the composition. It may also be accompanied by a medication for use in treatment. The unit dosage form may be, for example, present in a sealed vial containing sterile contents or a syringe for injection into a subject, or may be lyophilized for subsequent solubilization and administration, or may be a solid or controlled release dosage form.

[0134] Polypeptide: A chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). "Polypeptide" refers to amino acid polymers, including naturally occurring and non-naturally occurring amino acid polymers, in which one or more amino acid residues are unnatural amino acids, e.g., artificial chemical mimetics of the corresponding naturally occurring amino acids. "Residue" refers to an amino acid or amino acid mimetic incorporated into the polypeptide by an amide bond or amide bond mimetic. Polypeptides have an amino terminus (N-terminus) and a carboxy terminus (C-terminus). "Polypeptide" is used interchangeably with peptide or protein and is used herein to refer to a polymer of amino acid residues.

[0135] Polypeptide Modifications: Polypeptides and peptides, such as the antibodies disclosed herein, can be modified by a variety of chemical techniques to produce derivatives that have essentially the same activity as the unmodified peptide, and optionally, other desired properties. For example, carboxylic acid groups of the protein, whether at the carboxyl terminus or at the side chain, can be provided in the form of a salt with a pharmaceutically acceptable cation, or C1-C 16 may be esterified to form an ester, or may be of the formula NR1R2 (where R1 and R2 are each independently H or C1-C 16 The amide group of the peptide, whether amino-terminal or side chain, may be provided in the form of a pharmaceutically acceptable acid addition salt such as HCl, HBr, acetate, benzoate, toluenesulfonate, maleate, tartrate, and other organic salts, or may be converted to an amide of a C1-C 16 It may be modified to alkyl or dialkylamino, or further converted to an amide.

[0136] The peptide side chain hydroxyl groups are derivatized from C1-C using well-recognized techniques. 16 Alkoxy or C1-C 16The phenyl and phenol rings of the peptide side chains can be converted to esters by one or more halogen atoms such as F, Cl, Br, or I, or by C1-C 16 Alkyl, C1-C 16 The substituted carboxylic acid may be substituted with alkoxy, carboxylic acids and their esters, or amides of such carboxylic acids. Methylene groups in peptide side chains may be extended to homologous C2-C4 alkylenes. Thiols may be protected with any of a number of well-recognized protecting groups, such as acetamide groups. Those skilled in the art will also recognize methods for introducing ring structures into the peptides of the present disclosure to select and provide conformational constraints to the structure that result in enhanced stability. For example, a C- or N-terminal cysteine ​​can be added to a peptide so that, upon oxidation, the peptide contains a disulfide bond, generating a cyclic peptide. Other methods of peptide cyclization include the formation of thioethers and carboxyl- and amino-terminal amides and esters.

[0137] Purified: The term purified does not require absolute purity; rather, it is intended to be a relative term. Thus, for example, a purified peptide preparation is one in which a peptide or protein (such as an antibody) is enriched relative to the peptide or protein in its natural environment within a cell. In one embodiment, the preparation is purified so that the protein or peptide represents at least 50% of the total peptide or protein content of the preparation.

[0138] Recombinant: A recombinant nucleic acid is one that has a sequence that does not occur in nature or that has a sequence created by the artificial combination of two otherwise separate sequence segments. This artificial combination can be achieved by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acid, e.g., by genetic engineering techniques. A recombinant protein is one that has a sequence that does not occur in nature or that has a sequence created by the artificial combination of two otherwise separate sequence segments. In some embodiments, a recombinant protein is encoded by a heterologous (e.g., recombinant) nucleic acid introduced into a host cell, such as a bacterial cell or a eukaryotic cell. The nucleic acid can be introduced, for example, into an expression vector with a signal capable of expressing the protein encoded by the introduced nucleic acid, or the nucleic acid can be integrated into a host cell chromosome.

[0139] Sequence identity: The similarity between amino acid sequences is expressed by the similarity between the sequences, also called sequence identity. Sequence identity is often measured by the percentage of identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs, orthologs, or variants of a polypeptide will have a relatively high degree of sequence identity when aligned using standard methods.

[0140] Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8,155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents a detailed discussion of sequence alignment methods and homology calculations.

[0141] After alignment, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue occurs in both sequences. The percent sequence identity is determined by dividing the number of matches by either the length of the sequence shown in the identified sequence or by a segmented length (such as 100 consecutive nucleotides or amino acid residues from the sequence shown in the identified sequence), and then multiplying the resulting value by 100. For example, a peptide sequence that has 1166 matches when aligned with a subject sequence having 1554 amino acids is 75.0 percent identical to the subject sequence (1166 divided by 1554). * (100=75.0). Percent sequence identity values ​​are rounded to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, and 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. Length values ​​are always integers.

[0142] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD), and on the Internet, for use with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. A description of how to use this program to determine sequence identity is available on the Internet at the NCBI website.

[0143] Polypeptide homologs and variants are typically characterized by at least about 75% sequence identity, e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, when counted in a full-length alignment with the amino acid sequence of interest. Proteins with even greater similarity to the reference sequence will exhibit increasing percentages of identity, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, when assessed by this method. When sequences less than full length are compared for sequence identity, homologs and variants typically share at least 80% sequence identity in a short window of 10 to 20 amino acids, and may share at least 85%, or at least 90%, or 95% sequence identity, depending on the similarity to the reference sequence. Methods for determining sequence identity in such short windows are available on the internet at the NCBI website. Those skilled in the art will understand that these sequence identity ranges are provided for guidance only; it is entirely possible that strong and significant homologs will be available that do not fall within the ranges provided.

[0144] For the sequence comparison of nucleic acid sequence, typically, one sequence acts as the reference sequence with which test sequence is compared.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, and partial sequence coordinates are designated as necessary, and sequence algorithm program parameters are designated.Default program parameters are used.The method of aligning sequences for comparison is well known in the art. Optimal alignment of sequences for comparison can be achieved, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482, 1981, the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443, 1970, the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or manual alignment and visual inspection (e.g., Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4 thed, Cold Spring Harbor, New York, 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, through supplement 104, 2013). One example of a useful algorithm is PILEUP. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351-360, 1987. The method used is similar to that described by Higgins & Sharp, CABIOS 5:151-153, 1989. Using PILEUP, a reference sequence is compared to other test sequences to determine percent sequence identity using the following parameters: a default gap weight (3.00), a default gap length weight (0.10), and weighted end gaps. PILEUP can be obtained from the GCG sequence analysis software package, for example, version 7.0 (Devereaux et al., Nuc. Acids Res. 12:387-395, 1984).

[0145] Another example of a suitable algorithm for determining percent sequence identity and sequence similarity is the BLAST and BLAST 2.0 algorithms described in Altschul et al., J. Mol. Biol. 215:403-410, 1990 and Altschul et al., Nucleic Acids Res. 25:3389-3402, 1977. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov). The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, alignment (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands. The BLASTP program (for amino acid sequences) uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989). Oligonucleotides are linear polynucleotide sequences up to about 100 nucleotide bases in length.

[0146] As used herein, a reference to "at least 80% identity" refers to "at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity" to a specified reference sequence. As used herein, a reference to "at least 90% identity" refers to "at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity" to a specified reference sequence.

[0147] Specific binding: When referring to an antibody or antigen-binding fragment, this refers to a binding reaction that determines the presence of a target protein in the presence of a heterogeneous population of proteins and other biologics. Thus, under specified conditions, the antibody preferentially binds to a specific target protein, peptide, or polysaccharide (e.g., an antigen present on the surface of tumor cells, e.g., EGFRvIII) and does not bind in significant amounts to other proteins present in the sample or subject, such as wild-type EGFR expressed in wild-type (non-tumor) cells derived from the same tissue. In other embodiments, the antibody can specifically bind to EGFRvIII and forms of EGFR overexpressed in tumor cells, but does not bind to wild-type EGFR expressed in wild-type (non-tumor) cells from the same tissue. Specific binding can be determined by standard methods. For a description of immunoassay formats and conditions that can be used to determine specific immune reactivity, see Harlow & Lane, Antibodies, A Laboratory Manual, 2004, pp. 111-114, 2004. nd ed., Cold Spring Harbor Publications, New York (2013).

[0148] For antibody-antigen complexes, the specific binding between the antigen and the antibody is approximately 10 -7 Less than m, e.g., about 10 -8 Under M, 10 -9 , or even about 10 -10 K less than M D K D refers to the dissociation constant for a particular interaction, such as a polypeptide-ligand interaction or an antibody-antigen interaction. For example, for a bimolecular interaction between an antibody or antigen-binding fragment and an antigen, it is the concentration of the individual components of the bimolecular interaction divided by the concentration of the complex.

[0149] An antibody that specifically binds to an epitope on EGFRvIII is an antibody that substantially binds to EGFRvIII protein, for example, cells or tissues expressing EGFRvIII, a substrate to which EGFRvIII is attached, or EGFRvIII in or isolated from a biological specimen. Naturally, some non-specific interaction may occur between an antibody and a non-target (such as cells of the same tissue type that do not express wild-type EGFR). Typically, specific binding results in a much stronger association between an antibody and a protein or cell bearing an antigen than between an antibody and a protein or cell lacking the antigen. Specific binding typically results in a more than 2-fold, e.g., more than 5-fold, more than 10-fold, or more than 100-fold increase in the amount of antibody binding (per unit time) to a protein containing the epitope or to cells or tissues expressing the target epitope, compared to a protein, cell, or tissue lacking this epitope. Specific binding to a protein under such conditions requires an antibody selected for its specificity for a particular protein. A variety of immunoassay formats are suitable for selecting antibodies or other ligands specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein.

[0150] Subject: A living multicellular vertebrate organism, a category that includes humans and non-human mammals. In one example, the subject is a human. In a specific example, the subject has cancer. In an additional example, the subject is selected to be in need of inhibition of tumor growth or metastasis. For example, the subject has been diagnosed with a tumor that expresses EGFRvIII, such as head and neck cancer, breast cancer, or bladder cancer, and is in need of treatment.

[0151] T cells: White blood cells important for immune response. T cells contain CD4 + T cells and CD8 + These include, but are not limited to, T cells. CD4 +T lymphocytes are immune cells that express CD4 on their surface. These cells, also known as helper T cells, help orchestrate immune responses, including antibody responses and killer T cell responses. Th1 and Th2 cells are functional subsets of helper T cells. Th1 cells secrete a set of cytokines, including interferon-gamma, and their main function is to stimulate phagocyte-mediated defense against infections, particularly those involving intracellular microorganisms. Th2 cells secrete a set of cytokines, including interleukin (IL)-4 and IL-5, and their main function is to stimulate IgE- and eosinophil / mast cell-mediated immune responses and downregulate Th1 responses.

[0152] Therapeutic Agent: Used in a comprehensive sense to include treatment agents, prophylactic agents, and supplemental agents. Therapeutic agents are used to ameliorate a specific set of symptoms in a subject with a disease or disorder.

[0153] Treatment or prevention of disease: For example, inhibiting the full development of a disease or condition in a subject at risk of or with a disease, such as a tumor. "Treatment" refers to a therapeutic intervention that ameliorates the signs or symptoms of a disease or pathological condition after its onset. The term "amelioration" refers to an observable beneficial effect of the treatment with respect to the disease or pathological condition. A beneficial effect can be evidenced, for example, by a delay in the onset of clinical symptoms of the disease in a susceptible subject, a reduction in the severity of some or all clinical symptoms of the disease, a slowing of the progression of the disease, an improvement in the overall health or well-being of the subject, or other parameters well known in the art specific to a particular disease. "Prophylactic" treatment is a treatment administered to a subject who does not show signs of the disease or who shows only early signs, with the aim of reducing the risk of developing the pathology.

[0154] Toxin: An effector molecule that induces cytotoxicity upon contact with a cell. Specific, non-limiting examples of toxins include abrin, ricin, auristatins (e.g., monomethylauristatin E (MMAE; see, e.g., Francisco et al., Blood, 102:1458-1465, 2003) and monomethylauristatin F (MMAF; see, e.g., Doronina et al., BioConjugate Chem., 17:114-124, 2006)), maytansinoids (e.g., DM1; see, e.g., Phillips et al., Cancer Res., 68:9280-9290, 2008), Pseudomonas exotoxin (PE, e.g., PE35, PE37, PE38, and PE40), diphtheria toxin (DT), botulinum toxin, saporin, restrictocin, or gelonin, or modified toxins thereof, or other toxic agents that directly or indirectly inhibit cell growth or kill cells. For example, PE and DT are highly toxic compounds that typically cause death through liver toxicity. However, PE and DT can be modified into forms for use as immunotoxins by removing the native targeting component of the toxin (such as domain Ia of PE and the B chain of DT) and replacing it with a different targeting moiety, such as an antibody.

[0155] Transformed: A transformed cell is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. As used herein, terms such as transformed (e.g., transformation, transfection, transduction, etc.) include all techniques by which a nucleic acid molecule can be introduced into such a cell, such as transduction with a viral vector, transformation with a plasmid vector, and introduction of DNA by electroporation, lipofection, and particle gun acceleration.

[0156] Vector: An entity containing a nucleic acid molecule (such as a DNA molecule or an RNA molecule) operably linked to a coding sequence for a protein of interest and carrying a promoter capable of expressing the coding sequence. Non-limiting examples include naked DNA or DNA packaged (in lipid and / or protein), naked RNA or packaged RNA, viruses or bacteria or other microorganisms that may be replication-incompetent, or subcomponents of viruses or bacteria or other microorganisms that may be replication-competent. A vector is sometimes also called a construct. A recombinant DNA vector is a vector that contains recombinant DNA. A vector may contain a nucleic acid sequence that enables replication in a host cell, such as an origin of replication. A vector may also contain one or more selectable marker genes and other genetic elements. A viral vector is a recombinant nucleic acid vector that contains at least some nucleic acid sequences derived from one or more viruses. In some embodiments, the viral vector contains a nucleic acid molecule encoding the disclosed antibody or antigen-binding fragment that specifically binds to EGFRvIII.

[0157] Under conditions sufficient for: A phrase used to describe an environment that allows for a desired activity.

[0158] II. Description of Certain Embodiments Provided are isolated monoclonal antibodies and antigen-binding fragments thereof that specifically bind to EGFRvIII. In some embodiments, the antibodies and antigen-binding fragments can be used to treat tumors that express EGFRvIII, such as, but not limited to, head and neck cancer, breast cancer, or bladder cancer. Also disclosed herein are compositions comprising the antibodies and antigen-binding fragments and a pharmaceutically acceptable carrier. Nucleic acids encoding the antibodies or antigen-binding fragments, expression vectors containing these nucleic acids, and isolated host cells expressing the nucleic acids are also provided.

[0159] Compositions comprising monoclonal antibodies and antigen-binding fragments thereof that specifically bind to EGFRvIII can be used for research, diagnostic, and therapeutic purposes. For example, monoclonal antibodies can be used to diagnose or treat subjects with tumors that express EGFRvIII.

[0160] A. Antibodies and Antigen-Binding Fragments Provided herein are isolated monoclonal antibodies and antigen-binding fragments that specifically bind to an epitope of EGFRvIII. In some embodiments, the antibodies and antigen-binding fragments can inhibit the biological function or properties of EGFRvIII protein in vivo (for example, but not limited to, reducing and / or inhibiting tumor growth, or reducing and / or inhibiting metastatic tumors). Disclosed herein are several monoclonal antibodies that bind to EGFRvIII but not to wild-type EGFR. Furthermore, monoclonal antibody 40H3 binds to EGFR when overexpressed in tumor cells, but not in wild-type cells.

[0161] One structural element that is sterically unavailable under normal conditions is the disulfide-restricted loop at positions 287-302 (mature receptor numbering or 301-326 in the full-length receptor), which is exposed in EGFRvIII. In some embodiments, D290 and E293 are present in the EGFRvIII region, where the antibody or antigen-binding fragment binds to the EGFRvIII region. 287-302 In other embodiments, E293 is required for the antibody or antigen-binding fragment to bind to the EGFR loop. Exemplary antibodies having this binding characteristic are provided (such as 1D9, 3D10, 4A4, 9G11, and 11E3). 287-302 In a further embodiment, R300 is required for binding to the EGFR loop. Exemplary antibodies (such as 11E11) having this binding characteristic are provided. 287-302The 40H3 region is required for binding to the 40H3 loop. Exemplary antibodies (such as 40H3) with this binding characteristic are provided. The disclosed antibodies may be chimeric or humanized, and thus may contain one or more heterologous framework regions.

[0162] In some embodiments, the EGFRvIII-specific antibody comprises a variable heavy chain region (V H ) and the variable light chain region (V L In some embodiments, the monoclonal antibody comprises a heavy chain complementarity determining region (HCDR) 1, HCDR2, and HCDR3, and specifically binds to EGFRvIII. H , and V comprising light chain complementarity determining regions (LCDR) 1, LCDR2, and LCDR3 L Includes:

[0163] In some embodiments, the antibody or antigen-binding fragment is 287-302 In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to an epitope of EGFR, such as a V loop, comprising HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3, respectively, of one of the disclosed antibodies, and is neutralizing. H and V L Includes:

[0164] The following description of monoclonal antibodies includes V sequences each containing at least one CDR. H and V LThe term "monoclonal antibody" refers to an isolated monoclonal antibody comprising a CDR comprising a CDR sequence of a target molecule. Those skilled in the art will understand that various CDR numbering schemes (such as the Kabat, Chothia, or IMGT numbering schemes) can be used to determine the location of the CDRs. The amino acid sequences and CDR locations of the heavy and light chains of the disclosed monoclonal antibodies are presented herein using the IMGT numbering system. However, those skilled in the art will readily understand the use of various CDR numbering schemes when referring to specific amino acids of the antibodies disclosed herein. Programs for identifying CDRs using Chothia and Kabbat are available on the internet.

[0165] In some embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof comprises: (a) V, VIII, VIV, VIVV ... H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L [40H3]; (b) V, shown as SEQ ID NOs: 11 and 12, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L [3D10 / 9G11 consensus]; (c) V, shown as SEQ ID NOs: 13 and 14, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L [1D9 / 4A4 consensus]; or (d) V, shown as SEQ ID NOs: 15 and 16, respectively H and V LV, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L [11E11 / 11G3 consensus] The CDRs can be identified using, for example, IMGT, Kabat, or Chothia. In other embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof comprises one of: (a) V, shown as SEQ ID NOs: 17 and 12, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L [3D10]; (b) V, shown as SEQ ID NOs: 26 and 12, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L [9G11]; (c) V, shown as SEQ ID NOs: 29 and 30, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L [1D9]; (d) V, shown as SEQ ID NOs: 39 and 40, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L [4A4]; (e) V, shown as SEQ ID NOs: 43 and 44, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and VL [11E11]; or (f) V, shown as SEQ ID NOs: 53 and 54, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L [11G3] CDRs can be identified using, for example, IMGT, Kabat, or Chothia.

[0166] In some non-limiting examples, (a) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences shown as SEQ ID NOs:5, 6, 7, 8, 9, and 10, respectively [40H3]; (b) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences shown as SEQ ID NOs:20, 21, 22, 23, 24, and 25, respectively [3D10]; (c) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences shown as SEQ ID NOs: 20, 28, 22, 23, 24, and 25, respectively [9G11]; (d) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth as SEQ ID NOs: 33, 34, 35, 36, 37, and 38, respectively [1D9 and 4A4]; (e) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth as SEQ ID NOs: 47, 48, 49, 50, 51, and 52, respectively [11E11]; or (f) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth as SEQ ID NOs: 47, 57, 49, 58, 51, and 52, respectively [11G3]. In these embodiments, the monoclonal antibody or antigen-binding fragment specifically binds to EGFRvIII. In one non-limiting example, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth as SEQ ID NOs: 5, 6, 7, 8, 9, and 10 [40H3], and the monoclonal antibody or antigen-binding fragment also binds to EGFR overexpressed in tumor cells.

[0167] In some embodiments, V H and V L comprises an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 1 and 2, respectively [40H3]. H and V L comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 17 and 12, respectively [3D10]. H and V L comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 26 and 12, respectively [9G11]. H and V L comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 29 and 30, respectively [1D9]. H and VL comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 39 and 40, respectively [4A4]. H and V L comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 43 and 44, respectively [11E11]. H and V L and V comprise an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 53 and 54, respectively [11G3]. In these embodiments, the monoclonal antibody or antigen-binding fragment specifically binds to EGFRvIII. In one non-limiting example, V H and V L comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NOs: 1 and 2, and the monoclonal antibody or antigen-binding fragment also binds to EGFR overexpressed in tumor cells.

[0168] In some embodiments, V H and V L comprises an amino acid sequence that is 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences set forth as SEQ ID NOs: 1 and 2, respectively [40H3]. H and V L comprises an amino acid sequence that is 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences set forth as SEQ ID NOs: 17 and 12, respectively [3D10]. H and VL comprises an amino acid sequence that is 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences set forth as SEQ ID NOs: 26 and 12, respectively [9G11]. H and V L comprises an amino acid sequence that is 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences set forth as SEQ ID NOs: 29 and 30, respectively [1D9]. H and V L comprises an amino acid sequence that is 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences set forth as SEQ ID NOs: 39 and 40, respectively [4A4]. H and V L comprises an amino acid sequence that is 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences set forth as SEQ ID NOs: 43 and 44, respectively [11E11]. H and comprise amino acid sequences that are 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences set forth as SEQ ID NOs:53 and 54, respectively [11G3]. In these embodiments, the monoclonal antibody or antigen-binding fragment specifically binds to EGFRvIII. In one non-limiting example, V H and V L comprises an amino acid sequence that is 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NOs: 1 and 2, and the monoclonal antibody or antigen-binding fragment also binds to EGFR that is overexpressed in tumor cells.

[0169] In a further embodiment, V H and V Lcomprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NOs: 1 and 2, and HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth as SEQ ID NOs: 5, 6, 7, 8, 9, and 10, respectively [40H3]. H and V L comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NOs: 17 and 12, and HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth as SEQ ID NOs: 20, 21, 22, 23, 24, and 25, respectively [3D10]. H and V L comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NOs: 26 and 12, and HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth as SEQ ID NOs: 20, 28, 22, 23, 24, and 25, respectively [9G11]. H and V L comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NOs: 29 and 30, and HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth as SEQ ID NOs: 33, 34, 35, 36, 37, and 38, respectively [1D9]. H and V Lcomprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NOs: 39 and 40, and HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth as SEQ ID NOs: 33, 34, 35, 36, 37, and 38, respectively [4A4]. H and V L comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NOs: 43 and 44, and HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth as SEQ ID NOs: 47, 48, 49, 50, 51, and 52, respectively [11E11]. H and V L comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NOs: 53 and 54, and HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth as SEQ ID NOs: 47, 57, 49, 58, 51, and 52, respectively [11G3]. In these embodiments, the monoclonal antibody or antigen-binding fragment specifically binds to EGFRvIII. In one non-limiting example, V H and V Lcomprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NOs: 1 and 2, and HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth as SEQ ID NOs: 5, 6, 7, 8, 9, and 10, respectively, and the monoclonal antibody or antigen-binding fragment also binds to EGFR overexpressed in tumor cells.

[0170] In some embodiments, the monoclonal antibody or antigen-binding fragment comprises the amino acid sequence shown as SEQ ID NOs: 1 and 2, respectively. H and V L In other embodiments, the monoclonal antibody or antigen-binding fragment comprises V, ... H and V L In a further embodiment, the monoclonal antibody or antigen-binding fragment comprises the amino acid sequence set forth as SEQ ID NOs: 26 and 12, respectively. H and V L In additional embodiments, the monoclonal antibody or antigen-binding fragment comprises V, VB, VC, VD, VE, VF, VH, VF ... H and V L In a further embodiment, the monoclonal antibody or antigen-binding fragment comprises V, which comprises the amino acid sequences set forth as SEQ ID NOs: 39 and 40, respectively. H and V L In a further embodiment, the monoclonal antibody or antigen-binding fragment comprises V, which comprises the amino acid sequences set forth as SEQ ID NOs: 43 and 44, respectively. H and V L In other embodiments, the monoclonal antibody or antigen-binding fragment comprises V, ... H and V LIn these embodiments, the monoclonal antibody or antigen-binding fragment specifically binds to EGFRvIII. H and V L comprise the amino acid sequences shown as SEQ ID NO:1 and SEQ ID NO:2, respectively, and the monoclonal antibodies or antigen-binding fragments also bind to EGFR overexpressed in tumor cells.

[0171] 1. Additional Description of Antibodies and Antigen-Binding Fragments The antibody or antigen-binding fragment may be a humanized antibody or an antigen-binding fragment thereof. The antibody or antigen-binding fragment may comprise any suitable framework region, such as, but not limited to, one or more human framework regions. Human framework regions and mutations that can be made in human antibody framework regions are known in the art (see, for example, U.S. Patent No. 5,585,089, incorporated herein by reference). Chimeric antibodies are also provided. Heterologous framework regions, such as, but not limited to, different murine framework regions, can be included in the heavy or light chains of the antibody (see, e.g., Jones et al., Nature 321:522, 1986; Riechmann et al., Nature 332:323, 1988; Verhoeyen et al., Science 239:1534, 1988; Carter et al., Proc. Natl. Acad. Sci. USA 89:4285, 1992; Sandhu, Crit. Rev. Biotech. 12:437, 1992; and Singer et al., J. Immunol. 150:2844, 1993).

[0172] The antibody may be of any isotype. The antibody may be, for example, an IgM antibody or an IgG antibody, for example, IgG1, IgG2, IgG3, or IgG4. The class of an antibody that specifically binds to EGFRvIII may be switched to another class. In one aspect, V L or V HThe nucleic acid molecule encoding V is isolated using methods well known in the art so that it is free of nucleic acid sequences encoding the light or heavy chain constant regions, respectively. L or V H The nucleic acid molecules encoding the C L or C H This is operably linked to a nucleic acid sequence encoding a C L Chain or C H This can be achieved using vectors or nucleic acid molecules containing the chains. For example, an antibody that specifically binds to EGFRvIII, originally an IgG, can be class-switched. Class switching can be used to convert one IgG subclass to another, for example, IgG1 to IgG2, IgG3, or IgG4.

[0173] In some examples, the disclosed antibodies are oligomers of antibodies, such as dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, and the like.

[0174] (a) Binding affinity In some embodiments, the antibody or antigen-binding fragment is 1.0 x 10 -8 M or less, 5.0×10 -8 M or less, 1.0×10 -9 M or less, 5.0×10 -9 M or less, 1.0×10 -10 M or less, 5.0×10 -10 M, or 1.0 x 10 -11 M or less (e.g., K d It can specifically bind to EGFRvIII protein with an affinity (measured by K d can be measured, for example, by a radiolabeled antigen binding assay (RIA) performed with a Fab version of the antibody of interest and its antigen, using known methods. In one assay, the solution binding affinity of a Fab for an antigen is determined by the lowest concentration of ( 125The Fab is equilibrated with I)-labeled antigen, followed by capturing the bound antigen with a plate coated with an anti-Fab antibody (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish the conditions for the assay, MICROTITER® multiwell plates (Thermo Scientific) are coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), followed by blocking with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 μM or 26 pM [ 125 [I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., corresponding to the anti-VEGF antibody Fab-12 in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight; however, incubation may be continued for a longer period (e.g., about 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., 1 hour). The solution is then removed, and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. After drying, 150 μl / well of scintillant (MICROSCINT-20™; Packard) is added, and the plate is counted for 10 minutes in a TOPCOUNT™ gamma counter (Packard). The concentration of each Fab that gives 20% or less of maximum binding is selected for use in competitive binding assays.

[0175] In a separate assay, K dcan be measured using a surface plasmon resonance assay with a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C with an antigen CM5 chip immobilized at approximately 10 response units (RU). Briefly, a carboxymethylated dextran biosensor chip (CM5, BIACORE®, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) in 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 l / min to achieve approximately 10 response units (RU) of coupled protein. After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) in PBS containing 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) are injected at a flow rate of approximately 25 l / min at 25° C. By simultaneously fitting the association and dissociation sensorgrams, the association rate (k) was calculated using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2). on ) and dissociation rate (k off ) is calculated. The equilibrium dissociation constant (Kd) is calculated by the ratio k off / k on See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). The surface plasmon resonance assay showed an association rate of 106 M -1 s -1above 20°C, the association rate can be determined by using a fluorescence quenching technique to measure the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) in the presence of increasing concentrations of antigen at 25°C, as measured in a spectrometer such as a stop-flow equipped spectrophotometer (Aviv Instruments) or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirred cuvette.

[0176] (b) Multispecific antibody In some embodiments, the antibody or antigen-binding fragment is included in a multispecific antibody, such as a bispecific antibody. Such multispecific antibodies can be produced by known methods, such as crosslinking two or more antibodies or antigen-binding fragments (such as scFvs) of the same or different types. Exemplary methods for making multispecific antibodies include those described in PCT Publication No. WO2013 / 163427, incorporated herein by reference in its entirety. Suitable crosslinkers include heterobifunctional ones, having two different reactive groups separated by a suitable spacer (such as m-maleimidobenzoyl-N-hydroxysuccinimide ester), or homobifunctional ones (such as disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company (Rockford, Ill.).

[0177] In some embodiments, the antibody or antigen-binding fragment is comprised in a bispecific antibody that specifically binds to EGFRvIII and also specifically binds to a tumor antigen, such as Her-2, or a checkpoint inhibitor, such as programmed cell death (PD)-1 or PD ligand (PD-L1) or PD-L2.

[0178] Various types of multispecific antibodies are known. Bispecific single-chain antibodies can be encoded by a single nucleic acid molecule. Examples of bispecific single-chain antibodies and methods for constructing such antibodies are known in the art (see, for example, U.S. Patent Nos. 8,076,459, 8,017,748, 8,007,796, 7,919,089, 7,820,166, 7,635,472, 7,575,923, 7,435,549, 7,332,168, 7,323,440, 7,235,641, 7,229,760, 7,112,324, 6,723,538, which are incorporated herein by reference). Additional examples of bispecific single chain antibodies can be found in PCT Application No. WO 99 / 54440; Mack, J. Immunol., 158:3965-3970, 1997; Mack, PNAS, 92:7021-7025, 1995; Kufer, Cancer Immunol. Immunother., 45:193-197, 1997; Loffler, Blood, 95:2098-2103, 2000; and Bruhl, J. Immunol., 166:2420-2426, 2001. The generation of bispecific Fab-scFv ("bibody") molecules is described, for example, in Schoonjans et al. (J. Immunol. 165:7050-57, 2000) and Willems et al. (J Chromatogr B Analyt Technol Biomed Life Sci. 786:161-76, 2003). For bibodies, for example, to generate a bibody in which one scFv is fused to the C-terminus of a Fab chain, the scFv molecule can be fused to one of the VL-CL(L) or VH-CH1 chains.

[0179] (c) Antigen-binding fragment Antigen-binding fragments, such as Fab, F(ab'), and Fv, that contain the variable regions of the heavy and light chains and specifically bind to EGFRvIII are encompassed by the present disclosure. These antibody fragments retain the ability to selectively bind to an antigen and are "antigen-binding" fragments. These fragments include: (1) Fab, a fragment containing a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain. (2) A fragment of an antibody molecule, Fab', can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule. (3) The fragment of an antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction, (Fab')2; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds. (4) A genetically engineered fragment, Fv, containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains, as well as its disulfide-linked form (dsFv). (5) Single-chain antibodies (such as scFvs) are defined as genetically engineered molecules containing a light chain variable region and a heavy chain variable region linked by a suitable polypeptide linker as a genetically fused single-chain molecule. scFvs are fusion proteins in which an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region are linked by a linker (see, for example, Ahmad et al., Clin. Dev. Immunol., 2012, doi:10.1155 / 2012 / 980250; Marbry, IDrugs, 13:543-549, 2010). The V in scFvs H Domains and V L The intramolecular orientation of the domains is not critical for the provided antibodies (e.g., the provided multispecific antibodies). Therefore, both possible configurations (V H Domain-Linker Domain-V L Domain;V L Domain-Linker Domain-V H scFvs having a nucleotide sequence (domain) can be used. Other formats, such as ds-scFvs, are also useful. (6) Single-chain antibody dimers (scFV2), defined as dimers of scFVs, also named "miniantibodies."

[0180] Methods for generating these fragments are known in the art (e.g., Harlow and Lane, Antibodies: A Laboratory Manual, 2002). nd , Cold Spring Harbor Laboratory, New York, 2013).

[0181] In a further group of embodiments, the antibody-binding fragment may be an Fv antibody, which is typically about 25 kDa and contains a complete antigen-binding site comprising three CDRs for each heavy and light chain. To produce an Fv antibody, a V H and V L can be expressed from two separate nucleic acid constructs. H and V L When expressed discontinuously, the chains of an Fv antibody are typically linked by noncovalent interactions. However, because these chains tend to dissociate upon dilution, methods for cross-linking the chains have been developed using glutaraldehyde, intramolecular disulfides, or peptide linkers. Thus, in one example, the Fv can be a disulfide-stabilized Fv (dsFv), in which the heavy chain variable region and the light chain variable region are chemically linked by a disulfide bond.

[0182] In additional examples, the Fv fragment comprises V fragments connected by a peptide linker. H Chain and V L These single-chain antigen-binding proteins (scFv) contain two V chains connected by an oligonucleotide. H Domains and V LscFvs are prepared by constructing a nucleic acid molecule encoding the domains. The nucleic acid molecule is inserted into an expression vector, which is then introduced into a host cell, such as a mammalian cell. The recombinant host cell synthesizes a single polypeptide chain containing a linker peptide bridging the two V domains. Methods for producing scFvs are known in the art (see Whitlow et al., Methods: a Companion to Methods in Enzymology, Vol. 2, page 97, 1991; Bird et al., Science 242:423, 1988; U.S. Patent No. 4,946,778; Pack et al., Bio / Technology 11:1271, 1993; Ahmad et al., Clin. Dev. Immunol., 2012, doi:10.1155 / 2012 / 980250; Marbry, IDrugs, 13:543-549, 2010). Single chain antibody dimers (scFV2) are also contemplated.

[0183] Antigen-binding fragments can be prepared by proteolytic hydrolysis of the antibody or by expression in host cells (such as E. coli cells) of DNA encoding the fragment. Antigen-binding fragments can also be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antigen-binding fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment designated F(ab')2. This fragment can be further cleaved using a thiol reducing agent, optionally with a blocking group for the sulfhydryl groups resulting from cleavage of disulfide bonds, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly (see U.S. Pat. Nos. 4,036,945 and 4,331,647 and references contained therein; Nisonhoff et al., Arch. Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959; Edelman et al., Methods in Enzymology, Vol. 1, page 422, Academic Press, 1967; and Coligan et al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4).

[0184] Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of the fragments, or other enzymatic, chemical, or genetic techniques, can also be used so long as the fragments bind to the antigen recognized by the intact antibody.

[0185] Antigen-binding single V domain antibodies (dAbs) H The domain was also amplified from the genomic DNA of immunized mice. HThey have been identified from gene libraries (Ward et al. Nature 341:544-546, 1989). Human single immunoglobulin variable domain polypeptides capable of binding to antigens with high affinity have also been described (see, e.g., PCT Publication Nos. WO 2005 / 035572 and WO 2003 / 002609). The CDRs disclosed herein may be contained in a dAb.

[0186] In some embodiments, one or more of the heavy and / or light chain complementarity determining regions (CDRs) from the disclosed antibodies are expressed on the surface of another protein, such as a scaffold protein. Expression of antibody domains on the surface of a scaffold protein is known in the art (see, for example, Liu et al., J. Virology 85(17):8467-8476, 2011). Such expression creates a chimeric protein that retains binding to EGFRvIII. In some specific embodiments, one or more heavy chain CDRs, such as one or more of heavy chain CDR1, CDR2, and / or CDR3, are grafted onto a scaffold protein. One or more CDRs may be included in a diabody or another type of single-chain antibody molecule.

[0187] (d) Variant In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletion and / or insertion and / or substitution of residues in the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct retains the desired characteristics, for example, antigen binding.

[0188] In some embodiments, antibody variants are provided that have one or more amino acid substitutions.The sites of interest for substitution mutagenesis include CDR and framework regions.Amino acid substitutions can be introduced into the antibody of interest, and the product can be screened for desired activity, such as maintaining / improving antigen binding, reducing immunogenicity, or improving ADCC or CDC.

[0189] Variants typically lack the correct folding and V H Area and V L The amino acid residues necessary for stabilization between the V and V domains are retained, and the charge characteristics of the residues are retained to preserve the low pI and low toxicity of the molecule. H Area and V L Conservative amino acid substitution tables providing functionally similar amino acids are well known to those skilled in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another: (1) alanine (A), serine (S), threonine (T); (2) aspartic acid (D), glutamic acid (E); (3) asparagine (N), glutamine (Q); (4) arginine (R), lysine (K); (5) isoleucine (I), leucine (L), methionine (M), valine (V); and (6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0190] In some embodiments, the heavy chain of the antibody comprises up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NOs: 1, 11, 13, 15, 17, 26, 29, 39, 43, or 53. In some embodiments, the light chain of the antibody comprises up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NOs: 2, 12, 14, 16, 30, 40, 44, or 54.

[0191] In some embodiments, the antibody or antigen-binding fragment may contain up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) in the framework regions of the antibody heavy chain, and / or the antibody light chain, or the heavy and light chains of the antibody, compared to a known framework region or compared to the framework regions of an antibody disclosed herein, and retain specific binding activity to an EGFRvIII protein.

[0192] In certain embodiments, substitution, insertion, or deletion can be present in one or more CDRs, as long as such changes do not substantially reduce the antigen-binding ability of the antibody.For example, conservative changes (such as those provided herein) that do not substantially reduce binding affinity can be made in CDRs.In certain embodiments of the variant VH sequence and variant VL sequence provided above, each CDR is unchanged or contains no more than one, no more than two, or no more than three amino acid substitutions.

[0193] To increase the binding affinity of the antibody, V-CDRs can be introduced, for example, in the H-CDR3 or L-CDR3 regions, in a process similar to the in vivo somatic mutation process responsible for affinity maturation of antibodies in natural immune responses. L Segment and V H The V segment can be randomly mutated using PCR primers complementary to the H-CDR3 or L-CDR3, respectively. H Area and V L In vitro affinity maturation can be achieved by amplifying the region. In this process, the resulting PCR product is H and / or V L V with random mutations introduced into the CDR3 region of H Segment and V L A random mixture of four nucleotide bases is "spiked" into the primer at several positions to encode the V segment. H Segment and V L The segments can be tested to determine their binding affinity to EGFRvIII. Methods for in vitro affinity maturation are known (see, for example, Chowdhury, Methods Mol. Biol. 207:179-196 (2008) and Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)).

[0194] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis, as described by Cunningham and Wells (1989) Science, 244:1081-1085, is called "alanine scanning mutagenesis." In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody's interaction with the antigen 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 the antigen. Such contact residues and neighboring residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they contain the desired properties.

[0195] In certain embodiments, the antibody or antigen-binding fragment may be altered to increase or decrease the degree of glycosylation of the antibody or antigen-binding fragment. Addition or deletion of glycosylation sites may be conveniently accomplished by altering the amino acid sequence so that one or more glycosylation sites are created or removed.

[0196] If an antibody contains an Fc region, the carbohydrate attached thereto can be altered. Native antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides, generally N-linked to Asn297 in the CH2 domain of the Fc region. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in an antibody can be made to generate antibody variants with certain improved properties.

[0197] In one embodiment, antibody variants are provided that lack fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycans attached to Asn297 (e.g., complex, hybrid, and high-mannose structures) as measured by MALDI-TOF mass spectrometry, for example, as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region; however, due to minor antibody sequence variations, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function. See, for example, U.S. Patent Publication Nos. US 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US ​​2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lec 13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US 2003 / 0157108 A1 (Presta, L); and WO 2004 / 056312 A1 (Adams et al.), in particular, Example 11), and knockout cell lines such as alpha-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0198] Further provided are antibody variants comprising bisected oligosaccharides, for example, where the biantennary oligosaccharide attached to the Fc region of the antibody comprises a bisecting GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Also provided are antibody variants comprising at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0199] In some embodiments, the constant region of the antibody contains one or more amino acid substitutions to optimize the in vivo half-life of the antibody. The serum half-life of IgG Abs is controlled by the neonatal Fc receptor (FcRn). Thus, in some embodiments, the antibody contains amino acid substitutions that increase binding to FcRn. Substitutions in the IgG constant region include T250Q and M428L (see, e.g., Hinton et al., J Immunol., 176:346-356, 2006); M428L and N434S ("LS" mutations, see, e.g., Zalevsky, et al., Nature Biotechnology, 28:157-159, 2010); N434A (see, e.g., Petkova et al., Int. Immunol., 18:1759-1769, 2006); T307A, E380A, and N434A (see, e.g., Petkova et al., Int. Immunol., 18:1759-1769, 2006); and M252Y, S254T, and T256E (see, e.g., Dall'Acqua et al., J Immunol., 176:346-356, 2006). Some such substitutions are known to those of skill in the art (see, for example, J. Biol. Chem., 281:23514-23524, 2006).

[0200] In some embodiments, the constant region of the antibody contains one or more amino acid substitutions to optimize antibody-dependent cellular cytotoxicity (ADCC). ADCC is primarily mediated through a set of closely related Fcγ receptors. In some embodiments, the antibody contains one or more amino acid substitutions that increase binding to FcγRIIIa. Some such substitutions are known to those skilled in the art, such as substitutions in IgG constant regions, S239D and I332E (see, e.g., Lazar et al., Proc. Natl., Acad. Sci. USA, 103:4005-4010, 2006); and S239D, A330L, and I332E (see, e.g., Lazar et al., Proc. Natl., Acad. Sci. USA, 103:4005-4010, 2006).

[0201] Combinations of the above substitutions may be included to generate an IgG constant region with increased binding to FcRn and FcγRIIIa. Combinations increase the half-life and ADCC of the antibody. For example, such combinations include antibodies with the following amino acid substitutions in the Fc region: (1) S239D / I332E and T250Q / M428L; (2) S239D / I332E and M428L / N434S; (3) S239D / I332E and N434A; (4) S239D / I332E and T307A / E380A / N434A; (5) S239D / I332E and M252Y / S254T / T. 256E; (6) S239D / A330L / I332E and T250Q / M428L; (7) S239D / A330L / I332E and M428L / N434S; (8) S239D / A330L / I332E and N434A; (9) S239D / A330L / I332E and T307A / E380A / N434A; or (10) S239D / A330L / I332E and M252Y / S254T / T256E.

[0202] In some instances, antibodies or antigen-binding fragments thereof are modified to be directly cytotoxic to infected cells or to utilize natural defenses such as complement, antibody-dependent cellular cytotoxicity (ADCC), or phagocytosis by macrophages.

[0203] In certain embodiments, the antibodies provided herein can be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. Suitable moieties for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, propropylene glycol homopolymer, prolypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have manufacturing advantages due to its stability in water. The polymers can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and when multiple polymers are attached, they can be the same molecule or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used therapeutically under defined conditions, etc.

[0204] The antibody or antigen-binding fragment may be derivatized or linked to another molecule (such as another peptide or protein). Generally, the antibody or antigen-binding fragment is derivatized so that the derivatization or labeling does not adversely affect the binding to EGFRvIII. For example, the antibody or antigen-binding fragment may be functionally linked (by chemical coupling, genetic fusion, non-covalent association, or other methods) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or diabody), a detectable marker, an effector molecule, or a protein or peptide that can mediate the association of the antibody or a portion of the antibody with another molecule (such as a streptavidin core region or a polyhistidine tag).

[0205] Also included are antibodies that bind to the same epitope on EGFRvIII as the disclosed antibodies provided herein. 287-302 Antibodies that bind to such epitopes on the loops can be identified based on their ability to compete with (e.g., competitively inhibit binding in a statistically significant manner) the antibodies provided herein in binding assays (such as those described in the Examples). 6 ×K d In the presence of a competing antibody at a higher concentration of the competing antibody, the EGFR 287-302 In certain embodiments, an antibody "competes" for binding to the same EGFR loop as an antibody of the present disclosure if it inhibits loop binding by more than 50%. 287-302 Antibodies that bind to epitopes on the loops are human monoclonal antibodies. Such human monoclonal antibodies can be prepared and isolated as described herein.

[0206] B. Conjugates Human monoclonal antibodies or antigen-binding fragments thereof specific for EGFRvIII can be conjugated to agents such as effector molecules or detectable markers using a variety of methods known to those skilled in the art. Both covalent and non-covalent attachment methods can be used. Conjugates include, but are not limited to, molecules in which an effector molecule or detectable marker is covalently bonded to an antibody or antigen-binding fragment that specifically binds to EGFRvIII. Chemotherapeutic agents, anti-angiogenic agents, toxins, 125 I, 32 P, 14 C. 3 H, and 35 Those skilled in the art will appreciate that a variety of effector molecules and detectable markers may be used, including, but not limited to, radioactive agents such as S, as well as other labels, targeting moieties, and ligands.

[0207] The choice of a particular effector molecule or detectable marker will depend on the particular target molecule or target cell and the desired biological effect. Thus, for example, the effector molecule can be a cytotoxin used to bring about the death of a particular target cell (such as a tumor cell).

[0208] Effector molecules and detectable markers can be linked to the antibody or antigen-binding fragment of interest using numerous means known to those skilled in the art. Both covalent and non-covalent attachment means can be used. The method for attaching an effector molecule or detectable marker to an antibody or antigen-binding fragment varies depending on the chemical structure of the effector. Polypeptides typically contain various functional groups, such as carboxylic acid (COOH) groups, free amine (-NH) groups, or sulfhydryl (-SH) groups, available for reaction with appropriate functional groups on the antibody, resulting in the attachment of the effector molecule or detectable marker. Alternatively, the antibody or antigen-binding fragment can be derivatized to expose or attach additional reactive functional groups. Derivatization can include the attachment of numerous known linker molecules, such as those available from Pierce Chemical Company (Rockford, IL). The linker can be any molecule used to conjugate an antibody or antigen-binding fragment with an effector molecule or detectable marker. The linker can form a covalent bond with both the antibody or antigen-binding fragment and the effector molecule or detectable marker. Suitable linkers are known to those skilled in the art and include, but are not limited to, linear or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers. When the antibody or antigen-binding fragment and the effector molecule or detectable marker are polypeptides, the linker may be bonded to a constituent amino acid through a side chain (e.g., to cysteine ​​through a disulfide bond) or to the amino and carboxyl groups of the alpha carbon of the terminal amino acid.

[0209] Additionally, in some embodiments, the linker may comprise a spacer element, which, when present, increases the size of the linker so as to increase the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment. Exemplary spacers are known to those of skill in the art and are described in U.S. Patent Nos. 7,964,5667, 498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,700, and the like, each of which is incorporated herein by reference in its entirety. 25, 5,530,097, 5,521,284, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444, and 4,486,414, as well as those listed in U.S. Patent Publication Nos. 20110212088 and 20110070248.

[0210] Thus, in some embodiments, the conjugate comprises a linker connecting the effector molecule or detectable marker to the EGFRvIII-specific antibody or antigen-binding fragment thereof. In some embodiments, the linker is cleavable under intracellular conditions such that cleavage of the linker releases the effector molecule or detectable marker from the antibody or antigen-binding fragment in the intracellular environment. In still other embodiments, the linker is not cleavable, and the effector molecule or detectable marker is released, for example, by antibody degradation. In some embodiments, the linker is cleavable by a cleaving agent present in the intracellular environment (e.g., lysosomes, endosomes, or caveolae). The linker can be a peptide linker that is cleaved by intracellular enzyme peptidases or proteases, including, but not limited to, lysosomal or endosomal proteases. In some embodiments, the peptide linker is at least two amino acids long or at least three amino acids long. However, the linker can be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length, e.g., 1-2, 1-3, 2-5, 3-10, 3-15, 1-5, 1-10, or 1-15 amino acids in length. Proteases include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives within target cells, resulting in the release of the active drug (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). For example, a peptide linker cleavable by the thiol-dependent protease, cathepsin B, can be used (e.g., a phenylalanine-leucine or glycine-phenylalanine-leucine-glycine linker). Other examples of such linkers are described, for example, in U.S. Patent No. 6,214,345, incorporated herein by reference.In specific embodiments, the peptide linker cleavable by an intracellular protease is a valine-citrulline linker or a phenylalanine-lysine linker (see, e.g., U.S. Patent No. 6,214,345, which describes the synthesis of doxorubicin containing a valine-citrulline linker).

[0211] In other embodiments, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at a certain pH value. Typically, pH-sensitive linkers are hydrolyzable under acidic conditions. For example, acid-labile linkers (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketals, etc.) that are hydrolyzable in lysosomes can be used (see, for example, U.S. Patent Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661). Such linkers are relatively stable under neutral pH conditions, such as in blood, but are unstable at pH levels below 5.5 or 5.0, which is approximately the pH of lysosomes. In certain embodiments, the hydrolyzable linker is a thioether linker, such as, for example, a thioether attached to the therapeutic agent via an acylhydrazone bond (see, eg, US Pat. No. 5,622,929).

[0212] In still other embodiments, the linker is cleavable under reducing conditions (e.g., a disulfide linker). A variety of disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene), SPDB, and SMPT (see, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugate: Antibody Conjugates in Radioimagery and Therapy of Cancer (C.W. Vogel ed., Oxford U. Press, 1987); Phillips et al., Cancer Res. 68:92809290, 2008. See also U.S. Patent No. 4,880,935.

[0213] In yet other specific embodiments, the linker is a malonic acid linker (Johnson et al., 1995, AntiCancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).

[0214] In still other embodiments, the linker is not cleavable and the effector molecule or detectable marker is released by antibody degradation (see U.S. Publication No. 2005 / 0238649, incorporated herein by reference in its entirety).

[0215] In some embodiments, the linker is resistant to cleavage in an extracellular environment.For example, when the conjugate is present in an extracellular environment (e.g., in plasma), about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 3% or less, or about 1% or less of the linker in the sample of the conjugate is cleaved.Whether the linker is resistant to cleavage in an extracellular environment can be determined, for example, by incubating the conjugate containing the linker of interest with plasma for a predetermined period (e.g., 2, 4, 8, 16, or 24 hours), and then quantifying the amount of effector molecule or detectable marker present in plasma.A variety of exemplary linkers that can be used in the conjugate are described in WO 2004-010957, US Publication No. 2006 / 0074008, US Publication No. 2005 / 0238649, and US Publication No. 2006 / 0024317, each of which is incorporated herein by reference in its entirety.

[0216] The antibodies or antigen-binding fragments disclosed herein can be derivatized, for example, by crosslinking two or more antibodies (of the same type or of different types, e.g., to create bispecific antibodies). Suitable crosslinkers include heterobifunctional, having two different reactive groups separated by a suitable spacer (such as m-maleimidobenzoyl-N-hydroxysuccinimide ester), or homobifunctional (such as disuccinimidyl suberate). Such linkers are commercially available.

[0217] Given the numerous reported methods for attaching a variety of radiodiagnostic and radiotherapeutic compounds, labels (such as enzymes or fluorescent molecules), toxins, and other agents to antibodies, one of skill in the art would be able to determine an appropriate method for attaching a given agent to an antibody or antigen-binding fragment or other polypeptide. For example, an antibody or antigen-binding fragment can be conjugated to a low-molecular-weight drug, such as monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), maytansine, a maytansine derivative, e.g., the maytansine derivative known as DM1 (also known as mertansine), or other chemotherapeutic agents, to create an antibody-drug conjugate (ADC). In some embodiments, the various chemotherapeutic agents described herein can be conjugated to a provided antibody to generate a conjugate.

[0218] In some embodiments, conjugates of an antibody or antigen-binding fragment and one or more small molecule toxins, such as calicheamicin, maytansinoids, dolastatins, auristatins, trichothecenes, and CC1065, and derivatives of these toxins that have toxin activity, are provided.

[0219] Maytansine compounds suitable for use as maytansinoid toxin moieties are available and may be isolated from natural sources according to known methods, produced using genetic engineering techniques (see Yu et al. (2002) PNAS 99:7968-7973), or maytansinol and maytansinol analogs prepared synthetically according to known methods. Maytansinoids are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from the East African shrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, certain microorganisms were also found to produce maytansinol and maytansinoids, such as C-3 maytansinol esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol and its derivatives and analogs are described, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308, 269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533. Conjugates containing maytansinoids, methods for making them, and therapeutic uses thereof are disclosed, for example, in U.S. Pat. Nos. 5,208,020; 5,416,064; and 6,441,163, and European Patent EP 0 425 235 B1, the disclosures of which are specifically incorporated herein by reference.

[0220] In one example, the conjugate comprises a monoclonal antibody (or antigen-binding fragment thereof) that specifically binds to EGFRvIII, a non-reducible thioester linker, and a maytansinoid toxin DM1; for example, the conjugate may comprise the structure shown below ("mAb" refers to a monoclonal antibody or antigen-binding fragment thereof): TIFF0007815099000039.tif55138

[0221] In some embodiments, the effector molecule is an auristatin, such as auristatin E (also known in the art as a derivative of dolastatin-10) or a derivative thereof. The auristatin can be, for example, an ester formed between auristatin E and a keto acid. For example, auristatin E can be reacted with paraacetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other exemplary auristatins include AFP, MMAF, and MMAE. The synthesis and structures of exemplary auristatins are described in U.S. Patent Application Publication No. 2003 / 0083263; International Patent Publication No. WO 04 / 010957; International Patent Publication No. WO 04 / 010958; and U.S. Pat. Appl. Pub. No. 2004 / 010959, each of which is incorporated herein by reference in its entirety. 02 / 088172, as well as U.S. Patent Nos. 7,498,298; 6,884,869; 6,323,315; 6,239,104; 6,034,065; 5,780,588; 5,665,860; 5,663,149; 5,635,483; 5,599,902; 5,554,7 25; 5,530,097; 5,521,284; 5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,816,444; and 4,486,414. Additional description of antibody-drug conjugates containing auristatin MMAE and methods of making such conjugates is provided, for example, in U.S. Publication Nos. 2011 / 0268751, 2008 / 0305044, and 2007 / 0258987, each of which is incorporated by reference in its entirety. Auristatins have been shown to interfere with microtubule dynamics and nuclear and cellular division, and have anti-cancer activity. Auristatins can bind to tubulin and exert cytotoxic or cytostatic effects on cells. There are many different assays known in the art for determining whether an auristatin or the resulting conjugate exerts cytostatic or cytotoxic effects on a desired cell line.

[0222] In one example, the conjugate includes a monoclonal antibody (or antigen-binding fragment thereof) that specifically binds to EGFRvIII, a cleavable linker comprising a valine-citrulline (Val-Cit) peptide cleavable site, a spacer, and the toxin MMAE; for example, the conjugate may include the structure shown below ("mAb" refers to a monoclonal antibody or antigen-binding fragment thereof): TIFF0007815099000040.tif35162

[0223] In one preferred embodiment, the conjugate comprises: TIFF0007815099000041.tif38153 (where n is an integer between 0 and 10 (such as an even number) (e.g., 0 to 8, 0 to 4, 2 to 4, 2 to 8, 1 to 10, 1 to 8, or 1 to 4, or 2, 4, 6, or 8); A is a monoclonal antibody or antigen-binding fragment thereof disclosed herein; and S is a sulfur atom derived from the antibody). In one embodiment, preferably, n is an even number between 0 and 8, preferably between 0 and 4. The S moiety can be exposed by reduction or partial reduction of the interchain disulfides of the antibody (e.g., by treatment with a reducing agent such as DTT or TCEP).

[0224] In one non-limiting embodiment, the conjugate comprises: TIFF0007815099000042.tif38153, where n is 4 and A is a monoclonal antibody or antigen-binding fragment thereof disclosed herein.

[0225] Additional toxins may be utilized in conjunction with antibodies that specifically bind to EGFRvIII and antigen-binding fragments of these antibodies. Exemplary toxins include Pseudomonas exotoxin (PE), ricin, abrin, diphtheria toxin and its subunits, ribotoxin, ribonuclease, saporin, and calicheamicin, as well as botulinum toxins A-F. These toxins are well known in the art, and many are readily available from commercial sources (e.g., Sigma Chemical Company, St. Louis, MO). Contemplated toxins also include toxin variants (see, e.g., US Patent Nos. 5,079,163 and 4,689,401). In some embodiments, these conjugates are useful for the treatment of cancer, such as head and neck cancer, breast cancer, or bladder cancer.

[0226] Saporin is a toxin derived from the soap plant Saponaria officinalis that disrupts protein synthesis by inactivating the 60S portion of the ribosomal complex (Stirpe et al., Bio / Technology, 10:405-412, 1992). However, this toxin lacks a mechanism for specific cell entry and therefore requires conjugation with an antibody or antigen-binding fragment that recognizes an internalized cell surface protein for efficient uptake by cells.

[0227] Diphtheria toxin is isolated from Corynebacterium diphtheriae. Typically, diphtheria toxin for use in immunotoxins is mutated to reduce or eliminate nonspecific toxicity. A mutant known as CRM107, which has full enzymatic activity but significantly reduced nonspecific toxicity, has been known since the 1970s (Laird and Groman, J. Virol. 19:220, 1976) and has been used in human clinical trials. See U.S. Patent Nos. 5,792,458 and 5,208,021.

[0228] Ricin is the lectin RCA60 derived from the castor bean (Ricinus communis). For examples of ricin, see U.S. Patent Nos. 5,079,163 and 4,689,401. Castor bean agglutinin (RCA) has a molecular weight of approximately 65 kD and 120 kD, respectively, making it a popular choice for RCA. 60 and RCA 120 The A chain is responsible for inactivating protein synthesis and killing the cell (Nicholson & Blaustein, J. Biochim. Biophys. Acta 266:543, 1972). The B chain binds lysine to cell surface galactose residues, facilitating transport of the A chain into the cytosol (Olsnes et al., Nature 249:627-631, 1974 and U.S. Patent No. 3,060,165).

[0229] Ribonucleases have also been conjugated with targeting molecules for use as immunotoxins (see Suzuki et al., Nat. Biotech. 17:265-70, 1999). Exemplary ribonucleases, such as α-sarcin and restrictocin, are described, for example, in Rathore et al., Gene 190:31-5, 1997; and Goyal and Batra, Biochem. 345 Pt 2:247-54, 2000. Calicheamicin, originally isolated from Micromonospora echinospora, is a member of the enediyne antitumor antibiotic family that induces double-strand breaks in DNA, leading to apoptosis (see, for example, Lee et al., J. Antibiot. 42:1070-87, 1989). This drug is the toxic moiety of an immunotoxin in clinical trials (see, eg, Gillespie et al., Ann. Oncol. 11:735-41, 2000).

[0230] Abrin includes toxic lectins derived from the jequirity bean (Abrus precatorius). The toxic components, abrins a, b, c, and d, have molecular weights of approximately 63-67 kD and are composed of two polypeptide chains, A and B, linked by disulfides. The A chain inhibits protein synthesis; the B chain (abrin-b) binds D-galactose residues (see Funatsu et al., Agr. Biol. Chem. 52:1095, 1988; and Olsnes, Methods Enzymol. 50:330-335, 1978).

[0231] In one embodiment, the toxin is Pseudomonas exotoxin (PE) (U.S. Patent No. 5,602,095). As used herein, PE includes full-length native (naturally occurring) PE or modified PE. Such modifications may include, but are not limited to, elimination of domain Ia, various amino acid deletions in domains Ib, II, and III, single amino acid substitutions, and the addition of one or more sequences at the carboxyl terminus (see, e.g., Siegall et al., J. Biol. Chem. 264:14256-14261, 1989). PE utilized with the provided antibodies may include the native sequence, cytotoxic fragments of the native sequence, and conservatively modified variants of native PE and their cytotoxic fragments. Cytotoxic fragments of PE include those that become cytotoxic through or without subsequent proteolytic or other processing in the target cell. Cytotoxic fragments of PE include PE25, PE40, PE38, and PE35. For additional description of PE and its variants, see, e.g., U.S. Patent Nos. 4,892,827; 5,512,658; 5,602,095; 5,608,039; 5,821,238; and 5,854,044; PCT Publication No. WO 99 / 51643; Pai et al., Proc. Natl. Acad. Sci. USA, 88:3358-3362, 1991; Kondo et al., J. Biol. Chem., 263:9470-9475, 1988; Pastan et al., Biochim. Biophys. Acta, 1333:C1-C6, 1997.

[0232] Protease-resistant PE variants and PE variants with reduced immunogenicity, such as, but not limited to, PE-LR, PE-6X, PE-8X, PE-LR / 6X, and PE-LR / 8X, are also contemplated herein (see, e.g., Weldon et al., Blood 113(16):3792-3800, 2009; Onda et al., Proc. Natl. Acad. Sci. USA, 105(32):11311-11316, 2008; and PCT Publication Nos. WO 2007 / 016150, WO 2009 / 032954, and WO 2011 / 032022, which are incorporated herein by reference). The PE variant can be PE25 (see Weldon et al., Blood 2009;113:3792-3800, incorporated herein by reference).

[0233] In some instances, the PE is a variant that is resistant to lysosomal degradation, such as PE-LR (Weldon et al., Blood 113(16):3792-3800, 2009; PCT Publication No. WO 2009 / 032954). In other instances, the PE is a variant designated PE-LR / 6X (PCT Publication No. WO 2011 / 032022). In other instances, the PE is a variant designated PE-LR / 8M (PCT Publication No. WO 2011 / 032022).

[0234] A monoclonal antibody (or antigen-binding fragment thereof) that specifically binds to EGFRvIII may be conjugated with a detectable marker; for example, a detectable marker that can be detected by imaging diagnostic techniques such as ELISA, spectrophotometry, flow cytometry, microscopy, or imaging diagnostic techniques (such as computed tomography (CT), computerized axial tomography (CAT) scan, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance tomography (MTR), ultrasound, fiberoptic examination, and laparoscopy). Specific non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzyme linkages, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide for detection by MRI). For example, useful detectable markers include fluorescent compounds such as fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, etc. Bioluminescent markers such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP) are also useful. Antibodies or antigen-binding fragments may be conjugated to enzymes useful for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, and the like. When antibodies or antigen-binding fragments are conjugated to detectable enzymes, they can be detected by adding additional reagents that the enzyme uses to generate a discernible reaction product. For example, in the presence of the agent horseradish peroxidase, the addition of hydrogen peroxide and diaminobenzidine results in a visually detectable colored reaction product. Antibodies or antigen-binding fragments may be conjugated to biotin and detected through indirect measurement of avidin or streptavidin binding. It should be noted that avidin itself may be conjugated to an enzyme or fluorescent label.

[0235] Antibodies or antigen-binding fragments may be conjugated with superparamagnetic agents such as gadolinium. Superparamagnetic agents such as superparamagnetic iron oxide are also useful as labels. Antibodies may be conjugated with lanthanides (such as europium and dysprosium) and manganese. Antibodies or antigen-binding fragments may be labeled with a predetermined polypeptide epitope recognized by a secondary reporter (such as a leucine zipper pair sequence, a binding site for a secondary antibody, a metal binding domain, or an epitope tag).

[0236] The antibody or antigen-binding fragment may be conjugated with a radiolabeled amino acid. Radiolabels can be used for both diagnostic and therapeutic purposes. For example, radiolabels can be used to detect EGFRvIII and EGFRvIII-expressing cells by x-ray, radiation spectroscopy, or other diagnostic techniques. Furthermore, radiolabels can be used therapeutically as toxins to treat tumors in subjects, for example, cancers, such as head and neck cancer, breast cancer, or bladder cancer, that express EGFRvIII. Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionucleotides: 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I.

[0237] Means for detecting such detectable markers are known to those skilled in the art. Thus, for example, radiolabels can be detected using photographic film or scintillation counters, fluorescent markers can be detected using a photodetector to detect emitted light, enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.

[0238] The antibody or antigen-binding fragment may be derivatized with chemical groups such as polyethylene glycol (PEG), methyl or ethyl groups, or carbohydrate groups. These groups may be useful to improve the biological characteristics of the antibody or antigen-binding fragment, for example, to increase serum half-life or to increase tissue binding.

[0239] The average number of effector molecules or detectable marker moieties for each antibody or antigen-binding fragment in a conjugate can range, for example, from 1 to 20 moieties for each antibody or antigen-binding fragment. In some conjugates, the average number of effector molecules or detectable marker moieties for each antibody or antigen-binding fragment can be limited by the number of attachment sites on the antibody or antigen-binding fragment. For example, if the attachment is by cysteine ​​thiol, the antibody or antigen-binding fragment may have only one or a few cysteine ​​thiol groups, or may have only one or a few sufficiently reactive thiol groups to which a linker can be attached. In certain embodiments, the average number of effector molecules or detectable marker moieties for each antibody or antigen-binding fragment in the conjugate ranges from 1 to about 8; from about 2 to about 6; from about 3 to about 5; from about 3 to about 4; from about 3.1 to about 3.9; from about 3.2 to about 3.8; from about 3.2 to about 3.7; from about 3.2 to about 3.6; from about 3.3 to about 3.8; or from about 3.3 to about 3.7. See, e.g., U.S. Patent No. 7,498,298, incorporated herein by reference in its entirety. The average number of effector molecules or detectable marker moieties for each antibody or antigen-binding fragment in a conjugate preparation can be characterized by conventional means, such as mass spectrometry and ELISA assays. The loading of the conjugate (e.g., effector molecule / antibody ratio) can be controlled in various ways, for example, by (i) limiting the molar excess of effector molecule-linker intermediate or linker reagent relative to antibody, (ii) limiting the time or temperature of the conjugation reaction, (iii) partial or limited reducing conditions for cysteine ​​thiol modification, or (iv) recombinantly modifying the amino acid sequence of the antibody so that the number and position of cysteine ​​residues are modified to control the number or position of linker-effector molecule attachments (e.g., thioMabs or thioFabs prepared as disclosed in WO2006 / 03448, which is incorporated herein by reference in its entirety).

[0240] C. Chimeric Antigen Receptor (CAR) Also disclosed herein is a chimeric antigen receptor (CAR), an artificially constructed chimeric protein comprising an extracellular antigen-binding domain (e.g., a single-chain variable fragment (scFv)) that specifically binds to EGFRvIII, linked to a transmembrane domain, linked to one or more intracellular T cell signaling domains. Features of the disclosed CAR include the ability to redirect T cell specificity and reactivity to EGFRvIII-expressing cells in a non-MHC-restricted manner. Non-MHC-restricted EGFRvIII recognition confers the ability to recognize antigens independently of antigen processing to T cells expressing the disclosed CAR.

[0241] The intracellular T cell signaling domain can include, for example, a T cell receptor signaling domain, a T cell costimulatory signaling domain, or both.The T cell receptor signaling domain refers to a portion of CAR that includes the intracellular domain of a T cell receptor, such as the intracellular portion of the CD3 zeta protein.The costimulatory signaling domain refers to a portion of CAR that includes the intracellular domain of a costimulatory molecule, which is a cell surface molecule other than an antigen receptor or its ligand, that is required for the efficient response of lymphocytes to antigens.

[0242] 1. Extracellular region Some embodiments provide a CAR comprising an antigen-binding domain that specifically binds to EGF VIII as disclosed herein. For example, the antigen-binding domain can be an scFv comprising the heavy chain variable region and the light chain variable region of the antibody or its antigen-binding fragment disclosed above.

[0243] In some embodiments, the antigen-binding domain may comprise the heavy and light chain variable regions, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3, respectively, of one of the 40H3, 1D9, 3D10, 4A4, 9G3, 11E11, or 11G3 antibodies (see supra). For example, the antigen-binding domain may comprise the HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3, of SEQ ID NOs: 1 and 2, SEQ ID NOs: 11 and 12, SEQ ID NOs: 13 and 14, or SEQ ID NOs: 15 and 16. The antigen-binding domain may comprise HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 17 and 12, SEQ ID NOs: 26 and 12, SEQ ID NOs: 29 and 30, SEQ ID NOs: 39 and 40, SEQ ID NOs: 43 and 44, or SEQ ID NOs: 53 and 54. Any of the antibodies or antigen-binding fragments disclosed herein can be used in CARs. In one embodiment, the antibody or antigen-binding fragment is humanized.

[0244] In some embodiments, the antigen binding domain can be an scFv. In some embodiments, the scFv comprises: It comprises a heavy chain variable region and a light chain variable region joined by a peptide linker, such as a linker comprising the amino acid sequence shown as TIFF0007815099000043.tif5128.

[0245] The CAR may comprise a signal peptide sequence, for example, at the N-terminus of the antigen-binding domain. The signal peptide sequence may comprise any suitable signal peptide sequence. In one embodiment, the signal peptide sequence is The amino acid sequence of the CAR may be a human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor sequence, such as the amino acid sequence comprising or consisting of TIFF0007815099000044.tif5128. Although the signal peptide sequence can facilitate the expression of the CAR on the cell surface, the presence of the signal peptide sequence in the expressed CAR is not necessary for the CAR to function. Upon expression of the CAR on the cell surface, the signal peptide sequence can be cleaved from the CAR. Thus, in some embodiments, the CAR lacks a signal peptide sequence.

[0246] Between the antigen-binding domain and the transmembrane domain of the CAR, there may be a spacer domain comprising a polypeptide sequence. The spacer domain may comprise up to 300 amino acids, preferably 10-100 amino acids, and most preferably 25-50 amino acids. In some embodiments, the spacer domain may comprise an immunoglobulin domain, such as a human immunoglobulin sequence. In one embodiment, the immunoglobulin domain comprises the CH2 and CH3 domain sequence (CH2CH3) of immunoglobulin G (IgG1). In this regard, the spacer domain may comprise a sequence similar to SEQ ID NO:61: It may comprise an immunoglobulin domain comprising or consisting of the amino acid sequence shown as TIFF0007815099000045.tif24160.

[0247] Without being bound by any particular theory, it is believed that the CH2CH3 domain may distance the antigen-binding domain of the CAR from the membrane of the CAR-expressing cell, more accurately mimicking the size and domain structure of the native TCR.

[0248] 2. Transmembrane domain Regarding the transmembrane domain, the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain that is naturally associated with one of the domains in the CAR is used.

[0249] The transmembrane domain can be derived from natural or synthetic origin.When the origin is natural, the domain can be derived from membrane-bound or transmembrane protein.Exemplary transmembrane domains for use in the disclosed CAR can include at least the transmembrane region of the alpha chain, beta chain, or zeta chain of T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.Alternatively, the transmembrane domain can be synthetic, and in this case, it will mainly contain hydrophobic residues such as leucine and valine.In some embodiments, a triplet of phenylalanine, tryptophan, and valine will be found at each end of the synthetic transmembrane domain.

[0250] Optionally, a short oligopeptide or polypeptide linker, preferably 2-10 amino acids in length, can form the link between the transmembrane domain of the CAR and the intracellular T cell signaling domain and / or T cell costimulatory domain. Exemplary linker sequences include one or more glycine-serine doublets.

[0251] In some embodiments, the transmembrane domain comprises a T cell receptor transmembrane domain, such as a CD8 transmembrane domain. Thus, the CAR comprises SEQ ID NO:62: TIFF0007815099000046.tif11158. In another embodiment, the transmembrane domain comprises the transmembrane domain of a T cell costimulatory molecule, such as CD137 or CD28. Thus, the CAR may comprise a CD8 transmembrane domain comprising or consisting of SEQ ID NO:63: It may comprise a CD28 transmembrane domain comprising or consisting of TIFF0007815099000047.tif4158.

[0252] 3. Intracellular region The intracellular region of the CAR comprises one or more intracellular T cell signaling domains that are responsible for activating at least one of the normal effector functions of the T cell in which the CAR is expressed or placed. Exemplary T cell signaling domains are provided herein and known to those of skill in the art.

[0253] Although the entire intracellular T cell signaling domain may be used in a CAR, in many cases it is not necessary to use the complete chain. If a truncated portion of the intracellular T cell signaling domain is used, such a truncated portion can be used in place of the complete chain as long as it transmits the relevant T cell effector function signal.

[0254] Examples of intracellular T cell signaling domains for use in CARs include cytoplasmic sequences of T cell receptors (TCRs) and costimulatory molecules that act cooperatively to initiate signal transduction after antigen receptor engagement, as well as derivatives or variants of these sequences, and synthetic sequences with the same functional capabilities.

[0255] The T cell receptor signaling domain controls the primary activation of the T cell receptor complex in either a stimulatory or inhibitory manner. The disclosed CARs can include a primary cytoplasmic signaling sequence that acts in a stimulatory manner and can contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of ITAM-containing primary cytoplasmic signaling sequences that can be included in the disclosed CARs include those derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, and CD66d proteins. In some embodiments, the cytoplasmic signaling molecule in the CAR includes an intracellular T cell signaling domain derived from CD3 zeta.

[0256] The intracellular region of the CAR can comprise an ITAM-containing primary cytoplasmic signaling domain (such as CD3-zeta), alone or in combination with other desired cytoplasmic domains useful for the CAR. For example, the cytoplasmic domain of the CAR can comprise a CD3 zeta chain portion and an intracellular costimulatory signaling domain. The costimulatory signaling domain refers to a portion of the CAR that contains the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for efficient lymphocyte response to antigens. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3. An additional example of a signaling domain that can be included in the disclosed CARs is the signaling domain of tumor necrosis factor receptor superfamily member 18 (TNFRSF18; also known as glucocorticoid-inducible TNFR-related protein, GITR).

[0257] In some embodiments, the CAR may comprise a CD3-zeta signaling domain, a CD8 signaling domain, a CD28 signaling domain, a CD137 signaling domain, or a combination of two or more thereof. In one embodiment, the cytoplasmic domain comprises the CD3-zeta signaling domain and the CD28 signaling domain. In another embodiment, the cytoplasmic domain comprises the CD3-zeta signaling domain and the CD137 signaling domain. In yet another embodiment, the cytoplasmic domain comprises the CD3-zeta signaling domain and the CD28 and CD137 signaling domains. The order of one or more T cell signaling domains on the CAR can be varied as needed by those skilled in the art. Exemplary amino acid sequences for such T cell signaling domains are provided. For example, The CD3 zeta signaling domain TIFF0007815099000048.tif17158, The CD8 signaling domain TIFF0007815099000049.tif18157, The CD28 signaling domain TIFF0007815099000050.tif5128, The CD137 signaling domain It may comprise or consist of the amino acid sequence shown as TIFF0007815099000051.tif23136.

[0258] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the present invention may be linked to each other in a random or specified order. Optionally, a short polypeptide linker, preferably 2-10 amino acids in length, may form the linkage. A glycine-serine doublet provides a particularly suitable linker. Additionally, a spacer domain comprising a polypeptide sequence may be present between the signaling domain and the transmembrane domain of the CAR. The spacer domain may comprise up to 300 amino acids, preferably 10-100 amino acids, and most preferably 25-50 amino acids.

[0259] 4. Additional explanation of CAR Also provided are functional portions of the CARs described herein. The term "functional portion," when used in reference to a CAR, refers to a portion or fragment of a CAR that retains the biological activity of the CAR that contains it (the parent CAR). Functional portions include, for example, portions of a CAR that retain the ability to recognize target cells or detect, treat, or prevent disease to a similar, identical, or greater extent than the parent CAR. Based on the parent CAR, a functional portion can, for example, comprise about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more of the parent CAR.

[0260] A CAR or functional portion thereof can include additional amino acids at the amino or carboxy terminus, or at both ends, that are not found in the amino acid sequence of the parent CAR. Desirably, the additional amino acids do not interfere with the biological function of the CAR or functional portion, such as target cell recognition, cancer detection, cancer treatment or prevention, etc. More desirably, the additional amino acids enhance the biological activity compared to the biological activity of the parent CAR.

[0261] Also provided are functional variants of the CARs described herein that have substantial or significant sequence identity or similarity with the parent CAR and retain the biological activity of the CAR from which the variant is derived. Functional variants include, for example, variants of the CARs described herein (parent CARs) that retain the ability to recognize target cells to a similar, identical, or higher degree than the parent CAR. Based on the parent CAR, the functional variant can be, for example, at least about 30%, about 50%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more amino acid sequence identical to the parent CAR.

[0262] A functional variant can, for example, comprise the amino acid sequence of a parent CAR containing at least one conservative amino acid substitution. Alternatively, or in addition, a functional variant can comprise the amino acid sequence of a parent CAR containing at least one non-conservative amino acid substitution. In this case, the non-conservative amino acid substitution preferably does not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution can enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased compared to the parent CAR.

[0263] CARs (including functional portions and functional variants) can be of any length, i.e., contain any number of amino acids, provided that the CAR (or functional portion or functional variant thereof) retains biological activity, such as the ability to specifically bind to an antigen, detect diseased cells in a mammal, or treat or prevent disease in a mammal. For example, a CAR can be about 50 to about 5000 amino acids in length, e.g., 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000 amino acids in length or longer.

[0264] CARs (including functional portions and functional variants of the invention) can contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3-hydroxyproline, trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycerin ... Lysine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, oc-aminocycloheptanecarboxylic acid, -(2-amino-2-norbornane)-carboxylic acid, γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0265] CARs (including functional moieties and functional variants) may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized, e.g., via disulfide bridges, or converted into acid addition salts, and / or optionally dimerized or polymerized or conjugated.

[0266] Chimeric antigen receptors, methods for generating T cells containing such receptors, and their uses (e.g., for the treatment of cancer) are known in the art and are further described herein (e.g., Brentjens et al., 2010, Molecular Therapy, 18:4, 666-668; Morgan et al., 2010, Molecular Therapy, published online February 23, 2010, pages 1-9; Till et al., 2008, Blood, 112:2261-2271; Park et al., Trends Biotechnol., 29:550-557, 2011; Grupp et al., N Engl J Med., 368:1509-1518, 2013; Han et al., J. Hematol., 2013, 1:1509-1518, 2013, each of which is incorporated herein by reference in its entirety). Oncol., 6:47, 2013; PCT Publications WO2012 / 079000, WO2013 / 126726; and U.S. Publication No. 2012 / 0213783). For example, nucleic acid molecules encoding the disclosed chimeric antigen binding receptors can be included in an expression vector (such as a lentiviral vector) for expression in a host cell, such as a T cell, to generate the disclosed CARs. In some embodiments, a method of using a chimeric antigen receptor includes isolating T cells from a subject, transforming the T cells with an expression vector (such as a lentiviral vector) encoding the chimeric antigen receptor, and administering the modified T cells expressing the chimeric antigen receptor to the subject for treatment, e.g., for treatment of a tumor in the subject.

[0267] D. Polynucleotides and Expression Nucleic acids are provided that encode the amino acid sequences of antibodies, antibody-binding fragments, conjugates, and CARs that specifically bind to EGFRvIII.The nucleic acids that encode these molecules can be easily produced by those skilled in the art using the amino acid sequences provided herein (such as CDR sequences, heavy and light chain sequences), sequences available in the art (such as framework sequences), and genetic code.Nucleic acids that encode the same antibody sequence but have different sequences, or V L and / or V H One skilled in the art can readily use the genetic code to construct a variety of functionally equivalent nucleic acids, such as nucleic acids encoding conjugates or fusion proteins comprising the nucleic acid sequence of

[0268] Nucleic acid sequences encoding antibodies, antibody-binding fragments, conjugates, and CARs that specifically bind to EGFRvIII can be prepared by any suitable method, e.g., cloning appropriate sequences or by using the phosphotriester method of Narang et al., Meth. Enzymol. 68:90-99, 1979; the phosphodiester method of Brown et al., Meth. Enzymol. 68:109-151, 1979; the diethylphosphoramidite method of Beaucage et al., Tetra. Lett. 22:1859-1862, 1981; or an automated synthesizer as described, e.g., in Needham-VanDevanter et al., Nucl. Acids Res. 12:6159-6168, 1984, by Beaucage & Co. It can be prepared by direct chemical synthesis using methods such as the solid-phase phosphoramidite triester method described by Caruthers, Tetra. Letts. 22(20):1859-1862, 1981; and the solid support method of U.S. Pat. No. 4,458,066. Chemical synthesis produces single-stranded oligonucleotides, which can be converted into double-stranded DNA by hybridization with a complementary sequence or by polymerization with DNA polymerase using the single strand as a template. Chemical synthesis of DNA is generally limited to sequences of about 100 bases, but those skilled in the art will recognize that longer sequences can be obtained by ligating shorter sequences.

[0269] Exemplary nucleic acids can be prepared by cloning techniques. Examples of suitable cloning and sequencing techniques, as well as instructions sufficient to guide one of skill through many cloning exercises, are known (e.g., Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4 thed., Cold Spring Harbor, New York, 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, through supplement 104, 2013). Product information from manufacturers of biological reagents and laboratory equipment also provides useful information. Such manufacturers include SIGMA Chemical Company (Saint Louis, MO), R&D Systems (Minneapolis, MN), Pharmacia Amersham (Piscataway, NJ), CLONTECH Laboratories, Inc. (Palo Alto, CA), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, WI), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersburg, MD), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen (Carlsbad, CA), and Applied BioSystems (Foster City, CA), as well as many other commercial sources known to those of skill in the art.

[0270] Nucleic acids may be prepared by amplification methods, including polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification systems (TAS), and self-sustained sequence replication systems (3SR). A variety of cloning methods, host cells, and in vitro amplification methodologies are well known to those skilled in the art.

[0271] In some embodiments, the nucleic acid molecule encodes the CAR provided herein for expression in T cells to generate chimeric antigen receptor T cells. The nucleic acid molecule encoding the chimeric antigen binding receptor can be included in a vector (such as a lentiviral vector) for expression in host cells such as T cells. Exemplary cells include T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), and regulatory T cells. Nucleic acid molecules encoding chimeric antigen receptors and methods for generating T cells containing such receptors are known in the art (e.g., Brentjens et al., 2010, Molecular Therapy, 18:4, 666-668; Morgan et al., 2010, Molecular Therapy, published online February 23, 2010, pages 1-9; Till et al., 2008, Blood, 112:2261-2271; Park et al., Trends Biotechnol., 29:550-557, 2011; Grupp et al., N Engl J Med., 368:1509-1518, 2013; Han et al., J. Hematol., 2013, pp. 1111-1112, 2013; each of which is incorporated herein by reference in its entirety). Oncol., 6:47, 2013; PCT Publications WO2012 / 079000, WO2013 / 126726; and U.S. Publication 2012 / 0213783).

[0272] Nucleic acid molecules can be expressed in recombinantly engineered cells, such as bacterial, plant, yeast, insect, and mammalian cells. Antibodies, antigen-binding fragments, and conjugates can be expressed as individual V (optionally linked to an effector molecule or detectable marker) antibodies. H Chain and / or V LThe V may be expressed as a chain or as a fusion protein. Methods for expressing and purifying antibodies and antigen-binding fragments are known and further described herein (see, e.g., Al-Rubeai (ed.), Antibody Expression and Production, Springer Press, 2011). Immunoadhesins may also be expressed. Thus, in some instances, V H and V L , as well as nucleic acids encoding the immunoadhesin. The nucleic acid sequence may optionally encode a leader sequence.

[0273] To create an scFv, V is joined by a flexible linker. L Domains and V H As a continuous single-chain protein containing the V domain, H Array and V L V so that the sequence can be expressed H and V L A DNA fragment encoding the nucleotide sequence (Gly4-Ser) can be operably linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser)3 (see, e.g., Bird et al., Science 242:423-426, 1988; Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988; McCafferty et al., Nature 348:552-554, 1990; Kontermann and Dubel (Eds.), Antibody Engineering, Vols. 1-2, 2002). nd Ed., Springer Press, 2010; Harlow and Lane, Antibodies: A Laboratory Manual, 2 nd , Cold Spring Harbor Laboratory, New York, 2013). Optionally, a cleavage site, such as a furin cleavage site, may be included in the linker.

[0274] V H and / or VL The nucleic acid encoding may optionally encode an Fc domain (immunoadhesin). The Fc domain can be an IgA, IgM, or IgG Fc domain. The Fc domain can be an optimized Fc domain, such as those described in U.S. Published Patent Application No. 2010 / 093979, which is incorporated herein by reference. In one example, the immunoadhesin is an IgG1 Fc.

[0275] Single-chain antibodies contain a single V H and V L If only V is used, it can be monovalent, and if only two V H and V L When used, it can be bivalent and three or more V H and V L When the encoded V is used, it can be multivalent. Bispecific or multivalent antibodies can be generated that specifically bind to EGF R VIII and also specifically bind to another antigen, such as, but not limited to, CD3. H and V L optionally, a furin cleavage site is inserted into V H Domains and V L It may be included between the domain.

[0276] Numerous expression systems are known to those of skill in the art that are available for the expression of proteins, including E. coli, other bacterial hosts, yeast, and various higher eukaryotic cells such as COS, CHO, HeLa, and myeloma cell lines.

[0277] One or more DNA sequences encoding antibodies, antibody-binding fragments, conjugates, and CARs can be expressed in vitro by DNA transfer into suitable host cells.Cells can be prokaryotic or eukaryotic.This term also includes the descendants of the original host cell.It is understood that not all descendants are identical to the parent cell because mutations that occur during replication may exist.Stable transfer methods, i.e., foreign DNA is continuously maintained in the host, are known in the art.Hybridomas that express the antibody of interest are also included in the present disclosure.

[0278] A polynucleotide sequence encoding an antibody or antigen-binding fragment, or a conjugate thereof, can be operably linked to an expression control sequence. The expression control sequence operably linked to the coding sequence is ligated so that expression of the coding sequence is achieved under conditions compatible with the expression control sequence. Expression control sequences include, but are not limited to, an appropriate promoter, enhancer, transcription terminator, a start codon (i.e., ATG) in front of the protein-encoding gene, splicing signals for introns, maintenance of the correct reading frame of the gene to allow proper translation of mRNA, and a stop codon.

[0279] To obtain high-level expression of the cloned gene, it is desirable to construct an expression cassette containing, at a minimum, a strong promoter to direct transcription, a ribosome binding site for translation initiation (internal ribosome binding site), and a transcription / translation terminator. For E. coli, this includes a promoter such as the T7, trp, lac, or lambda promoter, a ribosome binding site, and preferably a transcription termination signal. For eukaryotic cells, regulatory sequences may include, for example, promoters and / or enhancers derived from immunoglobulin genes, HTLV, SV40, or cytomegalovirus, as well as polyadenylation sequences, and may further include splice donor and / or acceptor sequences (e.g., splice acceptor and donor sequences of CMV and / or HTLV). The cassette can be transferred into the selected host cell by well-known methods, such as transformation or electroporation for E. coli, or calcium phosphate treatment, electroporation, or lipofection for mammalian cells. Cells transformed with the cassette can be selected by antibiotic resistance conferred by genes contained in the cassette, such as the amp, gpt, neo, and hyg genes.

[0280] The polynucleotide sequence encoding the antibody or antigen-binding fragment, or a conjugate thereof, can be inserted into an expression vector, including but not limited to a plasmid, virus, or other vehicle that can be manipulated to allow the insertion or incorporation of the sequence, and expressed in either prokaryotic or eukaryotic cells. Hosts can include microorganisms, yeast, insects, and mammals. Methods for expressing DNA sequences containing eukaryotic or viral sequences in prokaryotic cells are well known in the art. Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art.

[0281] When the host is a eukaryotic cell, conventional mechanical techniques such as DNA transfection methods (e.g., calcium phosphate coprecipitation), microinjection, electroporation, and insertion of liposome-encapsulated plasmids or viral vectors can be used. Eukaryotic cells may be cotransformed with a polynucleotide sequence encoding an antibody, a labeled antibody, or an antigen-binding fragment thereof, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is the use of eukaryotic viral vectors, such as simian virus 40 (SV40) or bovine papillomavirus, to transiently infect or transform eukaryotic cells and express proteins (see, e.g., Viral Expression Vectors, Springer Press, Muzyczka ed., 2011). Those skilled in the art can readily use expression systems, such as plasmids and vectors, useful for producing proteins in cells, including higher eukaryotic cells such as COS, CHO, HeLa, and myeloma cell lines.

[0282] The host cell for producing the recombinant CAR can be a mammalian cell. The host cell can be a human cell. In some embodiments, the host cell can be a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC) or a T cell. The T cell can be any T cell, such as a cultured T cell, e.g., a primary T cell, or a T cell derived from a cultured T cell line, e.g., Jurkat, SupTl, etc., or a T cell obtained from a mammal. When obtained from a mammal, the T cell can be obtained from a number of sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or body fluids. The T cell can be enriched or purified. The T cell can be a human T cell. The T cell can be a T cell isolated from a human. The T cell can be any type of T cell, and can be CD4 + / CD8 + Double positive T cells, CD4 + Helper T cells, such as Th1 and Th2 cells, CD8 + T cells can be at any stage of development, including, but not limited to, T cells (e.g., cytotoxic T cells), tumor-infiltrating cells, memory T cells, naive T cells, etc. T cells can be CD8 + T cells or CD4 + It may be a T cell.

[0283] Populations of cells comprising at least one host cell described herein are also provided. The population of cells can be a heterogeneous population, comprising host cells comprising any of the described recombinant expression vectors in addition to at least one other cell, e.g., a host cell (e.g., a T cell) that does not comprise any of the recombinant expression vectors, or a cell other than a T cell, e.g., a B cell, macrophage, neutrophil, erythrocyte, hepatocyte, endothelial cell, epithelial cell, muscle cell, brain cell, etc. Alternatively, the population of cells can be a substantially homogeneous population that primarily comprises (e.g., consists essentially of) host cells comprising the recombinant expression vector. The population can also be a clonal population of cells, in which all cells of the population are clones of a single host cell comprising the recombinant expression vector, such that all cells of the population comprise the recombinant expression vector. In one embodiment of the present invention, the population of cells is a clonal population comprising host cells comprising the recombinant expression vector described herein.

[0284] Nucleic acids encoding polypeptides can be modified without reducing their biological activity. Some modifications can be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein. Such modifications are known to those skilled in the art and include, for example, stop codons, a methionine added to the amino terminus to provide an initiation site, additional amino acids placed on either end to create conveniently placed restriction sites, or additional amino acids (such as polyHis) to aid in the purification process. In addition to recombinant methods, the immunoconjugates, effector moieties, and antibodies of the present disclosure may be constructed, in whole or in part, using standard peptide synthesis, as is well known in the art.

[0285] After expression, antibodies, antigen-binding fragments, and conjugates can be purified by standard techniques in the art, such as ammonium sulfate precipitation, affinity columns, column chromatography, etc. (See generally, Simpson ed., Basic methods in Protein Purification and Analysis: A laboratory Manual, Cold Harbor Press, 2008). Antibodies, antigen-binding fragments, and conjugates need not be 100% pure. If to be used therapeutically after purification, partially or to the desired homogeneity, the polypeptide should be substantially free of endotoxin.

[0286] Methods for the expression and / or refolding of antibodies, antigen-binding fragments, and conjugates into suitable active forms from mammalian cells and bacteria such as E. coli have been described and are well known and are applicable to the antibodies disclosed herein. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, 2002. nd , Cold Spring Harbor Laboratory, New York, 2013, Simpson ed., Basic methods in Protein Purification and Analysis: A laboratory Manual, Cold Harbor Press, 2008, and Ward et al., Nature 341:544, 1989.

[0287] Often, functional heterologous proteins from E. coli or other bacteria are isolated from inclusion bodies and require solubilization using strong detergents and subsequent refolding. As is well known in the art, a reducing agent must be present during the solubilization step to separate disulfide bonds. An exemplary buffer containing a reducing agent is 0.1 M Tris (pH 8), 6 M guanidine, 2 mM EDTA, and 0.3 M DTE (dithioerythritol). Reoxidation of disulfide bonds can be carried out in the presence of reduced and oxidized low-molecular-weight thiol reagents, as described in Saxena et al., Biochemistry 9:5015-5021, 1970, and specifically described in Buchner et al. (supra).

[0288] In addition to recombinant methods, antibodies, antigen-binding fragments, and / or conjugates may be constructed entirely or partially using standard peptide synthesis. Solid-phase synthesis of polypeptides can be achieved by attaching the C-terminal amino acid of the sequence to an insoluble support, followed by sequential addition of the remaining amino acids in the sequence. Techniques for solid-phase synthesis are described by Barany & Merrifield, *The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A. pp. 3-284; Merrifield et al., *J. Am. Chem. Soc.* 85: 2149-2156, 1963; and Stewart et al., *Solid Phase Peptide Synthesis,* 2nd ed., *Pierce Chem. Co., Rockford, Ill., 1984. Longer proteins can be synthesized by condensing the amino and carboxyl termini of shorter fragments. Methods for forming peptide bonds by activation of carboxyl termini (eg, by use of the coupling reagent N,N'-dicyclohexylcarbodiimide) are well known in the art.

[0289] E. Methods of Detection Methods are provided for detecting the presence of cells expressing EGFRvIII, such as tumor cells expressing EGFRvIII, in a subject. In some embodiments, the methods include contacting cells from the subject with one or more antibodies or conjugates thereof that specifically bind to EGFRvIII to form an immune complex. The presence (or absence) of the immune complex is then detected. The presence of the immune complex indicates the presence of cells expressing EGFRvIII in the subject. The detection method can include in vivo or in vitro detection of the immune complex. In some embodiments, detecting cells expressing EGFRvIII includes detecting cell surface expression of EGFRvIII on tumor cells. In some embodiments of the provided methods, detecting cells expressing EGFRvIII in a subject detects a tumor. In some non-limiting examples, the tumor is a cancer such as head and neck cancer, breast cancer, or bladder cancer. In some examples, the method can also detect tumor cells overexpressing EGFRvIII.

[0290] Various formats are useful for detecting cells expressing EGFRvIII, for example, tumor cells expressing EGFRvIII. In some embodiments, a subject is selected who has, is suspected to have, or is at risk of developing a tumor, for example, a cancer. For example, the subject has, is suspected to have, or is at risk of developing head and neck cancer, breast cancer, or bladder cancer. In some examples, the subject has, is suspected to have, or is at risk of developing head and neck cancer, breast cancer, or bladder cancer. Thus, the presence of cells expressing EGFRvIII can be detected in these subjects.

[0291] In some embodiments, the antibody or antigen-binding fragment is 287-302R300 is required to bind to the loop, and the method can also detect overexpression of EGFR. Overexpression can be measured to detect cells having more than about 50,000 receptors, more than about 60,000 receptors, more than about 70,000 receptors, more than about 80,000 receptors, more than about 90,000 receptors, or more than about 100,000 receptors. For example, such a method includes contacting tumor cells in a biological sample derived from a subject with one or more of the conjugates or antibodies or antigen-binding fragments thereof provided herein to form immune complexes. The presence (or absence) of the immune complex is then detected and / or quantified. The presence (or amount) of the immune complex in cells derived from the subject indicates the presence of tumor cells overexpressing EGFR in the subject.

[0292] In one embodiment, a sample is obtained from a subject, and the presence of tumor cells expressing EGFRvIII is assessed in vitro.For example, such a method includes contacting tumor cells in a biological sample from a subject with one or more of the conjugates or antibodies or antigen-binding fragments thereof provided herein that specifically bind to EGFRvIII to form immune complexes.Then, the presence (or absence) of immune complexes is detected.The presence of immune complexes in cells from a subject indicates the presence of tumor cells expressing EGFRvIII in the subject.For example, an increase in the presence of immune complexes in the sample compared to the formation of immune complexes in a control sample indicates the presence of tumor cells expressing EGFRvIII in the subject.In some embodiments, a control can be used.

[0293] Biological samples are typically obtained from mammalian subjects of interest, such as humans. Samples can be any sample, including, but not limited to, tissues from biopsies, autopsies, and pathology specimens. Biological samples also include sections of tissue, such as frozen sections taken for histological purposes.

[0294] In some examples of the disclosed methods, the antibody or antigen-binding fragment is conjugated with a detectable marker. In some examples, the method further comprises contacting the EGFRvIII-specific antibody with a secondary antibody, its antigen-binding fragment, or a conjugate comprising these molecules that specifically binds to the EGFRvIII-specific antibody for a sufficient time to form an immune complex, and detecting the immune complex. An increase in the presence of the immune complex in a biological sample from a selected subject (as described above) compared to the presence of the immune complex in a control sample or other standard detects the presence of EGFRvIII-expressing endothelial cells in the biological sample. In some examples, the secondary antibody is conjugated with a detectable marker.

[0295] Suitable detectable markers for antibodies or secondary antibodies have been described and are known to those skilled in the art. For example, various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents, and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase. Non-limiting examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Non-limiting examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin. A non-limiting exemplary luminescent material is luminol; a non-limiting exemplary magnetic agent is gadolinium; and non-limiting exemplary radioactive labels include 125 I, 131 I, 35 S, or 3 Contains H.

[0296] Antibodies and their conjugates that specifically bind to EGFRvIII can be used in immunohistochemical assays. These assays are known to those skilled in the art (for a description of immunoassay formats, see Harlow & Lane, Antibodies, A Laboratory Manual, 2004). nd ed., Cold Spring Harbor Publications, New York (2013).

[0297] The antibodies disclosed herein can also be used to detect tumor cells expressing EGFRvIII in vivo. In some examples, in vivo detection diagnoses the presence of a tumor in a subject. Accordingly, methods are disclosed for detecting a pathological condition in a subject, such as a tumor, e.g., cancer; e.g., head and neck cancer, breast cancer, or bladder cancer. In one embodiment, an effective amount of an antibody (or antigen-binding fragment thereof) or a conjugate thereof that specifically binds to EGFRvIII is administered to a subject for a time sufficient for the antibody or antigen-binding fragment to form an immune complex, and the immune complex can then be detected. Detection of the immune complex in a subject determines the presence of tumor cells expressing EGFRvIII. In one specific, non-limiting example, detection of the immune complex is performed by immunoscintigraphy. Other specific, non-limiting examples of immune complex detection include radiolocalization, radioimaging, magnetic resonance imaging (e.g., using biotinylated antibodies and avidin-iron oxide), and immunohistochemistry (e.g., immunohistochemistry). 111 These methods include positron emission tomography (using indium-labeled monoclonal antibodies), or fluorescence imaging (using antibodies labeled with luciferase or green fluorescent protein, for example). See Paty et al., Transplantation., 77:1133-1137, 2004, incorporated herein by reference. In some examples, the disclosed methods detect, for example, head and neck cancer, breast cancer, or bladder cancer.

[0298] In the context of magnetic resonance imaging, contrast agent detection can be significantly affected by the magnetic resonance scanner field strength. Increasing field strength provides orders of magnitude improvement in the ability to detect contrast agents (Hu et al., Ann. Rev. Biomed. Eng., 6:157-184, 2004; Wedeking et al., Magn. Reson. Imaging., 17:569-575, 1999). For example, the limit of detection for gadolinium at 2 Tesla (T) is approximately 30 μM. At 4 T, the limit of detection drops to approximately 1 μM. Newly available 7-12 T scanners are expected to detect low (10-100) nM concentrations of this contrast agent. Similar sensitivity can also be achieved using contrast agents such as iron oxide. Once detected, test results can be used to assist or guide surgical or other resection of the tumor.

[0299] In one embodiment, an effective amount of an antibody or antigen-binding fragment thereof that specifically binds to EGFRvIII, or a conjugate thereof, is administered to a subject with a tumor after anti-cancer treatment. After a sufficient time has elapsed to allow the administered antibody, antigen-binding fragment, or conjugate to form an immune complex with EGFRvIII on tumor cells, the immune complex is detected. For example, an antibody or conjugate thereof that specifically binds to EGFRvIII can be administered to a subject before or after tumor treatment. The tumor can be (but is not limited to) a cancer such as head and neck cancer, breast cancer, or bladder cancer. The presence (or absence) of immune complexes indicates the effectiveness of the treatment. For example, an increase in immune complexes compared to a control taken before treatment indicates that the treatment is ineffective, and a decrease in immune complexes compared to a control taken before treatment indicates that the treatment is effective.

[0300] F. Treatment Method A therapeutically effective amount of an antibody or antigen-binding fragment or conjugate thereof that specifically binds to EGFRvIII, or CAR T cells expressing an antigen-binding fragment that specifically binds to EGFRvIII, can be administered to a subject to treat tumors that express EGFRvIII, such as cancers such as head and neck cancer, breast cancer, or bladder cancer. In some embodiments, administration of a therapeutically effective amount of an antibody or antigen-binding fragment or conjugate thereof that specifically binds to EGFRvIII, or CAR T cells expressing an antigen-binding fragment that specifically binds to EGFRvIII, reduces signs or symptoms of cancers such as head and neck cancer, breast cancer, or bladder cancer. Thus, subjects who have, are suspected of having, or are at risk of developing a tumor that expresses EGFRvIII can be selected for treatment. Therapeutically effective amounts of the nucleic acid molecules and vectors disclosed herein are also useful.

[0301] In other embodiments, the tumor may overexpress EGFR and / or express misfolded EGFR. In some embodiments, a subject is selected who has a tumor that overexpresses EGFR and / or expresses misfolded EGFR. The compositions disclosed herein are useful for treating these tumors in subjects.

[0302] In a further embodiment, a therapeutically effective amount of the antibodies or antigen-binding fragments disclosed herein can also be used in a method for inhibiting an EGFR-overexpressing tumor in a subject. The method comprises administering an effective amount of an antibody, antigen-binding fragment, nucleic acid molecule, vector, T cell, or pharmaceutical composition to a subject having an EGFR-overexpressing tumor. In some non-limiting examples, the antibody or antigen-binding fragment inhibits a VHCDR comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NO:1. H and V comprising LCDR1, LCDR2, and LCDR3 of SEQ ID NO:2. HThe nucleic acid molecules, vectors, and CAR T cells comprising these antigen-binding fragments are also useful. The tumor can be cancer, for example, head and neck cancer, breast cancer, or bladder cancer. Therefore, subjects who have, are suspected to have, or are at risk of developing EGFR-overexpressing tumors can be selected for treatment.

[0303] In some examples, the antibodies, antigen-binding fragments, CAR T cells, compositions, and conjugates disclosed herein can be administered to a subject to delay or inhibit tumor growth or metastasis, reduce tumor volume, or reduce metastasis. In these applications, a therapeutically effective amount of an antibody or antigen-binding fragment or conjugate or CAR T cell or composition that specifically binds to EGFRvIII is administered to a subject in an amount and under conditions sufficient to form an immune complex with EGFRvIII, thereby delaying or inhibiting tumor growth or metastasis, reducing tumor volume, or inhibiting signs or symptoms of the tumor. Examples of suitable subjects include subjects diagnosed with or suspected of having a tumor that expresses EGFRvIII, such as subjects with cancers such as breast cancer, lung cancer, colorectal cancer, or melanoma.

[0304] The therapeutically effective amount depends on the severity of the disease and the patient's overall condition. A therapeutically effective amount is one that provides either subjective symptom relief or objectively identifiable improvement as recognized by a physician or other qualified observer. In one embodiment, a therapeutically effective amount is an amount required to inhibit tumor growth (such as the growth of cancers such as head and neck cancer, breast cancer, and bladder cancer), inhibit metastasis, reduce tumor volume, or reduce tumor signs or symptoms. The therapeutically effective amount of the administered drug can vary depending on the desired effect and the subject to be treated. In some examples, a therapeutic amount is an amount that eliminates or reduces the patient's tumor burden, or prevents or reduces the growth of metastatic cells, or reduces tumor symptoms.

[0305] Subjects that can benefit from the disclosed methods include human and veterinary subjects. Subjects can be screened, for example, before starting the disclosed treatment to determine whether the subject has a tumor. The presence of a tumor that expresses EGFRvIII indicates that the tumor can be treated using the methods provided herein.

[0306] Any administration method, including local administration and systemic administration, can be used for the disclosed antibodies, antigen-binding fragments, conjugates, CAR T cells, compositions, and additional agents. For example, local administration, oral administration, intravascular administration, such as intravenous administration, intramuscular administration, intraperitoneal administration, intranasal administration, intradermal administration, intrathecal administration, and subcutaneous administration can be used. The specific administration mode and dosing regimen will be selected by the attending physician, taking into account the specifics of the case (e.g., the subject, the disease, the involved disease state, and whether the treatment is preventive). In cases where multiple agents or compositions are administered, one or more administration routes can be used; for example, the chemotherapeutic agent can be administered orally, and the antibody or antigen-binding fragment or conjugate or composition can be administered intravenously. Methods of administration include injection, in which the conjugate, antibody, antigen-binding fragment, CAR T cell, nucleic acid molecule, or composition is provided in a pharmaceutically acceptable carrier such as water, saline, Ringer's solution, dextrose solution, 5% human serum albumin, fixed oil, ethyl oleate, or liposomes. In some embodiments, local administration of the disclosed compounds can be used, for example, by applying the antibody or antigen-binding fragment to an area of ​​tissue from which a tumor has been removed or to an area suspected of being prone to tumor development. In some embodiments, sustained release of a pharmaceutical preparation comprising a therapeutically effective amount of an antibody or antigen-binding fragment (or a conjugate thereof) within (or near) the tumor can be beneficial.

[0307] Compositions containing antibodies or antigen-binding fragments or conjugates thereof, or CAR T cells, can be formulated into suitable unit dosage forms for individual administration of precise dosages. Furthermore, the compositions can be administered in a single-dose or multiple-dose schedule. A multiple-dose schedule involves an initial course of treatment with multiple separate doses, e.g., 1 to 10 doses, followed by additional doses at subsequent intervals as needed to maintain or reinforce the effect of the composition. Treatment can involve daily or multiple daily administrations of the compound for a period ranging from several days to several months or even years. Thus, the dosing regimen will be determined, at least in part, based on the specific needs of the subject being treated and will be dependent on the judgment of the administering practitioner.

[0308] Typical dosages of antibodies, conjugates, compositions, or additional agents can range from about 0.01 to about 30 mg / kg, e.g., from about 0.1 to about 10 mg / kg. In some examples, dosages are at least about 0.1 mg / kg, at least about 0.2 mg / kg, at least about 0.3 mg / kg, at least about 0.4 mg / kg, at least about 0.5 mg / kg, at least about 1 mg / kg, at least about 4 mg / kg, at least about 3 mg / kg, at least about 5 mg / kg, at least about 6 mg / kg, at least about 7 mg / kg, at least about 8 mg / kg, at least about 9 mg / kg, at least about 10 mg / kg, at least about 11 mg / kg, at least about 12 mg / kg, at least about 13 mg / kg, or at least about 16 mg / kg. / kg, at least about 14 mg / kg, at least about 15 mg / kg, at least about 16 mg / kg, at least about 17 mg / kg, at least about 18 mg / kg, at least about 19 mg / kg, at least about 20 mg / kg, at least about 21 mg / kg, at least about 22 mg / kg, at least about 23 mg / kg, at least about 24 mg / kg, at least about 25 mg / kg, at least about 26 mg / kg, at least about 27 mg / kg, at least about 28 mg / kg, at least about 29 mg / kg, or at least about 30 mg / kg.

[0309] In a specific example, a subject is administered a therapeutic composition comprising one or more of the conjugate, antibody, composition, CAR T cells, or additional agent, e.g., over a period of weeks, months, or years, in a multiple daily dosing schedule, such as at least 2 consecutive days, 10 consecutive days, etc. In one example, the subject is administered the conjugate, antibody, composition, or additional agent for a period of at least 30 days, e.g., at least 2 months, at least 4 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months.

[0310] In some embodiments, the disclosed therapeutic agents are administered intravenously, subcutaneously, or by other modes daily or multiple times weekly for a period of time, followed by a treatment-free period, and then the cycle is repeated. In some embodiments, the initial period of treatment (e.g., daily or multiple weekly administration of the therapeutic agent) lasts for 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks. In related embodiments, the treatment-free period lasts for 3 days, 1 week, 2 weeks, 3 weeks, or 4 weeks. In certain embodiments, the dosing regimen for the therapeutic agent is 3 days daily, followed by 3 days off; or 1 week daily or multiple times weekly, followed by 3 days or 1 week off; or 2 weeks daily or multiple times weekly, followed by 1 or 2 weeks off; or 3 weeks daily or multiple times weekly, followed by 1, 2, or 3 weeks off; or 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks daily or multiple times weekly, followed by 1, 2, 3, or 4 weeks off.

[0311] The administration of the antibody, antigen-binding fragment, conjugate, CAR T cell, or composition may be accompanied by the administration of other anti-cancer or anti-angiogenic agents, or therapeutic treatments (such as surgical removal of a tumor or radiation therapy). For example, the subject can receive one or more additional treatments before, during, or after the administration of a therapeutic amount of the antibody or conjugate. In one example, the subject receives one or more treatments to remove or reduce the tumor before the administration of a therapeutic amount of one or more agents for tumor treatment. For example, the additional agents may include, but are not limited to, chemotherapeutic agents, anti-angiogenic agents, or a combination thereof. In another example, at least a portion of the tumor is surgically or otherwise removed or reduced in size or volume before the administration of a therapeutically effective amount of the antibody or antigen-binding fragment or conjugate.

[0312] Specific examples of additional therapeutic agents that can be used include microtubule binding agents, DNA intercalators or cross-linking agents, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors. These agents and treatments (administered in therapeutically effective amounts) can be used alone or in combination. For example, any suitable anti-cancer or anti-angiogenic agent can be used in combination with the antibodies and conjugates disclosed herein. The methods and therapeutic dosages of such agents are known to those skilled in the art and can be determined by a skilled physician. In one example, the chemotherapeutic agent includes 5-FU or IRT, or both.

[0313] Microtubule-binding agents refer to agents that interact with tubulin to stabilize or destabilize microtubule formation, thereby inhibiting cell division. Examples of microtubule-binding agents that can be used with the disclosed treatments include, but are not limited to, paclitaxel, docetaxel, vinblastine, vindesine, vinorelbine (navelbine), epothilone, colchicine, dolastatin 15, nocodazole, podophyllotoxin, and rhizoxin. Analogs and derivatives of such compounds can also be used and are known to those skilled in the art. For example, suitable epothilones and epothilone analogs are described in International Publication No. WO 2004 / 018478. Taxoids such as paclitaxel and docetaxel, as well as paclitaxel analogs taught in U.S. Patent Nos. 6,610,860; 5,530,020; and 5,912,264, can also be used.

[0314] Suitable regulators of DNA and RNA transcription, including, but not limited to, actinomycin D, daunorubicin, doxorubicin, and their derivatives and analogs, are also suitable for use in combination with the disclosed treatments. DNA intercalators and cross-linking agents that may be administered to a subject include, but are not limited to, cisplatin, carboplatin, oxaliplatin, mitomycins such as mitomycin C, bleomycin, chlorambucil, cyclophosphamide, and their derivatives and analogs. DNA synthesis inhibitors suitable for use as therapeutic agents include, but are not limited to, methotrexate, 5-fluoro-5'-deoxyuridine, 5-FU, and their analogs. Examples of suitable enzyme inhibitors include, but are not limited to, camptothecin, etoposide, formestane, trichostatin, and their derivatives and analogs. Suitable compounds that affect gene regulation include agents that result in an increase or decrease in expression of one or more genes, such as raloxifene, 5-azacytidine, 5-aza-2'-deoxycytidine, tamoxifen, 4-hydroxytamoxifen, mifepristone, and derivatives and analogs thereof.

[0315] Examples of commonly used chemotherapy drugs include Adriamycin, Alkeran, Ara-C, BiCNU, Bursufen, CCNU, Carboplatinum, Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5-FU, Fludarabine, Hydrea, Idarubicin, Ifosfamide, Methotrexate, Mithramycin, Mitomycin, Mitoxantrone, Nitrogen Mustard, and Taxol. These include benzodiazepine (or other taxanes such as docetaxel), Velban, vincristine, VP-16, and some newer agents include gemcitabine (Gemzar), Herceptin, IRT (Camptosar, CPT-11), leustatin, navelbine, Rituxan STI-571, Taxotere, topotecan (Hycamtin), Xeloda (capecitabine), Zevelin, and calcitriol.

[0316] Non-limiting examples of immunomodulatory agents that may be used include AS-101 (Wyeth-Ayerst Labs.), bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocyte-macrophage colony-stimulating factor; Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immunoglobulin (Cutter Biological), IMREG (manufactured by Imreg, New Orleans, La.), SK&F 106528, and TNF (tumor necrosis factor; Genentech).

[0317] Accordingly, non-limiting examples of chemotherapeutic agents for use in combination with the disclosed EGFRvIII-specific antibodies, antigen-binding fragments, or conjugates thereof, CAT T cells, and nucleic acid molecules include chemotherapeutic agents such as erlotinib (TARCEVA®, Genentech / OSI Pharm.), bortezomib (VELCADE®, Millennium Pharm.), fulvestrant (FASLODEX®, AstraZeneca), Sutent (SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis), PTK787 / ZK 222584 (Novartis), oxaliplatin (Eloxatin®, Sanofi), 5-FU (5-fluorouracil), leucovorin, rapamycin (Sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, GlaxoSmithKline), lonafarnib (SCH 66336), sorafenib (BAY43-9006, Bayer Labs.), and gefitinib (IRESSA®, AstraZeneca), AG1478, AG1571 (SU 5271; Sugen), alkylating agents such as thiotepa and CYTOXAN® cyclophosphamide; alkyl sulfonic acids such as busulfan, improsulfan, and piposulfan; antifolate antineoplastic agents such as pemetrexed (ALIMTA® Eli Lilly), aziridines, such as benzodopa, carboquone, metuledopa, and uredopa; ethyleneimines and methylamelamines, such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylomelanamine; acetogenins (e.g., bullatacin and bullatacinone); camptothecins (e.g., the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (e.g., its synthetic analogs adozelesin, carzelesin, and bizelesin); cryptophycins (e.g., cryptophycin 1 and cryptophycin 8);dolastatins; duocarmycins (e.g., synthetic analogs, KW-2189 and CB1-TM1); eluterobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, e.g., chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, alan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics, such as the enediyne antibiotics, calicheamicin, calicheamicin gamma 1I, and calicheamicin omega 1I; dynemicins, such as dynemicin A; bisphosphonates, such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-di Azo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (e.g., morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin , puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine miprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as frolinic acid; aceglatone;Aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, e.g., maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid acid); 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; schizophyllan; spirogermanium; tenuazonic acid; triazicon; 2,2',2''-trichlorotriethylamine; trichothecenes (specifically, T-2 toxin, verracurin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa;Taxoids, such as paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE™ cremophor-free, albumin-free nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; Xeloda; ibandronic acid; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

[0318] Non-limiting examples of antiangiogenic agents include molecules such as proteins, enzymes, polysaccharides, oligonucleotides, DNA, RNA, and recombinant vectors, as well as small molecules that function to reduce or even inhibit blood vessel growth. Examples of suitable angiogenesis inhibitors include, but are not limited to, angiostatin K1-3, staurosporine, genistein, fumagillin, medroxyprogesterone, suramin, interferon-alpha, metalloproteinase inhibitors, platelet factor 4, somatostatin, thrombospondin, endostatin, thalidomide, and derivatives and analogs thereof. For example, in some embodiments, the antiangiogenic agent is an antibody that specifically binds to VEGF (e.g., AVASTIN®, Roche) or VEGF receptor (e.g., VEGFR2 antibody). In one example, the anti-angiogenic agent includes a VEGFR2 antibody, or DMXAA (also known as Vadimezan or ASA404; commercially available, for example, from Sigma Corp. (St. Louis, MO)), or both. Exemplary kinase inhibitors include GLEEVAC®, IRESSA®, and TARCEVA®, which prevent the phosphorylation and activation of growth factors. Antibodies that can be used include HERCEPTIN® and AVASTIN®, which block growth factors and angiogenesis pathways.

[0319] In some examples, the additional agent is a monoclonal antibody, e.g., 3F8, Abagovomab, Adecatumumab, Afutuzumab, Alacizumab, Alemtuzumab, Altumomab pentetate, Anatumomab mafenatox mafenatox, apolizumab, arcitumomab, bavituximab, bectumomab, belimumab, besilesomab, bevacizumab, bivatuzumab mertansine, blinatumomab, brentuximab vedotin, cantuzumab mertansine, capromab pendetide, catumaxomab, CC49, cetuximab, sitatuzumab bogatox, cizutumumab, clivatuzumab tertraxetan Tertraxetan, Conatumumab, Dacetuzumab, Detumomab, Ecromeximab, Eculizumab, Edrecolomab, Epratuzumab, Ertumaxomab, Etaracizumab, Farletuzumab, Figitumumab, Galiximab, Gemtuzumab Ozogamicin, Girentuximab, Glembatumumab Vedotin vedotin, Ibritumomab tiuxetan, Igovomab, Imciromab, Intetumumab, Inotuzumab ozogamicin, Ipilimumab, Iratumumab, Labetuzumab, Lexatumumab, Lintuzumab, Lorvotuzumab, Mertansine, Lucatumumab, Rumiliximab, Mapatuzumab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Mitumomab, Morolimumab, Nacolomab, Butafenatox tafenatox), naptumomab estafenatox, necitumumab, nimotuzumab, nofetumomab merpentane (Nofetumomabmerpentan, ofatumumab, olaratumab, oportuzumab monatox, oregovomab, panitumumab, pentumomab, pertuzumab, pintumomab, pritumumab, ramucirumab, rilotumumab, rituximab, robatumumab, satumomab pendetide, sibrotuzumab, sonepcizumab, sorafenib, sunitinib, tacatuzumab tetraxetan, taplitumomab paptox, tenatumomab, TGN1412, ticilimumab (=tremelimumab), tigatuzumab, TNX-650, trastuzumab, tremelimumab, tucotuzumab celmoleukin, veltuzumab, volociximab, votumumab, and zalutumumab.

[0320] Another common treatment for some types of cancer is surgical treatment, such as surgical removal of the cancer or a portion thereof. Another example of a treatment is radiation therapy, such as the administration of radioactive material or energy to the tumor site (e.g., external beam radiation therapy) to help eradicate or shrink the tumor prior to surgical removal.

[0321] Other therapeutic agents, e.g., anti-tumor agents, which may or may not fall into one or more of the above categories, are also suitable for administration in combination with the disclosed treatments. For example, such agents include adriamycin, apigenin, rapamycin, zebularine, cimetidine, and derivatives and analogs thereof.

[0322] Preparation and dosing schedules for additional agents may be used according to manufacturer's instructions or as empirically determined by one of skill in the art. Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service, (1992) Ed., M.C. Perry, Williams & Wilkins, Baltimore, Md.

[0323] Combination therapy can provide synergistic effects and be proven to be synergistic. That is, the effect achieved when active ingredients are used together is greater than the sum of the effects achieved when the compounds are used separately. Synergistic effects can be achieved when the active ingredients are (1) co-formulated and administered or delivered simultaneously as a combined unit dosage formulation; (2) delivered alternately or in parallel as separate formulations; or (3) by some other schedule. When delivered alternately, synergistic effects can be achieved when the compounds are administered or delivered sequentially, for example, by different injections using separate syringes. Generally, in alternation, effective dosages of the active ingredients are administered sequentially, i.e., consecutively, while in combination therapy, effective dosages of two or more active ingredients are administered simultaneously.

[0324] G. Composition Compositions are provided that include one or more of the disclosed conjugates, antibodies, or antigen-binding fragments, or nucleic acid molecules, or CAR T cells that specifically bind to EGFRvIII, in a carrier (such as a pharmaceutically acceptable carrier). The compositions can be prepared in unit dosage form for administration to a subject. The amount and timing of administration to achieve a desired outcome are at the discretion of the treating physician. The compositions can be formulated for systemic administration (such as intravenous) or local administration (such as intratumor). In one example, an antibody or antigen-binding fragment thereof that specifically binds to EGFRvIII, or a conjugate comprising such an antibody or antigen-binding fragment, or CAR T cells is formulated for parenteral administration, such as intravenous administration. Compositions comprising the conjugates, antibodies, or antigen-binding fragments, or CAR T cells disclosed herein are useful, for example, for the treatment and / or detection of tumors occurring in tumors, such as breast cancer, colorectal cancer, lung cancer, or skin cancer. In some examples, the compositions are useful for the treatment or detection of cancer.

[0325] Compositions for administration may include a solution of the conjugate, antibody, or antigen-binding fragment (or suspended CAR T cells) dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers may be used, such as buffered saline. These solutions are sterile and generally free of undesirable matter. These compositions may be sterilized by conventional, well-known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH-adjusting buffers, toxicity adjusters, etc., e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the antibody or antigen-binding fragment, conjugate, or CAR T cells in these formulations may vary widely and will be selected primarily based on the volume, viscosity, weight, etc. of the liquid, according to the selected mode of administration and the needs of the subject. Actual methods for preparing such dosage forms are known or will be apparent to those skilled in the art.

[0326] A typical composition for intravenous administration contains about 0.01 to about 30 mg / kg of antibody or antigen-binding fragment or conjugate (or an equivalent dose of a conjugate comprising the antibody or antigen-binding fragment) per subject per day. Actual methods for preparing administrable compositions are known or apparent to those skilled in the art and are described in more detail in publications such as Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, PA (1995). In some embodiments, the composition may be a liquid formulation containing one or more antibodies, antigen-binding fragments (such as antibodies or antigen-binding fragments that specifically bind to EGFRvIII) in a concentration range of about 0.1 mg / ml to about 20 mg / ml, or about 0.5 mg / ml to about 20 mg / ml, or about 1 mg / ml to about 20 mg / ml, or about 0.1 mg / ml to about 10 mg / ml, or about 0.5 mg / ml to about 10 mg / ml, or about 1 mg / ml to about 10 mg / ml.

[0327] Antibodies, antigen-binding fragments, or conjugates may be provided in lyophilized form and rehydrated with sterile water before administration, or may be provided as a sterile solution of known concentration. The antibody, antigen-binding fragment, or conjugate solution is then added to an infusion bag containing 0.9% sodium chloride (USP) and, in some cases, administered at a dosage of 0.5 to 15 mg / kg body weight. Considerable experience has been gained in the art with the administration of antibody, antigen-binding fragment, and conjugate drugs; for example, antibody drugs have been sold in the United States since the approval of RITUXAN® in 1997. Antibodies, antigen-binding fragments, and conjugates may be administered by slow infusion rather than intravenous push or bolus. In one example, a relatively high loading dose is administered, followed by maintenance doses at a relatively low level. For example, an initial loading dose of 4 mg / kg of antibody or antigen-binding fragment (or the equivalent dose of a conjugate containing the antibody or antigen-binding fragment) is infused over 90 minutes, followed by weekly maintenance doses of 2 mg / kg infused over 30 minutes for 4-8 weeks if the previous dose was well tolerated.

[0328] Controlled-release parenteral formulations can be formulated as implants, oily injections, or particulate systems. For a comprehensive review of protein delivery systems, see Banga, AJ, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA, (1995). Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain a therapeutic protein, such as a cytotoxin or drug, as a central core. In microspheres, the therapeutic agent is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 μm are commonly referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Because capillaries have a diameter of approximately 5 μm, only nanoparticles are administered intravenously. Microparticles are typically approximately 100 μm in diameter and are administered subcutaneously or intramuscularly. See, for example, Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pp. 315-339, (1992).

[0329] Polymers are used for ion-controlled release of the antibody or antigen-binding fragment or conjugate compositions disclosed herein. Various degradable and non-degradable polymer matrices for use in controlled drug delivery are known in the art (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, the block copolymer polaxamer 407 exists as a viscous but mobile liquid at low temperatures and forms a semi-solid gel at body temperature. It has been shown to be an effective vehicle for the formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. 9:425-434, 1992; and Pec et al., J. Parent. Sci. Tech. 44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for the controlled release of proteins (Ijntema et al., Int. J. Pharm. 112:215-224, 1994). In yet another aspect, liposomes are used for the controlled release and drug targeting of lipid-encapsulated drugs (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)).Numerous additional systems for controlled delivery of therapeutic proteins are known (see U.S. Patent Nos. 5,055,303; 5,188,837; 4,235,871; 4,501,728; 4,837,028; 4,957,735; 5,019,369; 5,055,303; 5,514,670; 5,413,797; 5,268,164; 5,004,697; 4,902,505; 5,506,206; 5,271,961; 5,254,342, and 5,534,496).

[0330] In some instances, a subject is administered DNA encoding an antibody, its antigen-binding fragment, or a conjugate (e.g., with a toxin), for example, to provide in vivo antibody production using the subject's cellular machinery. Immunization with nucleic acid constructs is well known in the art and is taught, for example, in U.S. Patent No. 5,643,578, U.S. Patent No. 5,593,972, and U.S. Patent No. 5,817,637. U.S. Patent No. 5,880,103 describes several methods for delivering encoding nucleic acids to organisms. These methods include liposomal delivery of nucleic acids. Such methods can be applied by those skilled in the art to produce antibodies or their antibody-binding fragments.

[0331] One approach to nucleic acid administration is direct administration of plasmid DNA, such as a mammalian expression plasmid. The nucleotide sequence encoding the disclosed antibody or antibody-binding fragment thereof can be placed under the control of a promoter to increase expression.

[0332] In another approach to using nucleic acids, the disclosed antibodies or antibody-binding fragments thereof may be expressed by attenuated viral hosts or vectors or bacterial vectors. Recombinant vaccinia virus, adeno-associated virus (AAV), herpesvirus, retrovirus, cytomegalovirus, or other viral vectors can be used to express antibodies. For example, vaccinia vectors and methods, useful protocols are described in U.S. Patent No. 4,722,848. BCG (Bacillus Calmette-Guerin) provides another vector for expressing the disclosed antibodies (see Stover, Nature 351:456-460, 1991).

[0333] In one embodiment, nucleic acids encoding the disclosed antibodies or antibody-binding fragments thereof can be directly introduced into cells. For example, nucleic acids can be loaded onto gold microspheres by standard methods and introduced into the skin using a device such as Bio-Rad's HELIOS™ Gene Gun. The nucleic acid can be "naked," consisting of a plasmid under the control of a strong promoter.

[0334] Typically, DNA is injected intramuscularly, but may also be injected directly into other sites. The dosage for injection is generally about 0.5 μg / kg to about 50 mg / kg, typically about 0.005 mg / kg to about 5 mg / kg (see, e.g., U.S. Patent No. 5,589,466).

[0335] H.Kit Kits are also provided, for example, kits for detecting tumor cells expressing EGFRvIII in a subject and for treating tumors in a subject. The kits typically include an antibody or antigen-binding fragment that specifically binds to EGFRvIII and / or a conjugate thereof.

[0336] A plurality of conjugates or antibodies or antigen-binding fragments that specifically bind to EGFRvIII may be included in the kit. Thus, the kit may include two or more antibodies that specifically bind to EGFRvIII, or antibodies or antigen-binding fragments that specifically bind to EGFRvIII, and conjugates thereof, or combinations thereof. In some embodiments, the kit includes an antigen-binding fragment such as an Fv fragment, or a conjugate comprising an antigen-binding fragment. In one example, for example, for in vivo use, the antibody may be an scFv fragment.

[0337] The kit may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container may be formed from a variety of materials, such as glass or plastic. The container typically holds a composition comprising one or more of the disclosed EGFRvIII-specific antibodies, antigen-binding fragments, or conjugates. In some embodiments, the container may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). The label or package insert indicates that the composition is used to treat a specific condition.

[0338] The label or package insert typically further includes instructions for using the disclosed EGFRvIII-specific antibody or fragment thereof, or conjugate thereof, for example, in a method for treating or preventing tumors. The package insert typically includes instructions customarily included in commercial packages of therapeutic products, containing information about the indications, usage, dosage, administration, contraindications, and / or warnings regarding the use of such therapeutic products. The instructional materials may be written, in electronic form (such as a computer diskette or compact disc), or visual (such as a video file). The kit may also include additional components to facilitate the specific application for which the kit is designed. Thus, for example, the kit may further contain a means for detecting the label (such as an enzyme substrate for an enzyme label, a filter set for detecting a fluorescent label, an appropriate secondary label such as a secondary antibody, etc.). The kit may further include buffers and other reagents routinely used for the implementation of a particular method. Such kits and their appropriate contents are known to those skilled in the art. [Example]

[0339] In the experimental studies disclosed herein, EGFR 287-302 The loop was generated as a disulfide-restricted peptide and coupled to the carrier protein keyhole limpet hemocyanin (KLH). The KLH-coupled peptide was injected into Balb / c mice. Serum from these mice was monitored for reactivity with the loop peptide, and when high titers were achieved, spleens were removed for hybridoma formation. Candidate hybridoma supernatants were then assayed for reactivity with wtEGFR and EGFRvIII. Antibodies with high reactivity to EGFRvIII and low reactivity to wtEGFR were isolated and characterized as described below.

[0340] Example 1 Selection of hybridomas reactive with EGFRvIII As targets for antibody-based therapy, amplified EGFR or mutant versions containing EGFRvIII are expressed on the surface of epithelial carcinomas and malignant gliomas. To generate antibodies against the disulfide-restricted loop 287-302 of the extracellular domain (ECD) of EGFR (numbering is as depicted in Figure 1A for mature human EGFR), five Balb / c mice were repeatedly immunized with a peptide conjugated to KLH containing residues 286-303 (Figure 1B). When the titer against the injected peptide exceeded background at a 1:128,000 dilution (Table 1), spleens were harvested and candidate hybridomas were generated through fusion with the myeloma cell line Sp2 / 0.

[0341] Table 1. Absorbance values ​​for the indirect ELISA assay measuring mouse antibody titers against loop peptides TIFF0007815099000052.tif15128

[0342] Two initially uncloned hybridomas that showed particularly high titers to the immunogen were established from two mice (Table 1). Approximately 40 hybridoma supernatants were generated from each fusion and evaluated in an ELISA format for binding to cells expressing the ECD of EGFRvIII-His and EGFRvIII. Follow-up experiments were performed to characterize seven promising hybridomas.

[0343] Individual supernatants from selected hybridomas with good cell-binding activity were assayed by ELISA for binding to the ECD of either wtEGFR-His or EGFRvIII-His. Nickel-coated 96-well plates were used to display His-tagged EGFR proteins. Results showed a strong binding preference for EGFRvIII-His over wtEGFR-His, confirming that the 287-302 loop is displayed in EGFRvIII but is less accessible in wtEGFR (Figure 2). Next, the same hybridoma supernatants (1:10 dilution) were assayed for binding to rat glioma F98 cells transfected with either EGFRvIII or wtEGFR. Again, binding reflected the strong interaction of the supernatants with the ECD of EGFRvIII (Figure 3) and the poor reactivity with wtEGFR (Figure 3). The monoclonal antibody ma528, which reacts with domain III of EGFR (see Fig. 1A ), was used as a positive control and was shown to bind equally well to the surface of both transfected cell lines ( Fig. 3 ).

[0344] The gene encoding EGFR is amplified and / or overexpressed in certain epithelial cancers. To assay binding to cells with overexpressed EGFR, supernatants (1:10 dilution) were added to either the triple-negative breast cancer cell line MDA-MB-468 or the epidermoid carcinoma line A431. Again, binding exceeding that of the pre-immune serum used as a negative control was observed for most supernatants. The antibody concentration of the supernatants was not determined at this point. Rather, the cells were cloned by limiting dilution and then grown as true "clonal" hybridomas.

[0345] Example 2 Characterization of purified antibodies Seven antibodies derived from the cloned hybridoma supernatants were purified using immobilized Protein A / G for further characterization. Two SDS-PAGE gels of the purified antibodies (one reduced and one non-reduced) were provided (Figure 4). When compared with the initial hybridoma supernatants, the purified antibodies reacted with qualitatively similar results in the ELISA assay. In fact, a relatively low concentration of antibody (5 ng / ml) produced a robust signal with EGFRvIII-His and showed little or no binding to wtEGFR-His (Figure 5). Flow cytometry was used to characterize the binding of each of the seven monoclonal antibodies in a cell-binding assay. Antibodies were added at 2 μg / ml. Again, ma528 was used as a positive control, and the same isotype antibody was used as a non-binding negative control. F98 EGFRWhen antibody binding to cells was characterized, histograms showed strong binding of ma528 (geometric mean of 3705), weak binding of 40H3 (geometric mean of 191), and lack of binding by the remaining six monoclonal antibodies or the isotype control (Figure 6B). When binding was assessed on F98-EGFRvIII, ma528 again showed the highest reactivity, but substantial binding from all seven monoclonal antibodies was close to it (geometric means ranging from approximately 1000 to 1300) (Figure 6B). The isotype control did not bind. Next, binding was assayed on the cancer lines MDA-MB-468 (Figure 6A), A431 (Figure 6B), and WI-38 (Figure 6C), noncancerous "normal" human cell lines. In MDA-MB-468 cells, ma528 and 40H3 showed substantial binding, while the other six antibodies showed minimal reactivity above the isotype control (Figure 6A). Substantially similar results were achieved in A431 cells (Figure 6A). However, in WI-38 cells, which express wtEGFR at physiological levels, only ma528 showed substantial binding, with a geometric mean of 2454 (Figure 6C). All other antibodies in WI-38 cells had geometric means below 100 (Figure 6C). Functionally, it was concluded that only antibodies 40H3 and ma528 exhibited binding to EGF-expressed by cancer. EGFR can be overexpressed either by gene amplification or loss of transcriptional regulation. High-level expression leads to either misfolding of the receptor or mutations in one or more of the gene copies.

[0346] However, 40H3 showed no reactivity with normal cells WI-38, and ma528 reacted strongly with both cancer and normal cells. Interestingly, when antibody binding to EGFRvIII-transfected F98 cells was assessed, the seven antibodies showed similar and substantial reactivity.

[0347] Example 3 EGFR 286-303 Location of the binding site within To aid in mapping the binding site of each antibody, 287-302Three charged residues in the loop, D290, E293, and R300, were each changed to alanine and prepared as his-tagged peptides (Figure 7). The wild-type sequence of the 287-302 loop was similarly prepared. Because the charged residues in the loop were spread out, the relative antibody binding activity could provide information about the location of key residues involved in each antibody interaction. The results showed that the antibodies fell into one of three groups: (1) binding was lost when residues D290 and E293 were changed to alanine; (2) binding was lost only by changing E293 to alanine; and (3) binding was lost when R300 was changed to alanine. Overall, the seven EGFRvIII-reactive antibodies fell into at least three groups based on their binding characteristics to the his-tagged peptides. Group 1 contained five of the seven antibodies: 1D9, 3D10, 4A4, 9G11, and 11E3. Group 2 had 11E11 and group 3 contained 40H3 (Table 2).

[0348] Table 2. Isotype determination and peptide reactivity of each monoclonal antibody TIFF0007815099000053.tif52128EGFR 287-302 Loop:CGADSYEMEEDGVRKC(SEQ ID NO:69)

[0349] Example 4 Antibody sequence analysis The variable regions of both the heavy and light chains of the seven antibodies were sequenced, and the deduced amino acids were determined (Figure 8). Sequence analysis of the cell-binding antibodies suggested that the seven antibodies could be clustered into four groups. 3D10 and 9G11 (group 1) share similar sequences, with V H 2 amino acids in the chain, V L There is one amino acid difference in the V chain between 1D9 and 4A4 (group 2). H 3 amino acids in the chain, V L There is one amino acid difference in the V chain, and finally, when comparing 11E11 and 11G3 (group 3), H 4 amino acids in the chain, V L There were two amino acid differences in the V chain.H Area or V L There were no differences in the amino acids located in the CDR3 of any of the regions. Sequence analysis suggested that 40H3 had the most distinct sequence compared to the other three groups and appeared to cluster separately. It had a significantly higher V H The V strands share 75% to 80% sequence similarity. L In contrast, the other six share 50% to 60% sequence similarity when compared with each other. H The V strands share 88% to 98% sequence similarity. L The sequence similarity is 85% to 98%. The amino acid sequence differences between 40H3 and the other three groups are V H Chain and V L It was found in all three CDR regions of both chains.

[0350] Table 3. Pairwise comparison of amino acid sequences, CDRs and framework (FR) regions with highest similarity TIFF0007815099000054.tif73153

[0351] Example 5 Cloned hybridoma-derived antibody cDNA sequences The DNA sequences for the heavy and light chains for each cloned antibody have been determined and are provided herein.

[0352] Example 6 Exemplary Antibody-Based Therapeutics With few exceptions, antibodies are rarely cytotoxic to tumor cells, even when they bind with good affinity. Therefore, strategies include adding a toxic payload to the antibody or modifying the parent antibody to attract immune cells to the tumor. Fusing the Fv portions (cDNA) of the heavy and light chains with PE38 provides a recombinant immunotoxin. 40H3 can be used to deliver the toxic payload, but other antibodies can also be used. The parent antibody of 40H3 is nontoxic to human tumor cells, but the immunotoxin is toxic in the nanomolar concentration range (Figure 9). Another exemplary construct, in which an antibody-binding fragment (such as an scFv) or a monoclonal antibody is modified to contain a cysteine ​​residue and a drug is conjugated to the antibody or antigen-binding fragment, is shown in Figure 19.

[0353] Example 7 Binding affinity of 40H3 to EGFR loops, EGFRwt ECD, and EGFRvIII ECD equilibrium dissociation constant K d The binding affinity of 40H3 to the 18 amino acid peptide loop (286-302 of the EGFR ECD; hereafter referred to as the "EGFR loop") described by [Delta] was approximately 1 nM (Figure 11 and Table 4). The binding affinity of 40H3 to the EGFRvIII ECD was approximately 270 pM K d The dissociation constants of 40H3 and EGFRwt ECD were significantly higher than those of 40H3 (Figure 11 and Table 4). This dissociation constant indicated that the 40H3 antibody exhibited high affinity for exposed EGFR loops. In contrast, the binding of 40H3 to EGFRwt ECD did not show a significant K due to its slow association rate. d No value was obtained (Figure 11).

[0354] Table 4. K of 40H3 antibody against EGFRwt ECD, EGFRvIII ECD, or EGFR loops d value TIFF0007815099000055.tif21139

[0355] Example 8 Cytotoxicity of 40H3-PE38 against EGFR- and EGFRvIII-expressing cell lines The 40H3-PE38 immunotoxin was generated by fusing the scFV region of 40H3 to a 38 kDa C-terminal fragment of Pseudomonas aeruginosa exotoxin A (PE). The binding affinity K of 40H3 PE38 to the EGFR loop d was 2.3 nM (Figure 12, Table 5).

[0356] Table 5. K of 40H3 DM1, 40H3 MMAE, or 40H3 PE38 against the EGFR loop d value TIFF0007815099000056.tif28139

[0357] The cytotoxicity of 40H3-PE38 was evaluated in cells expressing EGFRvIII or EGFR (F98 npEGFRvIII and F98 EGFR ) (Figure 10). 40H3-PE38 had an IC of less than 1 nM (approximately 0.4 nM). 50 In F98 npEGFRvIII It exhibits cytotoxic activity against erythrocytes with an IC of approximately 4 nM. 50 The same cells expressing wild-type EGFR, F98 EGFR It was 10 times more potent than the previous one (Table 6).

[0358] Table 6: IC of 40H3 PE38 or PE64 against MDA-MB-468, MDA-MB-468, or WI-38 50 value TIFF0007815099000057.tif28128

[0359] This result confirms the antibody's binding specificity to EGFRvIII, preferentially over wild-type EGFR. WI-38 cells, derived from lung fibroblasts and with normal EGFR expression, showed no loss of viability when incubated with 40H3-PE38 (IC 50 >10 nM) (Figure 10 and Table 6).

[0360] Example 9 Cytotoxicity of 40H3 MMAE against EGFRvIII-expressing or EGFR-overexpressing cell lines The 40H3 monoclonal antibody was conjugated to monomethyl auristatin E (MMAE) via the cleavable linker mc-vc-PAB. The resulting antibody-drug conjugate 40H3 MMAE exhibited strong binding to the EGFR loop, indicating that the conjugation process did not interfere with its binding ability (Figure 12, Table 5). The cytotoxicity of 40H3 MMAE was assessed in EGFRvIII-expressing cells (F98 npEGFRvIII ) or EGFR-overexpressing cancer cell lines (MDA-MB-468 and A431) (Figure 15, Table 7).

[0361] Table 7: IC for ADC and IT 50 Summary table of TIFF0007815099000058.tif47156 * Values ​​are the average of all data available at 72 hours (excluding experiments with only 1K cells / well)

[0362] 40H3 MMAE had an IC of approximately 8 nM against both cell lines, MDA-MB-468 and A431. 50 The F98 glioblastoma derived from rats showed cytotoxic activity (Table 7). npEGFRvIII Cells showed no loss of viability when incubated with 40H3 MMAE (IC 50 approximately 100 nM) (Figure 16 and Table 7).

[0363] Example 10 Cytotoxicity of 40H3 DM1 against EGFRvIII-expressing or EGFR-overexpressing cell lines The 40H3 monoclonal antibody was conjugated to maytansine (DM1) via a non-cleavable linker. The resulting antibody-drug conjugate, 40H3 DM1, showed strong binding to the EGFR loop, indicating that the conjugation process did not interfere with its binding ability (Figure 12, Table 5). The cytotoxicity of 40H3 DM1 was confirmed by immunohistochemistry using EGFRvIII-expressing cells (F98 npEGFRvIII ) or EGFR-overexpressing cancer cell lines (MDA-MB-468 and A431) (Figure 15, Table 7). 40H3 MMAE exhibited IC50 values ​​of approximately 75 nM and 30 nM against both MDA-MB-468 and A431, respectively. 50 The F98 glioblastoma derived from rats showed cytotoxic activity (Table 7). npEGFRvIII Cells did not show significant cell death when incubated with 40H3 DM1 (IC 50 >100 nM) (Figure 15 and Table 7).

[0364] Example 11 Cytotoxicity of 40H3-PE38 against patient-derived xenograft (PDX) glioblastoma cell lines We obtained a patient-derived xenograft (PDX) glioblastoma cell line, GBM39. This PDX cell line was shown to express both EGFR and EGFRvIII. Flow cytometry analysis demonstrated EGFR expression in the PDX via binding with the pan-EGFR monoclonal antibody 528 (Figure 13, Table 8).

[0365] Table 8. Flow cytometry data for 528 monoclonal antibody against GBM39 presented by median fluorescence intensity and total cell number (counts). TIFF0007815099000059.tif41128

[0366] Both 40H3 and 40H3 PE38 showed binding to PDX, suggesting the presence of EGFRvIII and / or misfolded, overexpressed EGFR (Figure 13, Tables 9 and 10). The sensitivity of GBM39 to 40H3-based antibody therapeutics was investigated by treatment with 40H3 PE38 (Figure 14).

[0367] Table 9. Flow cytometry data for 40H3 monoclonal antibody against GBM39 presented by median fluorescence intensity and total cell number (counts). TIFF0007815099000060.tif41128

[0368] Table 10. Flow cytometry data of 40H3 PE38 immunotoxin on GBM39 presented by median fluorescence intensity and total cell number (counts). TIFF0007815099000061.tif48128

[0369] Example 12 Cytotoxicity of 40H3-derived immunotoxins and antibody-drug conjugates against human glioblastoma cell lines expressing EGFRvIII The efficacy of 40H3-based antibody therapeutics was investigated in the EGRvIII-transfected human glioblastoma cell line DKMG. Flow cytometry analysis showed that both 40H3 and the pan-EGFR antibody 528 bound (Figure 17, Tables 11 and 12).

[0370] Table 11. Flow cytometry data for 40H3 antibody against DKMG-EGFRvIII presented by median fluorescence intensity and total cell number (counts). TIFF0007815099000062.tif41128

[0371] Table 12. Flow cytometry data for 528 antibodies against DKMG-EGFRvIII presented by median fluorescence intensity and total cell number (counts). TIFF0007815099000063.tif40128

[0372] Treatment with the antibody-drug conjugates 40H3 MMAE or 40H3 DM1 and the immunotoxin 40H3 PE38 demonstrated EGFR-directed cell death of DKMG-EGFRvIII cells. All three 40H3 antibody-derived variants induced cytotoxicity in the DKMG-EGFRvIII cell line (Figure 18, Table 7). Naked, unconjugated 40H3 antibody did not induce cytotoxic activity.

[0373] Example 13 Materials and Methods for Examples 7-12 Cytotoxicity assay (Figures 10, 13, 15, 16, and 9; Tables 6 and 7): 1000 cells were seeded per well in a volume of 100 μl in a 96-well plate. After 24 hours, antibody-drug conjugates (ADCs; 40H3 MMAE or 40H3 DM1), antibodies (40H3), immunotoxins (40H3 PE38), or Pseudomonas exotoxin (PE64) were added at the indicated concentrations (100, 10, 1, and 0.1 nM for ADCs and monoclonal antibodies; 10, 1, 0.1, and 0.01 nM for immunotoxins; and 1, 0.1, 0.01, and 0.001 nM for PE64). After 72 hours (48 hours for GBM39; Figure 3), viability was determined using the CellTiter-Glo Luminescent Cell Viability Assay kit (Promega, Madison, WI). This assay quantifies the amount of ATP present, which signals the presence of metabolically active cells. ATP was measured as fluorescence resulting from monooxygenation of luciferin catalyzed by Ultra-Glo-Luciferase. Fluorescence in each well was measured, and values ​​were presented as a percentage of untreated cells (control). Data were from at least two independent experiments with triplicate wells for each immunotoxin concentration.

[0374] Binding affinity assay (Figures 11 and 12; Tables 4 and 5): Binding affinity constants "K" of antibody drug conjugates (ADCs; 40H3 MMAE or 40H3 Dm1), antibody (40H3), or immunotoxin (40H3-PE38) against C-terminal His-tagged EGFRvIII ECD (Acrobiosystem, DE, USA) were measured. d " was measured using an Octet Red96 analyzer (Pall Life Sciences, New York, USA). EGFRvIII ECD-HIS was captured on a Ni-NTA biosensor and used as the "antigen." Briefly, all ligands and antibodies were diluted in a buffer consisting of 1x PBS, 1% BSA, and 0.05% Tween. 40H3-PE38 was diluted to 250 nM, 125 nM, 62.5 nM, 31.25 nM, 15.625 nM, and 7.813 nM. 2 μg / ml (in 200 μl) of EGFRlp-HIS, EGFRwt ECD-HIS, or EGFRvIII-HIS was used as the "antigen." K d The conditions for determination were as follows: 10 min presoak, 60 s baseline establishment, 120 s antigen loading, 120 s baseline re-establishment after antigen loading, 120 s 40H3 PE38 10 association, and finally, 20 min dissociation. The baseline, association, and dissociation buffers were pH 7.4. All procedures were performed at 30°C. Binding kinetics were analyzed using ForteBio Data Analysis 11.1 software. All K values ​​determined based on all different concentrations were used. d Combine the Global Fit options and d value was determined.

[0375] Flow cytometry assay (Figures 14 and 17; Tables 8–12): Antibodies or immunotoxins were incubated with suspension cells (1 × 10 cells per well) in a 96-well plate in FACS buffer consisting of PBS (KD Medical, MD, USA), 2 mM EDTA (KD Medical, MD, USA), 1% BSA (Sigma-Aldrich, MO, USA), and 0.1% sodium azide (Sigma-Aldrich, MO, USA) for 1 h at 4°C. Mouse anti-PE antibody M40-1 was used to detect immunotoxin binding. Bound antibodies were detected with R-phycoerythrin-conjugated F(ab')2 goat anti-mouse IgG Fcγ (catalog no. 115-116-071; Jackson ImmunoResearch, ME, USA) at a 1:250 dilution for 45–60 min at 4°C. Antibody binding was characterized using an SA3800 Spectral Analyzer (Sony Biotechnology, San Jose, CA, USA), and data were analyzed using FlowJo (Tree Star, Inc., Ashland, OR, USA) and displayed in a histogram format with median fluorescence intensity plotted.

[0376] In view of the many possible embodiments to which the principles of the present invention may be applied, it should be understood that the illustrated embodiments are merely examples of the invention and should not be considered as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

1. The heavy chain variable region (V) is shown as SEQ ID NO:1 H ) V including heavy chain complementarity determining regions (HCDRs) 1, HCDR2, and HCDR3 of H and a light chain variable region (V) shown as SEQ ID NO:

2. L ) V containing light chain complementarity-determining regions (LCDR) 1, LCDR2, and LCDR3 of L and 1. An isolated monoclonal antibody or antigen-binding fragment thereof comprising: An isolated monoclonal antibody or antigen-binding fragment thereof, wherein the monoclonal antibody specifically binds to epidermal growth factor receptor (EGFR) variant III (vIII).

2. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth as SEQ ID NOs: 5, 6, 7, 8, 9, and 10, respectively; The monoclonal antibody or antigen-binding fragment thereof according to claim 1.

3. V H and V L comprising an amino acid sequence that is at least 90% identical to the amino acid sequences set forth as SEQ ID NOs: 1 and 2, respectively; 3. The monoclonal antibody or antigen-binding fragment of claim 1 or claim 2.

4. 4. The monoclonal antibody or antigen-binding fragment of any one of claims 1 to 3, comprising a human framework region.

5. V H and V L comprising the amino acid sequences set forth as SEQ ID NOs: 1 and 2, respectively; The monoclonal antibody or antigen-binding fragment of any one of claims 1 to 3.

6. The monoclonal antibody of any one of claims 1 to 5, comprising a human constant domain.

7. The monoclonal antibody of any one of claims 1 to 6, which is an IgG.

8. 8. The monoclonal antibody of any one of claims 1 to 7, comprising a recombinant constant domain comprising a modification that increases the half-life of the antibody.

9. 9. The monoclonal antibody or antigen-binding fragment of any one of claims 1 to 8, conjugated to a toxin or chemotherapeutic agent.

10. 10. The monoclonal antibody or antigen-binding fragment thereof of claim 9, wherein the toxin is Pseudomonas exotoxin (PE), ricin, abrin, diphtheria toxin, ribotoxin, ribonuclease, saporin, calicheamicin, or botulinum toxin.

11. 11. The monoclonal antibody or antigen-binding fragment of claim 10, wherein the toxin is PE, and the PE is PE25, PE38, or PE40.

12. 10. The monoclonal antibody or antigen-binding fragment of claim 9, wherein the chemotherapeutic agent is monomethyl auristatin E or a maytansinoid.

13. An antigen-binding fragment according to any one of claims 1 to 5 or 9 to 12.

14. Fv fragment, dsFv fragment, ds-scFv fragment, Fab fragment, F(ab') 2 fragment, scFv fragment, or scFv 2 14. The antigen-binding fragment of claim 13, which is a fragment.

15. 15. The monoclonal antibody or antigen-binding fragment of any one of claims 1 to 14, conjugated to a detectable marker.

16. A chimeric antigen T-cell receptor comprising the antigen-binding fragment of any one of claims 1 to 5 or 9 to 15.

17. A bispecific antibody comprising the monoclonal antibody or antigen-binding fragment of any one of claims 1 to 15.

18. The monoclonal antibody or antigen-binding fragment of any one of claims 1 to 15, or the V of said monoclonal antibody or antigen-binding fragment H Or V L or the chimeric antigen T cell receptor of claim 16. An isolated nucleic acid molecule encoding

19. V, shown as SEQ ID NOs: 3 and 4, respectively H and / or V L The nucleotide sequence of 20. The nucleic acid molecule of claim 18, comprising:

20. 20. The nucleic acid molecule of claim 18 or claim 19, which is a cDNA.

21. 21. The nucleic acid molecule of any one of claims 18 to 20, operably linked to a promoter.

22. A vector comprising the nucleic acid molecule of any one of claims 18 to 21.

23. 23. An isolated host cell comprising the nucleic acid molecule or vector of any one of claims 18 to 22.

24. 17. An isolated T cell expressing the chimeric antigen T cell receptor of claim 16.

25. an effective amount of the monoclonal antibody or antigen-binding fragment of any one of claims 1 to 15, the bispecific antibody of claim 17, a nucleic acid molecule encoding said monoclonal antibody or antigen-binding fragment, a vector comprising a nucleic acid molecule encoding said monoclonal antibody or antigen-binding fragment, or a T cell of claim 24; a pharmaceutically acceptable carrier; 10. A pharmaceutical composition for use in treating an EGFRvIII-expressing cancer, comprising:

26. A method for producing a monoclonal antibody or antigen-binding fragment that specifically binds to EGFRvIII, or a bispecific antibody comprising said monoclonal antibody or antigen-binding fragment, comprising: Expressing in a host cell one or more nucleic acid molecules encoding the monoclonal antibody or antigen-binding fragment of any one of claims 1 to 15, or the bispecific antibody of claim 17; and purifying the monoclonal antibody, the antigen-binding fragment, or the bispecific antibody. A method comprising:

27. 1. A method for detecting the presence of EGFRvIII in a biological sample derived from a human subject, comprising: contacting the biological sample with an effective amount of the monoclonal antibody or antigen-binding fragment of any one of claims 1 to 15 under conditions sufficient to form an immune complex; and detecting the presence of immune complexes in the biological sample, wherein the presence of immune complexes in the biological sample indicates the presence of EGFRvIII in the sample. A method comprising:

28. 28. The method of claim 27, wherein the subject has glioma, head and neck cancer, breast cancer, or bladder cancer.

29. 29. The method of claim 28, wherein the biological sample is a biopsy sample from glioma, head and neck cancer, breast cancer, or bladder cancer, respectively.

30. 26. A pharmaceutical composition for inhibiting a tumor expressing EGFRvIII in a subject having such a tumor, comprising the monoclonal antibody or antigen-binding fragment of any one of claims 1 to 15, the bispecific antibody of claim 17, a nucleic acid molecule encoding said monoclonal antibody or antigen-binding fragment, a vector comprising a nucleic acid molecule encoding said monoclonal antibody or antigen-binding fragment, or the T cell of claim 24.

31. 31. The pharmaceutical composition of claim 30, wherein the tumor is glioma, head and neck cancer, breast cancer, or bladder cancer.

32. 32. The pharmaceutical composition of claim 30 or 31, wherein the subject is a human.

33. 33. The pharmaceutical composition of any one of claims 30-32, wherein inhibiting the tumor comprises reducing tumor growth, size, or metastasis.

34. 26. A pharmaceutical composition for use in inhibiting tumors that overexpress EGFR in a subject having a tumor that expresses EGFRvIII, comprising the monoclonal antibody or antigen-binding fragment of any one of claims 1 to 15, the bispecific antibody of claim 17, a nucleic acid molecule encoding said monoclonal antibody or antigen-binding fragment, a vector comprising a nucleic acid molecule encoding said monoclonal antibody or antigen-binding fragment, or the T cell of claim 24.

35. 35. The pharmaceutical composition of claim 34, wherein the tumor is glioma, head and neck cancer, breast cancer, or bladder cancer.

36. 36. The pharmaceutical composition of claim 34 or 35, wherein the subject is a human.

37. 37. The pharmaceutical composition of any one of claims 34 to 36, wherein inhibiting the tumor comprises reducing tumor growth, size, or metastasis.

38. In the manufacture of a medicament for inhibiting an EGFRvIII-expressing tumor in a subject; or For detecting the presence of EGFRvIII in a biological sample, Use of the monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 15, a nucleic acid molecule encoding said monoclonal antibody or antigen-binding fragment, a vector comprising a nucleic acid molecule encoding said monoclonal antibody or antigen-binding fragment, or a T cell according to claim 24.

Images