Humanized 40H3 antibodies

Abstract

Disclosed are antibodies (e.g., humanized monoclonal antibodies and antigen binding fragments) that specifically bind to epidermal growth factor receptor variant III (EGFRvIII) and / or genetically amplified EGFR, as well as conjugates and chimeric antigen receptors (CARs) that include such antibodies. Also provided are nucleic acid molecules encoding the antibodies, conjugates or CARs disclosed herein, as well as host cells expressing these nucleic acid molecules. Further disclosed are methods of using the disclosed compositions, for example, for treating or detecting tumors, such as tumors expressing EGFRvIII and / or gene amplified EGFR.

Description

Cross-references to related applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 525,493, filed July 7, 2023, which is incorporated herein by reference in its entirety.

[0002] public domain This application relates to the field of cancer biology, particularly to humanized monoclonal antibodies and antigen-binding fragments that specifically bind to epidermal growth factor receptor mutants / variants (e.g., EGFRvIII and / or gene-amplified EGFR) expressed by tumor cells.

[0003] Government Support Statement This invention was developed with government funding from the National Cancer Institute of the National Institutes of Health (NIH), under project number Z01#: Z1A BC 008757. The U.S. government holds certain rights to this invention.

[0004] sequence list reference The sequence list was submitted as an XML file named "Sequence.xml", which was created on July 8, 2024 (1,386,226 bytes) and is incorporated into this article by reference.

[0005] background EGFR is frequently involved in the oncogenic progression of human cancers. Various alterations in its expression, including gene amplification and activating mutations, contribute to tumorigenesis. The human form of this large receptor has a 621-amino acid extracellular domain (ECD), a 23-amino acid single transmembrane domain (TM), and a 542-amino acid intracellular domain for enzyme activity (ICD). EGFR is a member of the receptor tyrosine kinase family and was the first receptor to show a positive correlation with human cancer. Ligand binding leads to receptor dimer formation and activation of the kinase domain, signaling to one of several pathways that can promote mammalian cell growth, survival, and spread. Activating mutations can occur in either the ECD or the ICD. Gene amplification and deletion can also activate EGFR. Exemplary deletions include the deletion of exons 2–7 (which produces EGFR variant III (EGFRvIII)) and the deletion of exon 19, which generates a constitutively active enzyme mutant. EGFR gene amplification, EGFRvIII expression, or EGFR exon 19 deletion has only been reported in cancer cells.

[0006] Antibodies typically bind tightly and specifically to their target antigens, making them useful therapeutic agents for a wide range of diseases characterized by alterations in protein expression, such as EGFRvIII or gene-amplified EGFR. However, for many therapeutic applications, the efficacy and safety of nonhuman antibodies are compromised because nonhuman immunoglobulin (Ig) molecules are inherently immunogenic and can induce anti-Ig immune responses, making repeated dosing highly impaired. Therefore, for therapeutic use, modifying antibodies to reduce or eliminate their immunogenicity can be beneficial. There remains a need for humanized antibodies that can bind to EGFRvIII or gene-amplified EGFR but not wild-type EGFR to exhibit cancer cell specificity.

[0007] Public Overview Disclosed are isolated humanized monoclonal antibodies and their antigen-binding fragments, wherein the monoclonal antibody specifically binds to EGFRvIII and / or EGFR gene amplification. In some aspects, the monoclonal antibody or antigen-binding fragment includes a heavy chain variable region (V... H ) and light chain variable region (V L ), the V H and V L : a) Contains SEQ ID NO: 5 and SEQ ID NO: 8 (A10, VH3+VL3), respectively; b) Contains SEQ ID NO: 3 and SEQ ID NO: 6 (A2, VH1+VL1), respectively; c) Contains SEQ ID NO: 3 and SEQ ID NO: 7 (A3, VH1+VL2), respectively; d) Contains SEQ ID NO: 3 and SEQ ID NO: 8 (A4, VH1+VL3), respectively; e) Contains SEQ ID NO: 4 and SEQ ID NO: 6 (A5, VH2+VL1), respectively; f) Contains SEQ ID NO: 4 and SEQ ID NO: 7 (A6, VH2+VL2), respectively; g) contains SEQ ID NO: 4 and SEQ ID NO: 8 (A7, VH2+VL3), respectively; h) contains SEQ ID NO: 5 and SEQ ID NO: 6 (A8, VH3+VL1), respectively. i) Contains SEQ ID NO: 5 and SEQ ID NO: 7 (A9, VH3+VL2), respectively; j) Contains SEQ ID NO: 1 and SEQ ID NO: 9 (B1, 40H3 VH+VL-EG); k) respectively contains SEQ ID NO: 1 and SEQ ID NO: 10 (B2, 40H3 VH+VL-DA); l) respectively contain SEQ ID NO: 4 and SEQ ID NO: 11 (B3, VH2+VL1-DA); m) respectively contains SEQ ID NO: 4 and SEQ ID NO: 12 (B4, VH2+VL2-DA); n) respectively contain SEQ ID NO: 5 and SEQ ID NO: 11 (B5, VH3 and VL1-DA); o) respectively contains SEQ ID NO: 31 and SEQ ID NO: 32 (C10); p) contains SEQ ID NO: 5 and SEQ ID NO: 14 (D2) respectively; or q) contains SEQ ID NO: 5 and SEQ ID NO: 24 (D3) respectively.

[0008] Conjugates of these antibodies and antigen-binding fragments are also disclosed. Further disclosure includes chimeric antigen receptors (CARs), comprising the disclosed antibodies or antigen-binding fragments, and cells expressing such chimeric antigen receptors (e.g., immune cells) (e.g., CAR T cells).

[0009] It also discloses the V encoding any antibody disclosed herein. H and / or V L Nucleic acid molecules, vectors containing these nucleic acids, and host cells transformed with these nucleic acids and / or vectors.

[0010] In a further aspect, the use of the monoclonal antibodies or antigen-binding fragments disclosed herein for inhibiting tumors expressing EGFRvIII or amplified EGFR in a subject is disclosed. In some embodiments, patients with tumors or cancers expressing EGFRvIII or amplified EGFR are selected for treatment. In other aspects, the use of the monoclonal antibodies or antigen-binding fragments disclosed herein for detecting EGFRvIII or amplified EGFR is disclosed.

[0011] The foregoing and other features will become more apparent from the following detailed description with reference to the accompanying drawings.

[0012] Brief description of the attached diagram Figure 1A-Figure 1BAn antibody that binds to rat glioma cells expressing human EGFRvIII. A10 exhibited the highest MFI compared to other candidates, including chimeric antibodies (mouse VH and VL+ human IgG1CH / human IgκCL derived from 40H3 antibody). Cetuximab was used as a universal positive control, binding to both WT and cancer-expressed EGFR (EGFRvIII).

[0013] Figures 2A-2B : Compared with human tumor cells expressing amplified EGFR (especially MDA-MB-468 cells) Figure 2A ) and A431 cells ( Figure 2B Antibodies that bind to antibodies. Antibody A10 exhibited the highest MFI.

[0014] Figures 3A-3B : Compared with cells expressing WT EGFR (especially WI-38 cells) Figure 3A ) or U87MG cells ( Figure 3B The A10 antibody does not bind to cells expressing WT EGFR. However, evidence of surface WT EGFR binding was confirmed by cetuximab binding.

[0015] Figures 4A-4B The table shows the antibody properties, including variable region combination, dissociation constant (KD), cell binding, and EC. 50 .

[0016] Figures 5A-5B Comparative data on antibody binding of 40H3, A10 and cetuximab. Figure 5A 40H3 and A10 showed strong binding to cells expressing amplified EGFR (MDA-MB-468), but weak binding to cells with EGFR-WT (WI-38). Conversely, cetuximab showed strong binding to cells with EGFR-WT. Figure 5B The results showed that both A10 and cetuximab strongly bound to cells with EGFRvIII.

[0017] Figures 6A-6C Crystal structure of EGFR ring-A10 Fab. Figure 6B and Figure 6C yes Figure 6A A magnified view of a portion of the image to show more details of the EGRF ring-A10 interaction.

[0018] Figure 7 Summary of information about the cell lines shown ( See (Column 1).

[0019] Figures 8A-8B pMH289-A10-1 ( Figure 8A(SEQ ID NO: 51) and pMH289-A10-2 ( Figure 8B The vector spectrum of SEQ ID NO: 52.

[0020] Figure 9 Cell viability of A431-luc cells 24 or 48 hours after the treatment shown. "CAR-1" and "CAR-2" are A10 scFv-CAR-T cells (respectively...). See Figure 8A and Figure 8B The control group consisted of activated T cells lacking transduction of the chimeric antigen receptor.

[0021] Figure 10 Cell viability of MDA-MB-468 cells after treatment with two A10 antibody-drug conjugates (ADCs): A10 conjugated with deruxtecan (A10-Dxd) or A10 conjugated with monomethylauristatin E (A10-MMAE).

[0022] sequence The listed nucleic acid and amino acid sequences are displayed using the standard letter abbreviations for nucleotide bases and amino acids as defined in 37 CFR 1.822. Only one strand is shown for each nucleic acid sequence, but by referring to the strands shown, it can be understood that the complementary strand is also included. In the heavy and light chain variable domain sequences, the CDR sequence (determined by the Kabat, IMGT, or Chothia numbering scheme) is underlined. In SEQ ID NO: 3-32, the framework residues shown in bold are humanized residues, specifically those corresponding to SEQ ID NO: 1 (V H (Sequence) or SEQ ID NO: 2 (V L Compared to the sequence.

[0023] SEQ ID NO: 1 is the amino acid sequence of the variable domain of the 40H3 heavy chain.

[0024] QVQLKQSGPGLVQPSQSLSITCTVSGFSLT NYGIH WLRQSPGKGLEWLGM MWRGGGT DYNAAFISRLTITKDTKSSQVFFRMNNLQTNDTAIYYC ARKGVGMGLG YWGQGTSVTVSS SEQ ID NO: 2 is the amino acid sequence of the variable domain of the 40H3 light chain.

[0025] DIQMTQSPASQSASLGESVTITC LASQTIGTWVA WYQQKPGRSPQLLIY GATNLAD GVPSRFSGSGSGTKFSFKISSLQAEDFVSYYC QQLYSNPYT FGGGTKLEIK SEQ ID NO: 3 is the amino acid sequence of the variable domain of the VH1 heavy chain.

[0026] QVTLKESGPVLVKPTETLTLTCTVSGFSLT NYGIH WIRQPPGKALEWLA MMWRGGGTDYNAAFIS RLTISKDTSKSQVVLTTMTNMDPVDTATYYCAR KGVGMGLGY WGQGTLVTVSS SEQ ID NO: 4 is the amino acid sequence of the variable domain of the VH2 heavy chain.

[0027] QVTLKESGPVLVKPTETLTLTCTVSGFSLT NYGIH WIRQPPGKALEWLG MMWRGGGTDYNAAFIS RLTITKDTSKSQVVLTTMTNMDPVDTATYYCAR KGVGMGLGY WGQGTLVTVSS SEQ ID NO: 5 is the amino acid sequence of the VH3 heavy chain variable domain, which is also the amino acid sequence of the D2 and D3 heavy chain variable domains.

[0028] QVTLKESGPVLVKPTETLTLTCTVSGFSLT NYGIH WLRQPPGKALEWLG MMWRGGGTDYNAAFIS RLTITKDTSKSQVVFTMTNMDPVDTATYYCAR KGVGMGLGY WGQGTLVTVSS SEQ ID NO: 6 is the amino acid sequence of the variable domain of the VL1 light chain.

[0029] DIQMTQSPSSVSASVGDRVTITC LASQTIGTWVA WYQQKPGKAPKLLIY GATNLAD GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQLYSNPYT FGGGTKLEIK SEQ ID NO: 7 is the amino acid sequence of the variable domain of the VL2 light chain.

[0030] DIQMTQSPSSVSASVGDRVTITC LASQTIGTWVAWYQQKPGKSPQLLIY GATNLAD GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQLYSNPYT FGGGTKLEIK SEQ ID NO: 8 is the amino acid sequence of the VL3 light chain variable domain, which is also the amino acid sequence of the D1 light chain variable domain.

[0031] DIQMTQSPSSVSASVGDRVTITC LASQTIGTWVA WYQQKPGKSPQLLIY GATNLAD GVPSRFSGSGSGTKFTLTISSLQPEDFATYYC QQLYSNPYT FGGGTKLEIK SEQ ID NO: 9 is the amino acid sequence of the variable domain of the VL-EG light chain.

[0032] DIQMTQSPASQSASLGESVTITC LASQTIGTWVA WYQQKPGRSPQLLIY GATNLA EGVPSRFSGSGSGTKFSFKISSLQAEDFVSYYC QQLYSNPYT FGGGTKLEIK SEQ ID NO: 10 is the amino acid sequence of the variable domain of the VL-DA light chain.

[0033] DIQMTQSPASQSASLGESVTITC LASQTIGTWVA WYQQKPGRSPQLLIY GATNLAD AVPSRFSGSGSGTKFSFKISSLQAEDFVSYYC QQLYSNPYT FGGGTKLEIK SEQ ID NO: 11 is the amino acid sequence of the variable domain of the VL1-DA light chain.

[0034] DIQMTQSPSSVSASVGDRVTITC LASQTIGTWVA WYQQKPGKAPKLLIY GATNLAD AVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQLYSNPYT FGGGTKLEIK SEQ ID NO: 12 is the amino acid sequence of the variable domain of the VL2-DA light chain.

[0035] DIQMTQSPSSVSASVGDRVTITC LASQTIGTWVA WYQQKPGKSPQLLIY GATNLADAVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQLYSNPYT FGGGTKLEIK SEQ ID NO: 13 is the amino acid sequence of the C1 heavy chain variable domain, which is also the amino acid sequence of the D1 heavy chain variable domain.

[0036] QVQLQESGPGLVKPSETLSLTCTVSGGSIS NYGIH WIRQPPGKGLEWIG MMWRGGGTDYNAAFIS RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR KGVGMGLGY WGQGTLVTVSS SEQ ID NO: 14 is the amino acid sequence of the C1 light chain variable domain, which is also the amino acid sequence of the D2 light chain variable domain.

[0037] DIQMTQSPSSVSASVGDRVTITC LASQTIGTWVA WYQQKPGKAPKLLIY GATNLAD GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQLYSNPYT FGQGTKLEIK SEQ ID NO: 15 is the amino acid sequence of the C2 heavy chain variable domain.

[0038] QVTLKESGPVLVKPTETLTLTCTVSGFSLT NYGIH WLRQPPGKALEWLG MMWRGGGTDYNAAFIS RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR KGVGMGLGY WGQGTLVTVSS SEQ ID NO: 16 is the amino acid sequence of the C2 light chain variable domain.

[0039] DIQMTQSPSSVSASVGDRVTITC LASQTIGTWVA WYQQKPGKAPKLLIY GATNLAD GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQLYSNPYT FGQGTKLEIK SEQ ID NO: 17 is the amino acid sequence of the C3 heavy chain variable domain.

[0040] QVQLQESGPGLVKPSETLSLTCTVSGGSIS NYGIH WIRQPPGKGLEWIG MMWRGGGTDYNAAFISRLTITKDTSKSQVVFTMTNMDPVDTATYYCAR KGVGMGLGY WGQGTLVTVSS SEQ ID NO: 18 is the amino acid sequence of the variable domain of the C3 light chain.

[0041] DIQMTQSPSSVSASVGDRVTITC LASQTIGTWVA WYQQKPGKAPKLLIY GATNLAD GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQLYSNPYT FGQGTKLEIK SEQ ID NO: 19 is the amino acid sequence of the C4 heavy chain variable domain.

[0042] QVQLQESGPGLVKPSETLSLTCTVSGGSIS NYGIH WIRQPPGKGLEWIG MMWRGGGTDYNAAFIS RLTISKDTSKNQVSLKLSSVTAADTAVYYCAR KGVGMGLGY WGQGTLVTVSS SEQ ID NO: 20 is the amino acid sequence of the variable domain of the C4 light chain.

[0043] DIQMTQSPSSVSASVGDRVTITC LASQTIGTWVA WYQQKPGKAPKLLIY GATNLAD GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQLYSNPYT FGQGTKLEIK SEQ ID NO: 21 is the amino acid sequence of the C5 heavy chain variable domain.

[0044] QVQLQESGPGLVKPSETLSLTCTVSGFSLT NYGIH WLRQPPGKGLEWIG MMWRGGGTDYNAAFIS RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR KGVGMGLGY WGQGTLVTVSS SEQ ID NO: 22 is the amino acid sequence of the variable domain of the C5 light chain.

[0045] DIQMTQSPSSVSASVGDRVTITC LASQTIGTWVA WYQQKPGKAPKLLIY GATNLAD GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQLYSNPYTFGQGTKLEIK SEQ ID NO: 23 is the amino acid sequence of the variable domain of the C6 heavy chain.

[0046] QVQLQESGPGLVKPSETLSLTCTVS GFSLTNY GWSWIRQPPGKGLEWIGY MWRGGGT NYNPSLKSRVTISSVDTSKNQFSLKLSSVTAADTAVYYC ARKGVGMGLGY WGQGTLVTVSS SEQ ID NO: 24 is the amino acid sequence of the C6 light chain variable domain, which is also the amino acid sequence of the D3 light chain variable domain.

[0047] DIQMTQSPSSVSASVGDRVTITCRAS QTIGTW LAWYQQKPGKAPKLLIY GAT SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQLYSNPYT FGQGTKLEIK SEQ ID NO: 25 is the amino acid sequence of the C7 heavy chain variable domain.

[0048] QVQLQESGPGLVKPSETLSLTCTVS GFSLTNY YWSWIRQPPGKGLEWIGYI WRGGG TNYNPSLKSRVTISSVDTSKNQFSLKLSSVTAADTAVYYCAR KGVGMGLGY WGQGTLVTVSS SEQ ID NO: 26 is the amino acid sequence of the variable domain of the C7 light chain.

[0049] DIQMTQSPSSVSASVGDRVTITC LASQTIGTWVA WYQQKPGKAPKLLIY GATNLAD GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQLYSNPYT FGQGTKLEIK SEQ ID NO: 27 is the amino acid sequence of the variable domain of the C8 heavy chain.

[0050] QVQLQESGPGLVKPSQTLSLTCTVSGGSIS NYGIH WIRQSPGKGLEWIGY MMWRGGGTDYNAAFIS RLTISKDTSKSQVVLTTMTNMDPVDTATYYCAR KGVGMGLGY FGQGTRLTVSS SEQ ID NO: 28 is the amino acid sequence of the variable domain of the C8 light chain.

[0051] DIQMTQSPSSLSASVGDRVTITC LASQTIGTWVA WYLQKPGQSPQLLIY GATNLAD GVPSRFSGSGSGTEFTLTISSLQPEDFATYYC QQLYSNPYT FGQGTKLEIK SEQ ID NO: 29 is the amino acid sequence of the variable domain of the C9 heavy chain.

[0052] QVTLKESGPVLVKPTETLTLTCTVSGFSLS NYGIH WIRQPPGKALEWLA MMWRGGGTDYNAAFIS RLTISKDTSKSQVVLTTMTNMDPVDTATYYCAR KGVGMGLGY WGQGTLVTVSS SEQ ID NO: 30 is the amino acid sequence of the variable domain of the C9 light chain.

[0053] DIQMTQSPSSVSASVGDRVTITC LASQTIGTWVA WYQQKPGKAPKLLIY GATNLAD GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQLYSNPYT FGQGTKLEIK SEQ ID NO: 31 is the amino acid sequence of the C10 heavy chain variable domain.

[0054] QVQLQESGPGLVKPSETLSLTCTVSGFSLT NYGIH WLRQPPGKGLEWIG MMWRGGGTDYNAAFIS RLTISKDTSKNQVSLKLSSVTAADTAVYYCAR KGVGMGLGY WGQGTLVTVSS SEQ ID NO: 32 is the amino acid sequence of the variable domain of the C10 light chain.

[0055] DIQMTQSPSSVSASVGDRVTITC LASQTIGTWVA WYQQKPGKAPKLLIY GATNLAD GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQLYSNPYT FGQGTKLEIK SEQ ID NO: 33-42 is the amino acid sequence of the components of the chimeric antigen receptor.

[0056] SEQ ID NO: 43 is the amino acid sequence of A10 VH-VL scFv.

[0057] QVTLKESGPVLVKPTETLTLTCTVSGFSLTNYGIHWLRQPPGKALEWLGMMWRGGGTDYNAAFISRLTITKDTSKSQVVFTMTNMDPVDTATYYCARKGVGMGLGYWGQGTLVTVSSGG GGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCLASQTIGTWVAWYQQKPGKSPQLLIYGATNLADGVPSRFSGSGSGTKFTLTISSLQPEDFATYYCQQLYSNPYTFGGGTKLEIK SEQ ID NO: 44 is an exemplary nucleic acid sequence encoding A10 VH-VL scFv.

[0058] CAAGTCACCCTGAAAGAATCCGGTCCGGTGCTCGTGAAGCCGACTGAGACCCTGACTCTGACTTGTACTGTATCCGGCTTCAGTCTGACCAACTATGGCATTCATTGGCTGCGCCAGCCGCCGGGCAAAGCCCTCGAGTGGCTGGGTATGATGTGGAGGGGCGGCGGCACCGATTACAATGCGGCTTTCATCTCCAGGCTCACTATTACCAAAGACACCTCCAAGAGCCAGGTTGTCTTCACCATGACCAACATGGACCCGGTAGACACCGCGACCTATTATTGCGCACGCAAAGGCGTGGGTATGGGCCTGGGCTACTGGGGCCAGGGTACTCTGGTTACGGTATCTTCCGGTGGTGGTGGCTCAGGCGGTGGTGGCTCCGGCGGCGGTGGCTCCGACATCCAGATGACCCAATCCCCGTCTTCTGTTTCCGCGAGCGTAGGCGATCGGGTAACCATCACTTGTCTGGCTTCCCAAACCATTGGCACGTGGGTTGCCTGGTACCAGCAGAAACCCGGGAAATCCCCACAGCTGCTGATCTATGGTGCCACCAACCTGGCTGACGGTGTTCCTTCCCGATTTAGTGGTTCCGGTAGCGGCACCAAATTTACCCTGACCATCTCCTCCCTGCAGCCGGAAGACTTTGCAACTTACTATTGTCAACAGTTGTACTCCAACCCTTACACTTTCGGTGGTGGTACCAAACTGGAAATCAAA SEQ ID NO: 45 is the amino acid sequence of A10 VL-VH scFv.

[0059] DIQMTQSPSSVSASVGDRVTITCLASQTIGTWVAWYQQKPGKSPQLLIYGATNLADGVPSRFSGSGSGTKFTLTISSLQPEDFATYYCQQLYSNPYTFGGGTKLEIKGGGGSGGGGSGGGGSQVTLKESGPVLVKPTETLTLTCTVSGFSLTNYGIHWLRQPPGKALEWLGMMWRGGGTDYNAAFISRLTITKDTSKSQVVFTMTNMDPVDTATYYCARKGVGMGLGYWGQGTLVTVSS SEQ ID NO: 46 is an exemplary nucleic acid sequence encoding A10 VL-VH scFv.

[0060] GACATCCAGATGACCCAATCCCCGTCTTCTGTTTCCGCGAGCGTAGGCGATCGGGTAACCATCACTTGTCTGGCTTCCCAAACCATTGGCACGTGGGTTGCCTGGTACCAGCAGAAACCCGGGAAATCCCCACAGCTGCTGATCTATGGTGCCACCAACCTGGCTGACGGTGTTCCTTCCCGATTTAGTGGTTCCGGTAGCGGCACCAAATTTACCCTGACCATCTCCTCCCTGCAGCCGGAAGACTTTGCAACTTACTATTGTCAACAGTTGTACTCCAACCCTTACACTTTCGGTGGTGGTACCAAACTGGAAATCAAAGGTGGTGGTGGCTCAGGCGGTGGTGGCTCCGGCGGCGGTGGCTCCCAAGTCACCCTGAAAGAATCCGGTCCGGTGCTCGTGAAGCCGACTGAGACCCTGACTCTGACTTGTACTGTATCCGGCTTCAGTCTGACCAACTATGGCATTCATTGGCTGCGCCAGCCGCCGGGCAAAGCCCTCGAGTGGCTGGGTATGATGTGGAGGGGCGGCGGCACCGATTACAATGCGGCTTTCATCTCCAGGCTCACTATTACCAAAGACACCTCCAAGAGCCAGGTTGTCTTCACCATGACCAACATGGACCCGGTAGACACCGCGACCTATTATTGCGCACGCAAAGGCGTGGGTATGGGCCTGGGCTACTGGGGCCAGGGTACTCTGGTTACGGTATCTTCC SEQ ID NO: 47 is the amino acid sequence of the A10 Fab (heavy chain). The sequence terminates at the site where papain cleavage occurs.

[0061] QVTLKESGPVLVKPTETLTLTCTVSGFSLTNYGIHWLRQPPGKALEWLGMMWRGGGTDYNAAFISRLTITKDTSKSQVVFTMTNMDPVDTATYYCARKGVGMGLGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH SEQ ID NO: 48 is an exemplary nucleic acid sequence encoding the A10 Fab (heavy chain).

[0062] CAAGTCACCCTGAAGGAGAGTGGGCCAGTTTTAGTTAAGCCCACCGAGACACTGACCCTGACCTGTACCGTGTCCGGCTTCTCTCTGACCAACTATGGCATCCACTGGCTGCGGCAGCCTCCTGGCAAGGCACTGGAATGGCTGGGCATGATGTGGCGGGGCGGCGGCACAGACTACAATGCCGCCTTCATCAGCCGGCTGACCATCACCAAGGACACCTCCAAGAGCCAGGTGGTGTTCACCATGACCAACATGGACCCTGTGGACACCGCCACCTACTACTGTGCCCGGAAGGGCGTGGGCATGGGCCTGGGCTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCTGCCAGTACCAAGGGACCCTCTGTGTTTCCTCTGGCTCCTTCCAGCAAGTCTACCTCTGGCGGCACAGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTTCCTGAGCCTGTGACCGTGTCTTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCAC SEQ ID NO: 49 is the amino acid sequence of A10 Fab (light chain).

[0063] DIQMTQSPSSVSASVGDRVTITCLASQTIGTWVAWYQQKPGKSPQLLIYGATNLADGVPSRFSGSGSGTKFTLTISSLQPEDFATYYCQQLYSNPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 50 is an exemplary nucleic acid sequence encoding A10 Fab (light chain).

[0064] GACATCCAGATGACCCAGTCTCCTTCCTCCGTGTCTGCTTCTGTGGGCGACAGAGTGACCATCACCTGCCTGGCCTCTCAGACCATCGGCACCTGGGTGGCCTGGTATCAGCAGAAGCCTGGCAAGTCCCCCCAGCTGCTGATCTATGGCGCCACCAACCTGGCAGATGGAGTGCCCTCCAGATTCTCTGGCTCTGGCTCTGGCACCAAGTTCACCCTGACCATCTCCTCCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCAGCTGTACAGCAACCCTTACACCTTTGGCGGCGGCACCAAGCTGGAGATCAAGAGAACAGTGGCTGCTCCTTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACAGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCTCGGGAAGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGTCCGGCAACTCCCAAGAGTCTGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGTCCTCCACACTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCATCAGGGCCTGTCTAGCCCTGTGACCAAGTCTTTCAACCGGGGCGAGTGC SEQ ID NO: 51 is an exemplary sequence encoding the pMH289-A10-1 construct.

[0065] SEQ ID NO: 52 is an exemplary sequence encoding the pMH289-A10-2 construct.

[0066] Detailed description I. Summary This article discloses humanized monoclonal antibodies and their antigen-binding fragments that specifically bind to mutated / variant EGFRs expressed by cancer cells (e.g., EGFRvIII or gene-amplified EGFR), but not to wild-type EGFR expressed on normal cells. These humanized monoclonal antibodies not only exhibit low immunogenicity, but also bind more strongly to target EGFRvIII and / or gene-amplified EGFR.

[0067] Activation of wild-type EGFR involves dimerization, which requires ligand binding and a monomeric-to-dimer transition, accompanied by a change in receptor conformation. Several extracellular domain structures of EGFR in monomeric and dimeric conformations have been reported. Analysis of these structures indicates the presence of unexposed residues in the wild-type receptor. However, under oncogenic conditions, the receptor is highly expressed and may not be properly folded, or a mutant version of the receptor may be expressed, potentially exposing these hidden structures. One structural element that is spatially inaccessible under normal conditions is the 287-302 (the number for the mature receptor – or 301-326 for the full-length receptor) disulfide-restriction loop. This loop is exposed in EGFRvIII and may become exposed when receptor expression is very high or when ECD mutations alter the wild-type structure. In several instances, the antibody or antigen-binding fragments disclosed herein bind to EGFR. 287-302 Cycle binding. It also provides inhibition or treatment of EGFR with cellular exposure. 287-302 Methods for treating tumors with ring-shaped structures are also provided. Methods for inhibiting tumors that overexpress EGFR in subjects are also provided.

[0068] II. Terminology Unless otherwise stated, technical terms are used according to convention. Definitions of commonly used terms in molecular biology can be found in Benjamin Lewin's work. Genes X , published by Jones&Bartlett Publishers, 2009; and Meyers et al. (eds.), The Encyclopedia of Cell Biology and Molecular Medicine Published by Wiley-VCH in 16 volumes, 2008; and other similar references.

[0069] Unless the context clearly indicates otherwise, as used herein, the singular forms “a,” “an,” and “the” refer to both the singular and the plural. For example, the term “antigen (an antigen)” includes one or more antigens and can be considered equivalent to the phrase “at least one antigen.” As used herein, the term “comprising” means “including.” It should also be understood that, unless otherwise stated, any and all base sizes or amino acid sizes given for nucleic acids or polypeptides, as well as all molecular weight or molecular mass values, are approximate and provided for descriptive purposes. Although many methods and materials similar to or equivalent to those described and used herein may be used, particularly suitable methods and materials are described herein. In case of conflict, this specification (including the explanation of terms) shall prevail. Furthermore, these materials, methods, and examples are illustrative only and are not intended to be limiting.

[0070] To facilitate a comprehensive overview, the following terminology is explained: Approximately: Unless the context otherwise indicates, “approximately” means plus or minus 5% of a reference value. For example, “approximately” 100 means 95 to 105.

[0071] Administration: The composition is introduced into a subject via a chosen route. Administration can be local or systemic. For example, if the chosen route is intravenous, the composition (e.g., a composition comprising a disclosed humanized monoclonal antibody or antigen-binding fragment) is introduced into the subject's vein to administer the composition. Exemplary routes of administration include, but are not limited to, oral, injectable (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, intratumoral, and intravenous), sublingual, rectal, transdermal (e.g., topical), intranasal, vaginal, and inhalation routes. In one instance, administration is systemic. In one instance, administration is local, such as direct application to a tumor.

[0072] Reagent: Any substance or combination of substances useful in achieving an outcome or result; for example, a substance or combination of substances useful in inhibiting tumor growth or metastasis in a subject. Reagents include proteins, nucleic acid molecules, compounds, small molecules, organic compounds, inorganic compounds, or other molecules of interest. Reagents may include therapeutic agents (e.g., chemotherapeutic agents), diagnostic agents, or pharmaceutical agents. In some examples disclosed herein, the reagent is a humanized monoclonal antibody that specifically binds to EGFRvIII and / or gene-amplified EGFR, its antigen-binding fragment, its conjugate, or a chimeric antigen receptor (CAR) comprising such antibody or antigen-binding fragment. Those skilled in the art will understand that a particular reagent can be used to achieve more than one outcome.

[0073] Amino acid substitution: An amino acid in a polypeptide chain is replaced by a different amino acid, or is replaced by an amino acid that is absent (i.e., missing). 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 a humanized monoclonal antibody that specifically binds to EGFRvIII or its antigen-binding fragment can be substituted with the corresponding amino acid in another antibody that specifically binds to EGFRvIII or its antigen-binding fragment.

[0074] Antibody: An immunoglobulin, antigen-binding fragment, or derivative thereof, which specifically binds to and recognizes an analyte (antigen), such as EGFRvIII. The term “antibody” is used in the broadest sense herein 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, provided they exhibit the desired antigen-binding activity.

[0075] Non-limiting examples of antibodies include, for example, intact immunoglobulins and their variants, and antigen-binding fragments that retain binding affinity for the antigen. Examples of antigen-binding fragments include, but are not limited to, Fv, Fab, dsFv, Fab', Fab'-SH, F(ab')2; diabody; 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 generated by modifying intact antibodies or antigen-binding fragments synthesized de novo using recombinant DNA methods. See For example, Kontermann and Dübel (Eds.), Antibody Engineering Vols. 1-2, 2 nd ed., Springer-Verlag, 2010).

[0076] Antibodies also include genetically engineered forms, such as chimeric antibodies (e.g., humanized mouse antibodies) and heteroconjugated antibodies (e.g., bispecific antibodies).

[0077] Antibodies can have one or more binding sites. If there are more than one binding site, these binding sites can be the same or different from each other. For example, naturally occurring immunoglobulins have two identical binding sites, single-chain antibodies or Fab fragments have one binding site, while bispecific or bifunctional antibodies have two different binding sites.

[0078] Naturally occurring immunoglobulins typically consist of heavy (H) chains and light (L) chains linked together by disulfide bonds. Immunoglobulin genes include κ, λ, α, γ, δ, ε, and μ 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.

[0079] Each heavy and light chain contains a constant region (or constant domain) and a variable region (or variable domain). The variable regions of the heavy and light chains bind together to specifically bind antigens.

[0080] Mentioning "V" H "VH" or "VH" refers to the variable region of the antibody heavy chain, including the variable region of antigen-binding fragments (such as Fv, scFv, dsFv, or Fab). Mentioning "V"... L "VL" or "VL" refers to the variable region of the antibody light chain, including the variable regions of Fv, scFv, ds-scFv, or Fab.

[0081] V H and V L It contains a "framework" region separated by three high-variability regions (also known as "complementary determination regions" or "CDRs"). See For example, Kabat et al. , Sequences of Proteins of Immunological Interest , 5 th (ed., NIHP Publication No. 91-3242, Public Health Service, National Institutes of Health, US Department of Health and Human Services, 1991). The framework region sequences of different light or heavy chains are relatively conserved within species. The antibody's framework region, i.e., the combined framework region of the constitutive light and heavy chains, is used to locate and align CDRs in three-dimensional space.

[0082] CDRs are primarily responsible for binding to epitopes of antigens. The amino acid sequence boundaries of a given CDR can be readily determined using any of a variety of methods, including those employed by Kabat et al. (…). Sequences of Proteins of Immunological Interest , 5 thed., NIH Publication No. 91-3242, Public Health Service, National Institutes of Health, US Department of Health and Human Services, 1991; “Kabat” numbering scheme), Al-Lazikani et al. (“Standard conformations for the canonicalstructures 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; those schemes described by the “IMGT” numbering scheme). The CDRs of each chain are usually referred to as CDR1, CDR2, and CDR3 (from N-end to C-end), and are usually also identified by the chain in which the specific CDR is located. Therefore, V H CDR3 is derived from the V of its associated antibody. H CDR3, and V L CDR1 is derived from the V of its associated antibody. L The CDR1 of the region. Light chain CDRs are sometimes also referred to as LCDR1, LCDR2, and LCDR3. Heavy chain CDRs are sometimes referred to as HCDR1, HCDR2, and HCDR3.

[0083] In some instances, disclosed humanized monoclonal antibodies include heterologous constant domains. For example, the antibody may include a constant domain that differs from the natural constant domain, such as a constant domain that includes one or more modifications (e.g., "LS" mutations) to extend its half-life.

[0084] "Monoclonal antibody" is an antibody obtained from a substantially homogeneous group of antibodies, meaning that individual antibodies comprising that group are identical and / or bind to the same epitopes, except for possible variant antibodies, such as those containing naturally occurring mutations or variants generated during the production of the monoclonal antibody preparation, which are typically present in very small amounts. In contrast to polyclonal antibody preparations, which typically comprise different antibodies targeting different determinants (epitopes), each antibody in a monoclonal antibody preparation targets a single determinant on the antigen. Therefore, the modifier "monoclonal" indicates that the antibody is characterized by being obtained from a substantially homogeneous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies can be prepared 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 described herein for the preparation of monoclonal antibodies. In some instances, monoclonal antibodies are isolated from a subject, such as a mammal, like a human. Monoclonal antibodies may have conserved amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin function. See For example, Greenfield (Ed.), Antibodies: A Laboratory Manual , 2 nd ed. New York: Cold Spring Harbor Laboratory Press, 2014).

[0085] Compared to their counterparts, non-human (e.g., mouse, rat, or synthetic) antibodies or antigen-binding fragments, “humanized” antibodies or antigen-binding fragments include amino acid substitutions, typically in the human frame region. In some instances of this disclosure, the non-human antibody or antigen-binding fragment providing the CDR is referred to as the “donor,” and the human antibody or antigen-binding fragment providing the frame is referred to as the “recipient.” In some instances of this disclosure, all CDRs and many frame residues are derived from the donor immunoglobulin, but some frame residues at specific locations are substituted with amino acids from the corresponding locations in the human frame region. Constant regions are not required to be present, but if present, they can be substantially identical to the constant regions of human immunoglobulins, for example, at least about 85-90%, or about 95% or higher.

[0086] A chimeric antibody is an antibody that comprises sequences derived from two different antibodies that typically belong to different species. In some instances, a chimeric antibody includes one or more CDRs and / or frame regions from one antibody (e.g., a mouse antibody) and CDRs and / or frame regions from another antibody (e.g., a human antibody).

[0087] "Fully human antibody" or "human antibody" is an antibody that includes sequences from (or derived from) the human genome and excludes sequences from other species. For example, a human antibody may include the CDR, frame region, and (if present) Fc region from (or derived from) the human genome. Human antibodies can be identified and isolated using techniques for constructing antibodies based on sequences derived from the human genome, such as through phage display or using transgenic animals. See For example, Barbas et al. Phage display:A Laboratory Manuel .1 st Ed. New York: Cold Spring Harbor Laboratory Press, 2004. Print.; Lonberg, Nat. Biotech., 23: 1117-1125, 2005; Lonenberg, Curr. Opin. Immunol ., 20:450-459, 2008).

[0088] Biological samples: Samples obtained from a subject. Biological samples include all clinical samples that can be used to detect a disease or tumor in a subject (e.g., head and neck cancer, breast cancer, pancreatic cancer, colorectal cancer, bladder cancer, or glioma), including but not limited to cells, tissues, and body fluids such as blood, blood derivatives and fractions (e.g., serum), cerebrospinal fluid; and tissues obtained through biopsy or surgical removal, such as unfixed tissue, frozen tissue, or tissue fixed in formalin or paraffin. In specific instances, biological samples are obtained from subjects who have or are suspected of having a tumor (e.g., but not limited to head and neck cancer, breast cancer, pancreatic cancer, colorectal cancer, glioma, or bladder cancer).

[0089] Bispecific antibodies: Recombinant molecules composed of two distinct antigen-binding domains, thus binding to two different antigenic epitopes. Bispecific antibodies consist of molecules in which two antigen-binding domains are chemically or genetically linked. The antigen-binding domains can be linked using adapters. The antigen-binding domains can be monoclonal antibodies, antigen-binding fragments (e.g., Fab, scFv, ds-scFv), or combinations thereof. Bispecific antibodies may include one or more constant domains, but do not necessarily include constant domains.

[0090] Cancer: A malignant tumor including transformed epithelial cells. Non-limiting examples of cancer include adenocarcinoma, squamous cell carcinoma, undifferentiated carcinoma, as well as large cell carcinoma and small cell carcinoma. In some instances, cancer is breast cancer, head and neck cancer, pancreatic cancer, colorectal cancer, glioma, or bladder cancer.

[0091] Chemotherapy agents: Any chemical agent that is therapeutically useful in treating diseases characterized by abnormal cell growth. For example, chemotherapeutic agents can be used to treat cancers, including but not limited to head and neck cancer, breast cancer, pancreatic cancer, colorectal cancer, bladder cancer, and glioma. In a non-limiting example, a chemotherapeutic agent is an agent used to treat cancer. Specific examples of other therapeutic agents that can be used in combination with publicly disclosed humanized antibodies include one or more of microtubule binding agents, DNA intercalating agents or cross-linking agents, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors. In a non-limiting example, a chemotherapeutic agent is a radioactive compound. Other examples include the use of the antitumor drugs 5-fluorouracil (5-FU) and IRT. A person skilled in the art can readily identify the chemotherapeutic agents used. See For example, Slapak and Kufe, Principles of Cancer Therapy , Chapter 86 in Harrison's Principles of InternalMedicine, 14 th edition; Perry et al ., Chemotherapy , Ch. 17 in Abeloff, ClinicalOncology 2 nd ed., © 2000 Churchill Livingstone, Inc; Baltzer, L., Berkery, R.(eds): Oncology Pocket Guide to Chemotherapy , 2 nd ed. St. Louis, Mosby-YearBook, 1995; Fischer, DS, Knobf, MF, Durivage, HJ (eds): The Cancer Chemotherapy Handbook , 4 th ed. St. Louis, Mosby-Year Book, 1993; Chabner andLongo, Cancer Chemotherapy and Biotherapy: Principles and Practice (4 th ed.).Philadelphia: Lippincott Willians&Wilkins, 2005; Skeel, Handbook of Cancer Chemotherapy (6 th (Lippincott Williams & Wilkins, 2003). Combination chemotherapy involves administering more than one agent to treat cancer, such as one or more humanized antibodies and another chemotherapeutic agent. These agents can be administered sequentially or simultaneously.

[0092] Chimeric antigen receptor (CAR): An engineered T-cell receptor having an extracellular antibody-derived targeting domain (e.g., scFv) that is linked 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 preparing CARs (e.g., for cancer treatment) have been described. See For example, 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 Pubs. WO2012 / 079000, WO2013 / 059593; and U.S. Publication No. 2012 / 0213783).

[0093] Conditions sufficient for the formation of immune complexes: conditions that allow an antibody or antigen-binding fragment to bind detectably to its homologous epitope to a greater degree than to all other epitopes, and / or substantially exclude binding to all other epitopes. The conditions sufficient for the formation of immune complexes depend on the form of the binding reaction and are generally the conditions utilized in the immunoassay protocol or encountered in vivo. A description of the immunoassay forms and conditions is provided below. See For example, Harlow & Lane, Antibodies, A Laboratory Manual , 2 nd ed. Cold Spring Harbor Publications, New York (2013). The conditions used in these methods are “physiological conditions,” which include references to conditions typical of living mammals or mammalian cells (e.g., temperature, osmotic pressure, pH). While it is recognized that some organs suffer from extreme conditions, the environment within organisms and cells is generally at around pH 7 (e.g., pH 6.0 to 8.0, more typically pH 6.5 to 7.5), contains water as the primary solvent, and exists at temperatures above 0°C but below 50°C. Osmotic pressure is within the range that supports cell viability and proliferation.

[0094] Conjugates: Complexes of two molecules linked together (e.g., covalently). In one instance, an antibody is linked to an effector molecule; for example, an antibody or antigen-binding fragment disclosed herein is covalently linked to an effector molecule. This linking can be achieved chemically or through recombination. In one instance, this linking is chemical, where a reaction between the antibody portion and the effector molecule creates a covalent bond between the two molecules to form a single molecule. Optionally, a peptide linker (short peptide sequence) may be included between the antibody and the effector molecule. Because conjugates can be prepared from two molecules with different functions (e.g., an antibody and an effector molecule), they are sometimes referred to as “chimeric molecules.”

[0095] Conservative variants: Sequence variants resulting from conserved substitutions. "Conserved" amino acid substitutions do not substantially affect or reduce protein function, such as the protein's ability to interact with target proteins. For example, the EGFRvIII-specific and / or gene-amplified EGFR-specific humanized monoclonal antibodies disclosed herein may include up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conserved substitutions compared to a reference antibody sequence, and retain specific binding activity to either EGFRvIII or gene-amplified EGFR, respectively. The term "conservative variant" also includes the use of substituted amino acids in place of unsubstituted parental amino acids.

[0096] A single substitution, deletion, or addition of a single amino acid or a small fraction of amino acids (e.g., less than 5%, less than 3%, less than 1%, etc.) in the coding sequence is a conserved variation in which these changes result in the substitution of the amino acid by a chemically similar amino acid.

[0097] The following six groups are examples of amino acids that are considered to be conserved substitutes for each other: 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).

[0098] Non-conserved substitutions are substitutions that reduce antibody activity or function (e.g., the ability to specifically bind to EGFRvIII and / or amplified EGFR, or the ability to bind to cancer cells expressing EGFRvIII and / or amplified EGFR). For example, if a particular amino acid residue is essential for the function of a protein, even a normally conserved substitution may impair that activity. Therefore, conserved substitutions do not alter the fundamental function of the protein of interest.

[0099] Contact: refers to direct physical association; it includes both solid and liquid forms and can occur in vivo or in vitro. Contact includes the contact between one molecule and another, such as the contact between an amino acid on the surface of one polypeptide (e.g., a peptide) and another polypeptide. Contact can also include contact with cells, for example, by directly physically associating the polypeptide with a cell.

[0100] Control: A reference standard. In some instances, the control is a negative control sample obtained from healthy subjects (e.g., subjects without tumors) or subjects without tumors expressing EGFRvIII or EGFR gene amplification. In other instances, the control is a positive control sample obtained from subjects diagnosed with tumors expressing EGFRvIII and / or EGFR gene amplification or purified EGFRvIII recombinantly generated. In still other instances, the control is a historical control or a standard reference value or range of values ​​(e.g., a control sample previously tested, such as a group of subjects with a known prognosis or outcome, or a sample group representing baseline or normal values). A qualified professional can readily determine an appropriate control.

[0101] The difference between the test sample and the control can be an increase or, conversely, a decrease. The difference can be qualitative or quantitative, such as a statistically significant difference. In some instances, the difference is an increase or decrease of at least about 5% relative to the control, 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 greater than 500%. In some instances, treatment with the publicly available antibody resulted in a reduction in tumor growth, volume, and / or metastasis.

[0102] Reduction or decrease: reduction in quality, quantity, or intensity; for example, a reduction in tumor burden. In one instance, the therapy reduces the tumor (e.g., tumor size, number of tumors, tumor metastasis, or a combination thereof) or one or more symptoms associated with the tumor, compared to the response without treatment. In a specific instance, after receiving the therapy, the therapy reduces the size of the tumor, the number of tumors, tumor metastasis, or a combination thereof, for example, by 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 reductions can be measured using the methods disclosed herein.

[0103] Degenerate variants: In the context of this disclosure, a “degenerate variant” refers to a polynucleotide sequence variant resulting from redundancy in the genetic code (which contains 20 naturally occurring amino acids, most of which are specified by more than one codon). Thus, although degenerate variants have different nucleotide sequences, they encode the same peptide.

[0104] Detectable biomarkers: Detectable molecules (also called markers) that are directly or indirectly conjugated to a second molecule, such as humanized monoclonal antibodies, to facilitate the detection of the second molecule. For example, detectable biomarkers can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or diagnostic imaging techniques (e.g., CT scans, MRI, ultrasound, fiberoptic endoscopy, and laparoscopy). Specific, non-limiting examples of detectable biomarkers include fluorophores, chemiluminescent agents, enzyme-linked compounds, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for MRI detection). Methods for using detectable biomarkers and guidance on selecting detectable biomarkers suitable for various purposes are discussed, for example, in Green and Sambrook (…). Molecular Cloning: A Laboratory Manual , 4 th (ed., New York: ColdSpring Harbor Laboratory Press, 2012) and Ausubel et al. (editors) Current Protocols in Molecular Biology (New York: John Wiley and Sons, including supplement, 2017).

[0105] Detection: Identifying the existence, appearance, or fact of something.

[0106] Effective amount: The amount of a specific substance sufficient to achieve the desired effect in a subject who has been administered the substance, such as a therapeutically effective amount for treating a disease. For example, this could be the amount of humanized monoclonal antibody required to inhibit tumor growth and / or metastasis, or to measurably alter the external symptoms of a tumor.

[0107] In some respects, compared with a suitable control, the administration of an effective amount of a publicly available humanized monoclonal antibody or antigen-binding fragment (which binds to EGFRvIII and / or gene-amplified EGFR) reduces or inhibits tumor growth, tumor metastasis, or tumor volume by a desired amount, for example, at least 10%, at least 20%, at least 30%, at least 40%, 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% (tumor elimination).

[0108] The effective amount of humanized monoclonal antibody or antigen-binding fragment specifically binding to EGFRvIII and / or gene-amplified EGFR administered to a subject may vary depending on a number of factors associated with that subject, such as the subject's overall health and / or weight. The effective amount can be determined by varying the dose and measuring the resulting response (e.g., reduction in tumor burden). The effective amount can also be determined by various in vitro, in vivo, or in situ immunoassays or clinical trials.

[0109] Effective doses encompass fractions of the reagent that are combined with prior or subsequent administrations to contribute to an effective response. For example, over a course of treatment lasting several days or weeks, an effective dose of the reagent may be administered in single or multiple doses, such as daily. However, the effective dose can vary depending on the subject being treated, the severity and type of the condition being treated, and the method of administration. The reagent may be packaged in unit dosage forms of the effective dose or multiples thereof, for example, in vials with sterile components (e.g., with a puncture cap) or syringes.

[0110] Effector molecule: A molecule intended to have or produce a desired effect; for example, to produce a desired effect on cells targeted by the effector molecule. Effector molecules include molecules such as chemical compounds, peptides, 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 may have or produce more than one desired effect. In one example, the effector molecule includes a disclosed humanized monoclonal antibody or a fragment thereof. In another example, the effector molecule is a chimeric molecule that includes a disclosed humanized monoclonal antibody or a fragment thereof, intended to produce a desired effect on cells targeted by the chimeric molecule.

[0111] 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, EGF binds to its ligand and activates the RTK / RAS / PI(3)K pathway via receptor phosphorylation, leading to increased cell proliferation, angiogenesis, local tissue invasion, and anti-apoptosis. The nucleic acid sequence of human EGFR can be found in GENBANK® accession number NM_005228.5 (June 18, 2019) and GENBANK® accession number NC_000007.14 (EGFR in chromosomes) (June 14, 2019), both of which are incorporated herein by reference. EGFR has a 621-amino acid extracellular domain (ECD), a 23-amino acid single transmembrane domain (TM), and a 542-amino acid intracellular domain for enzyme activity (ICD). Ligand binding leads to receptor dimer formation and activation of the kinase domain, signaling to one of several pathways that promote mammalian cell growth, survival, and proliferation. In tumors, deletion of exons 2–7 produces EGFR variant III (EGFRvIII), which has constitutive activity. The cDNA sequence of EGFRvIII is available at GENBANK® accession number NM_001346941 (June 18, 2019), which is incorporated herein by reference, and the amino acid sequence is available at NP_001333870.1 (June 14, 2019), which is incorporated herein by reference. Activation of wild-type EGFR involves dimerization, which requires ligand binding and a monomer-to-dimer transition, accompanied by a change in receptor conformation. Activating mutations can occur in the ECD or ICD. Deletion of exon 19 produces a constitutively active enzyme mutant. EGFR can be overexpressed through gene amplification or by reducing transcriptional control. High levels of expression (e.g., more than approximately 50,000 receptors per cell) lead to receptor misfolding or mutations in one or more gene copies. Overexpression can result in a two-fold or greater increase in the presence of EGFR in cells compared to wild-type controls. One structural element that is spatially inaccessible under normal conditions is the 287-302 (the number for the mature receptor – or 301-326 for the full-length receptor) disulfide restriction loop. This loop is exposed in EGFRvIII and may become exposed when receptor expression is very high or when ECD mutations alter the wild-type structure.

[0112] EGFR and EGFRvIII are known to be expressed in cancers in non-human animals. See For example, Cho et al. Oncology 58(4): 674-682, 2021). Non-human primate EGFR has the same response loop sequence as human EGFR (EGFR...). 287-302(A ring), where the antibodies disclosed herein can bind. In some instances, the antibodies disclosed herein bind to mammalian EGFRvIII or genetically amplified EGFR. In some instances, the antibodies disclosed herein bind to human, non-human primate, canine, or feline EGFRvIII or genetically amplified EGFR. In non-limiting instances, the antibodies disclosed herein bind to human, non-human primate, canine, or feline EGFRvIII.

[0113] Epitopes: Antigenic determinants. These are specific chemical groups or peptide sequences on a molecule that are antigenic, causing them to elicit a specific immune response. For example, an epitope is an antigenic region to which B cells and / or T cells respond. Antibodies can bind to specific antigenic epitopes (e.g., epitopes on EGFRvIII).

[0114] 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, and in some instances, these RNA or RNA fragments are processed into mRNA. A gene is also expressed when its mRNA is translated into an amino acid sequence (e.g., a protein or protein fragment). In a specific instance, a heterologous gene is expressed when it is transcribed into RNA. In another instance, a heterologous gene is expressed when its RNA is translated into an amino acid sequence. In this document, the term “expression” is used to refer to transcription or translation. Regulation of expression can include control over transcription, translation, RNA transport and processing, control over the degradation of intermediate molecules (e.g., mRNA), or control over the activation, inactivation, compartmentalization, or degradation of a particular protein molecule after its production.

[0115] Expression control sequences: Nucleic acid sequences that regulate the expression of a heterologous nucleic acid sequence to which they are operatively linked. These expression control sequences are operatively linked to the nucleic acid sequence when they control and regulate transcription and (if applicable) translation of the nucleic acid sequence. Therefore, expression control sequences may include suitable promoters, enhancers, transcription terminators, start codons (ATGs) preceding the protein-coding gene, intron splicing signals that maintain the correct reading frame of the gene to allow correct mRNA translation, and stop codons. The term "control sequence" is intended to include at least the components whose presence can affect expression, and may also include additional components whose presence is advantageous, such as leader sequences and fusion coupler sequences. Expression control sequences may include promoters.

[0116] A promoter is the smallest sequence sufficient to direct transcription. It also includes promoter elements sufficient to make promoter-dependent gene expression cell-type specific, tissue-specific, or inducible to external signals or reagents; such elements can be located in the 5' or 3' region of a gene. These include constitutive promoters and inducible promoters (…). See For example, Bitter et al. , Methods in Enzymology (153:516-544, 1987). For example, when cloning in bacterial systems, exemplary inducible promoters can be used, including pL, plac, ptrp, ptac (ptrp-lac heterozygous promoters) of bacteriophage λ. When cloning in mammalian cell systems, non-restrictive exemplary promoters include promoters derived from mammalian cell genomes (e.g., metallothionein promoters) or mammalian viruses (e.g., retroviral long terminal repeat sequences; adenovirus late promoters; vaccinia virus 7.5K promoters). Promoters generated by recombinant DNA or synthetic techniques can also be used to provide nucleic acid sequences for transcription.

[0117] Polynucleotides can be inserted into expression vectors containing promoter sequences, which promotes the transcription of the inserted gene sequence in host cells. Expression vectors typically contain an origin of replication, a promoter, and specific nucleic acid sequences that allow for phenotypic selection in transformed cells.

[0118] Expression vectors: Vectors comprising expression control sequences (e.g., promoters, enhancers, terminators, etc.) operatively linked to the nucleotide sequence to be expressed. Expression vectors contain sufficient cis-acting expression elements; other expression elements may be provided by the host cell or an in vitro expression system. Expression vectors include all vectors known in the art, such as granules, plasmids (e.g., naked plasmids or plasmids contained in liposomes), and viral vectors (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0119] FC Region: The constant region of the antibody, excluding the first heavy chain constant domain. The Fc region typically 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-terminus of these domains. For IgA and IgM, the Fc region may or may not include a tailpiece and may or may not bind via the J chain. For IgG, the Fc region is generally understood to include immunoglobulin domains Cγ2 and Cγ3, and optionally the lower hinge portion between Cγ1 and Cγ2. Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is generally defined as including residues from C226 or P230 to the Fc carboxyl terminus, where numbering follows Kabat. For IgA, the Fc region includes immunoglobulin domains Cα2 and Cα3, and optionally the lower hinge portion between Cα1 and Cα2.

[0120] Frame regions: Amino acid sequences inserted between CDRs within the variable regions of the antibody heavy or light chain. These include the variable light chain and variable heavy chain frame regions. The role of the frame regions is to maintain the CDRs in their proper positioning.

[0121] Amplified EGFR: An increase in the EGFR gene copy number in cells (e.g., tumor cells) relative to normal cells (e.g., non-tumor cells) leads to EGFR overexpression. Generally, cells (e.g., tumor cells) containing more than 6 EGFR copies are considered to have amplified EGFR. Amplified EGFR has been described, for example, in French... et al ."Defining EGFR amplification status" Neuro-Oncology , 21(19): 1263-1272, 2019.

[0122] In some instances, the amplified EGFR gene includes EGFR genes with a copy number greater than 6, for example, EGFR genes with a copy number of at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30 or more. In non-limiting instances, the amplified EGFR gene includes EGFR genes with a copy number of at least 7. In some instances, the amplified EGFR gene comprises 6 to 200 copies of the EGFR gene, such as 6 to 150, 6 to 100, 6 to 75, 6 to 50, 6 to 25, 6 to 10, 7 to 150, 7 to 100, 7 to 75, 7 to 50, 7 to 25, 7 to 10, 8 to 150, 8 to 100, 8 to 75, 8 to 50, 8 to 25, 8 to 10, 10 to 150, 10 to 100, 10 to 75, 10 to 50, 10 to 25, 15 to 150, 15 to 100, 15 to 75, 15 to 50, 15 to 25, 20 to 150, 20 to 100, 20 to 75, or 20 to 50 copies of the EGFR gene. In non-limiting instances, the amplified EGFR gene comprises 7 to 100 copies of the EGFR gene. Gene amplification leads to EGFR overexpression in cells (e.g., tumor cells). In some instances, at least one copy of EGFR includes an activating mutation (e.g., a substitution, insertion, or deletion that activates EGFR).

[0123] Heterologous: derived from a different genetic source. A nucleic acid molecule heterologous to a cell originates from a genetic source other than that cell. In certain non-limiting instances, a heterologous nucleic acid molecule encoding a protein (e.g., a disclosed humanized monoclonal antibody or fragment thereof) is expressed in a cell (e.g., a mammalian cell). Methods for introducing heterologous nucleic acid molecules into cells or organisms are known in the art, such as nucleic acid transformation, including electroporation, liposomes, particle gun acceleration, and homologous recombination.

[0124] Host cell: The cell in which the vector can reproduce and express its DNA. The cell can be a prokaryotic or eukaryotic cell. The term also includes any progeny of the host cell. It should be understood that all progeny may not be identical to the parent cell due to mutations that can occur during replication. However, when the term "host cell" is used, such progeny is included.

[0125] IgG: A class of antibodies or isotypes of polypeptides that are essentially encoded by the well-known immunoglobulin γ gene. In humans, this class includes IgG1, IgG2, IgG3, and IgG4.

[0126] Immune complexes are formed when antibodies or antigen-binding fragments (e.g., scFv) bind to soluble antigens. The formation of immune complexes can be detected by conventional methods such as immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, ELISA, Western blotting (e.g., Western blotting), magnetic resonance imaging, CT scans, radiography, and affinity chromatography.

[0127] Immune response: The response of immune system cells (such as B cells, T cells, or monocytes) to a stimulus. This response can be specific to a particular antigen (“antigen-specific response”). In some instances, the immune response is a T cell response, such as a CD4+ or CD8+ response. In other instances, the response is a B cell response and results in the production of specific antibodies.

[0128] Immunogen: A compound, composition, or substance that can stimulate an animal to produce an antibody or T-cell response, including compositions that are injected or absorbed by an animal, such as EGFRvIII conjugated to a vector. Immunogens can be used to generate antibodies, such as those disclosed herein.

[0129] Suppressing or treating tumors: Therapeutic interventions that reduce the signs or symptoms of tumors (e.g., administration of a therapeutically effective amount of a humanized monoclonal antibody disclosed herein). In some instances, treatments reduce tumor size, decrease the number of tumors, or induce remission. In specific instances, treatments include suppressing metastasis.

[0130] The term "reduction" is a relative term, meaning that the agent reduces the disease or condition if, compared to a reference agent, the disease or condition is reduced in number after administration, or if the disease or condition is alleviated after administration. Reduction of signs or symptoms refers to any observable beneficial effect of treatment. Reduction of tumor-related signs or symptoms can be demonstrated, for example, by delayed onset of clinical symptoms of the disease in susceptible subjects (e.g., subjects with tumors that have not yet metastasized), reduction in the severity of some or all of the clinical symptoms of the disease, slowing disease progression (e.g., by prolonging the lifespan of subjects with tumors), reducing the number of tumors or the time between tumor resection and tumor recurrence, improving the overall health or well-being of the subject, or by other known parameters specific to a particular tumor.

[0131] "Prophylactic" treatment is administered to subjects who do not exhibit signs of cancer but have a genetic predisposition to cancer, or who only exhibit early signs (such as precancerous lesions), with the aim of reducing the risk of developing cancer. The term "prophylaxis" does not necessarily mean that the agent completely eliminates the disease or condition; eliminating at least one characteristic of the disease or condition is sufficient. Therefore, compositions that reduce or prevent cancer may, but do not necessarily completely prevent, the risk of developing cancer.

[0132] Isolated: Biological components (e.g., nucleic acids, peptides, proteins, or protein complexes, such as antibodies) that have been substantially separated, produced separately, or purified from other components in cells or samples. Therefore, isolated nucleic acids, peptides, and proteins include nucleic acids and proteins purified using standard purification methods. The term also covers nucleic acids, peptides, and proteins prepared through recombinant expression in host cells, as well as chemically synthesized nucleic acids. Isolated nucleic acids, peptides, or proteins (e.g., antibodies) may 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.

[0133] Kabat position: The amino acid sequence follows that of Kabat et al. ( Sequences of Proteins of Immunological Interest , 5 th The residue positions are based on the numbering convention described in the 1991 edition, Department of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda, NIHP Publication No. 91-3242.

[0134] Linkers: Bifunctional molecules that can be used to link two molecules into a single continuous molecule, for example, linking an effector molecule to an antibody or linking a detectable marker to an antibody. Non-limiting examples of peptide linkers include glycine-serine linkers.

[0135] The terms "joining," "linking," or "bonding" can refer to making two molecules into a single continuous molecule; for example, linking two polypeptides into a single continuous polypeptide, or covalently attaching an effector molecule, a detectable biomarker, a radionuclide, or other molecule to a polypeptide, such as scFv. This linking can be achieved chemically or through recombination. "Chemically" refers to a reaction between an antibody moiety and an effector molecule, resulting in a covalent bond between the two molecules to form a single molecule.

[0136] Tumor formation, cancer, or tumor: Tumor formation is the abnormal growth of tissue or cells caused by excessive cell division. Tumorigenic growth can produce a tumor. The amount of tumor in an individual is called the "tumor burden," which can be measured as the number, volume, or weight of the tumor. Tumors that have not metastasized are called "benign." Tumors that invade surrounding tissues or can metastasize (or both) are called "malignant."

[0137] Tumors of the same histological type are primary tumors originating from a specific organ or body part (such as the head and neck, breast, pancreas, colon or rectum, central nervous system (CNS), or bladder). Tumors of the same histological type can be further subdivided into different subtypes. For example, lung cancer can be classified as adenocarcinoma, small cell, squamous cell, or non-small cell tumors.

[0138] Examples of solid tumors, such as sarcomas (connective tissue carcinomas) and carcinomas (epithelial carcinomas), include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colorectal cancer, malignant tumors of the lymphatic system, 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, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, and Wilms' tumor. Tumors, cervical cancer, testicular tumors, seminomas, bladder cancer, and CNS tumors (such as gliomas, astrocytomas, medulloblastomas, craniopharyngiomas, ependymomas, pineal tumors, hemangioblastomas, acoustic neuromas, oligodendrogliomas, meningiomas, melanomas, neuroblastomas, and retinoblastomas).

[0139] Nucleic acids: polymers composed of nucleotide units (e.g., ribonucleotides, deoxyribonucleotides) linked by phosphodiester bonds, their associated naturally occurring structural variants and synthetic non-natural analogs. Therefore, the term includes nucleotide polymers, where the nucleotides and their linkages include non-natural synthetic analogs such as, but not limited to, phosphate thioesters, phosphoramides, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, peptide nucleic acids (PNAs), etc. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "oligonucleotide" generally refers to short polynucleotides, typically not exceeding about 50 nucleotides. It should be understood that when nucleotide sequences are represented using DNA sequences (i.e., A, T, G, C), this also includes RNA sequences (i.e., A, U, G, C), where "U" replaces "T".

[0140] "Nucleotide" includes, but is not limited to, monomers comprising a base linked to a sugar (such as pyrimidine, purine, or their synthetic analogues) or a base linked to an amino acid (such as in peptide nucleic acids (PNA)). A nucleotide is a monomer within a polynucleotide. A nucleotide sequence refers to the sequence of bases in a polynucleotide.

[0141] This article uses conventional symbols to describe nucleotide sequences: 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 called the 5' direction. The direction in which nucleotides are added to the nascent RNA transcript from 5' to 3' is called the transcription direction. The DNA strand with the same sequence as the mRNA is called the "coding strand"; the sequence on the DNA strand with the same sequence as the mRNA transcribed from this DNA and located at the 5' to 5' end of the RNA transcript is called the "upstream sequence"; the sequence on the DNA strand with the same sequence as the RNA and located at the 3' to 3' end of the coding RNA transcript is called the "downstream sequence".

[0142] “cDNA” refers to DNA in single-stranded or double-stranded form that is complementary to or identical to mRNA.

[0143] "Encoding" refers to the inherent characteristics of a specific nucleotide sequence in a polynucleotide (such as a gene, cDNA, or mRNA) that serves as a template for the synthesis of other polymers and macromolecules in biological processes. These polymers and macromolecules have defined nucleotide sequences (i.e., rRNA, tRNA, and mRNA) or defined amino acid sequences, and the resulting biological properties. Therefore, if the transcription and translation of mRNA produced by a gene results in a protein in a cell or other biological system, then the gene encodes that protein. Both the coding strand of a gene or cDNA (whose nucleotide sequence is identical to the mRNA sequence and is usually provided in the sequence listing) and the non-coding strand (used as a transcription template) can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise stated, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate variants of each other and encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNA may include introns.

[0144] If a polynucleotide with the first sequence hybridizes specifically with a polynucleotide with the second sequence, then the first sequence is the "antisense" sequence relative to the second sequence.

[0145] Operable ligation: When a first nucleic acid sequence and a second nucleic acid sequence are functionally related, the first nucleic acid sequence and the second nucleic acid sequence are operably ligated. For example, if a promoter (such as the CMV promoter) affects the transcription or expression of a coding sequence, then the promoter is operably ligated to the coding sequence. Typically, operably ligated DNA sequences are contiguous and, when two protein-coding regions need to be ligated, they are located within the same reading frame.

[0146] Pharmaceutically acceptable carrier: The pharmaceutically acceptable carrier used is conventional. Remington’s Pharmaceutical SciencesThis book, by EW Martin, Mack Publishing Co., Easton, PA, 19th Edition, 1995, describes compositions and formulations suitable for drug delivery of publicly disclosed humanized monoclonal antibodies and their antigen-binding fragments.

[0147] The nature of the carrier may depend on the specific route of administration employed. For example, parenteral preparations typically contain injectable liquids, including pharmaceutically and physiologically acceptable fluids as solvents, such as water, physiological saline, balanced salt solutions, glucose solutions, glycerol, etc. For solid compositions (e.g., in powder, pill, tablet, or capsule form), conventional nontoxic solid carriers may include, for example, pharmaceutical-grade mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical composition to be administered may also contain trace amounts of nontoxic excipients, such as wetting agents or emulsifiers, preservatives, and pH buffers, such as sodium acetate or sorbitan monolaurate. In specific instances, the carrier is sterile and / or suspended or otherwise contained in a unit dosage form containing one or more measured doses of the composition. For example, the unit dosage form may be in a sealed vial containing sterile contents or a syringe for injection into a subject, or may be lyophilized for subsequent dissolution and administration, or in a solid or controlled-release dosage form.

[0148] Polypeptide: Any chain of amino acids, regardless of its length or post-translational modifications (e.g., glycosylation or phosphorylation). "Polypeptide" includes polymers of naturally occurring amino acids and polymers of non-naturally occurring amino acids, such as polymers in which one or more amino acid residues are non-natural amino acids, for example, artificial chemical mimics of the corresponding naturally occurring amino acids. "Residue" refers to an amino acid or amino acid mimic incorporated into a polypeptide via an amide bond or an amide bond mimic. Polypeptides have an amino terminus (N-terminus) and a carboxyl terminus (C-terminus). "Polypeptide" is used interchangeably with peptide or protein and, herein, refers to a polymer of amino acid residues.

[0149] Peptide modification: Peptides and peptides (such as the antibodies disclosed herein) can be modified using a variety of chemical techniques to produce peptides with substantially the same activity as unmodified peptides, and optionally with other desired properties. For example, the carboxyl groups of proteins, whether carboxyl-terminal or side chains, can be provided in the form of pharmaceutically acceptable cationic salts, or esterified to form C1-C124 ... 16 Ester, or amide converted to the formula NR1R2, wherein R1 and R2 are each independently H or C1-C. 16Alkyl groups, or combinations thereof, form heterocyclic rings, such as 5- or 6-membered rings. The amino groups of the peptide, whether amino-terminal or side-chain, can be pharmaceutically acceptable acid addition salts, such as HCl, HBr, acetates, benzoates, toluenesulfonates, maleates, tartrates, and other organic salts, or can be modified to C1-C. 16 Alkyl or dialkylamino, or further converted into amide.

[0150] Using recognized techniques, the hydroxyl groups on the peptide side chains can be converted into C1-C2 groups. 16 Alkoxy or C1-C 16 Esters. The phenyl and phenolic rings of the peptide side chain can be substituted with one or more halogen atoms (e.g., F, Cl, Br, or I), or be C1-C. 16 Alkyl, C1-C 16 Alkoxy groups, carboxylic acids and their esters, or amide substitutions of such carboxylic acids can be used. The methylene group on the peptide side chain can be extended to a homologous C2-C4 alkylene group. Thiols can be protected with any of a variety of recognized protecting groups, such as acetamide groups. Methods for introducing cyclic structures into peptides have been described, such as selecting and providing conformational constraints to improve stability. For example, a C-terminal or N-terminal cysteine ​​residue can be added to the peptide so that, upon oxidation, the peptide will contain a disulfide bond, forming a cyclic peptide. Other peptide cyclization methods include the formation of thioethers and carboxyl-terminated and amino-terminated amides and esters.

[0151] Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide formulation is one in which peptides or proteins (e.g., antibodies or fragments thereof) are more enriched than peptides or proteins in their native intracellular environment. In one instance, the formulation is purified such that the protein or peptide constitutes at least 50%, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or higher of the total peptide or protein content of the formulation. In a non-limiting instance, the formulation is purified such that the protein (e.g., the antibody disclosed herein) constitutes at least 70% of the total protein content of the formulation.

[0152] Recombination: Recombinant nucleic acids are nucleic acids having a non-naturally occurring sequence or a sequence artificially composed of two originally separate sequence segments. This artificial combination can be achieved, for example, through chemical synthesis or by artificially manipulating the separated nucleic acid segments, such as through genetic engineering techniques. Recombinant proteins have a non-naturally occurring sequence or a sequence artificially composed of two originally separate sequence segments. In several instances, recombinant proteins are encoded by heterologous (e.g., recombinant) nucleic acids that have been introduced into host cells (e.g., bacteria or eukaryotic cells). For example, the nucleic acid can be introduced into an expression vector having a signal capable of inducing the expression of the encoded protein in the host cell. In some instances, recombinant nucleic acids can be integrated into the host cell chromosome.

[0153] Sequence identity: The degree of similarity between amino acid or nucleic acid sequences. Sequence identity is usually measured as a percentage (or similarity or homology); the higher the percentage, the more similar the two sequences are. When compared using standard methods, homologs, orthologs, or variants of peptides will have a relatively high degree of sequence identity.

[0154] Sequence alignment methods for comparison are known. Various programs and alignment algorithms are described in, for example, 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, discusses in detail sequence alignment methods and homology calculation.

[0155] Once aligned, the number of matches is determined by counting the number of identical nucleotide or amino acid residues in both sequences. The percentage of sequence identity is determined by dividing the number of matches by the sequence length shown in the identified sequence, or by a specified length (e.g., 100 consecutive nucleotide or amino acid residues in the identified sequence), and then multiplying the result by 100. For example, when aligned with a test sequence having 1554 amino acids, a peptide sequence with 1166 matches is 75.0% identical to the test sequence (1166 ÷ 1554). 100 = 75.0). Sequence percentage identity values ​​are rounded to one decimal place. For example, 75.11, 75.12, 75.13, and 75.14 are rounded to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded to 75.2. Length values ​​are always integers.

[0156] 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 the Internet, for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Descriptions of how to use this program to determine sequence identity are available on the NCBI website on the Internet. In some instances, sequence identity is determined using the BLAST program with default parameters.

[0157] Homologous and variant peptides are typically characterized by having at least about 75% sequence identity, calculated based on a full-length alignment with the amino acid sequence of interest, such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Proteins with even higher similarity to a reference sequence will show increased percentage identity when evaluated in this manner, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%. When comparing sequence identity for less than the entire sequence, homologous and variant peptides typically have at least 80% sequence identity within a short window of 10–20 amino acids, and may have at least 85%, at least 90%, or at least 95% sequence identity depending on their similarity to a reference sequence. Methods for determining sequence identity within such short windows are available on the NCBI website on the Internet. Those skilled in the art will understand that these sequence identity ranges are for reference only; it is entirely possible to obtain strongly significant homologous peptides beyond the provided ranges.

[0158] For nucleic acid sequence comparisons, a reference sequence is typically used and compared with the test sequence. When using a sequence comparison algorithm, the test and reference sequences are input into the computer, and subsequence coordinates and algorithm parameters are specified if necessary. Default parameters are usually used.

[0159] As used herein, "at least 80% identity" means having "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" with a specific reference sequence. As used herein, "at least 90% identity" means having "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" with a specific reference sequence.

[0160] Specific binding: When referring to antibody or antigen-binding fragments, this means a binding reaction that identifies the presence of a target protein in the presence of a heterogeneous population of proteins and other biological agents. Therefore, under specified conditions, antibodies preferentially bind to specific target proteins, peptides, or polysaccharides (e.g., antigens present on the surface of tumor cells, such as EGFRvIII) and do not bind in significant amounts to other proteins present in the sample or subject, including wild-type EGFR expressed on wild-type (non-tumor) cells from the same tissue. Antibodies (e.g., those disclosed herein) may specifically bind to EGFRvIII and / or overexpressed forms of EGFR on tumor cells (e.g., gene-amplified EGFR), but not to wild-type EGFR expressed on wild-type (non-tumor) cells from the same tissue. Specific binding can be determined by standard methods. For a description of the forms and conditions of immunoassays that can be used to determine specific immunoreactivity, see Harlow & Lane, Antibodies, A Laboratory Manual ,2 nd ed., Cold Spring Harbor Publications, New York (2013).

[0161] Regarding antibody-antigen complexes, the Kc of the specific binding of antigen and antibody... D Less than about 10 -7 moles, for example less than about 10 -8 mole, 10 -9 moles or even less than about 10 -10 Moore. K DThis refers to the dissociation constant of a given interaction, such as peptide-ligand interactions or antibody-antigen interactions. For example, for a bimolecular interaction between an antibody or antigen-binding fragment and an antigen, it is the concentration of each component of the bimolecular interaction divided by the concentration of the complex.

[0162] Humanized monoclonal antibodies that specifically bind to epitopes on EGFRvIII are those that substantially bind to EGFRvIII proteins, including cells or tissues expressing EGFRvIII, substrates to which EGFRvIII is attached, or EGFRvIII in or isolated from a biosample. It should be recognized that some degree of non-specific interaction may occur between the antibody and non-target proteins (e.g., cells of the same tissue type that do not express wild-type EGFR). Typically, specific binding results in a much stronger association between the antibody and a protein or cell carrying the antigen than with a protein or cell lacking the antigen. Specific binding typically results in a greater than 2-fold increase (per unit time) in the amount of antibody binding to a protein containing the epitope or to a cell or tissue expressing the target epitope compared to a protein or cell or tissue lacking the epitope. Under these conditions, specific binding to the protein requires selection of antibodies specific to the specific protein. Various immunoassays are available for selecting antibodies or other ligands that have a specific immunoreactivity with a particular protein. For example, solid-phase ELISA is often used to select monoclonal antibodies that have a specific immunoreactivity with proteins.

[0163] Subject: A living multicellular vertebrate organism, this category includes humans and non-human mammals. In some instances, the subject is a mammalian subject, such as a human, a non-human primate, a canine, or a feline. In a non-restrictive instance, the subject is a human. In a specific instance, the subject has cancer. In another instance, the selected subject needs to suppress tumor growth or metastasis. For example, the subject could be a subject who has been diagnosed with a tumor expressing EGFRvIII and / or EGFR gene amplification (e.g., head and neck cancer, breast cancer, pancreatic cancer, colon cancer, CNS cancer, or bladder cancer) and needs treatment.

[0164] T cells: White blood cells that are essential for the immune response. T cells include, but are not limited to, CD4+ cells. + T cells and CD8 + T. CD4 +T lymphocytes are immune cells that express CD4 on their surface. These cells, also known as helper T cells, help coordinate immune responses, including antibody responses and cytotoxic T cell responses. Th1 and Th2 cells are functional subsets of helper T cells. Th1 cells secrete a set of cytokines, including interferon-γ, and their primary function is to stimulate phagocytic-mediated defense against infection, particularly that involving intracellular microbes. Th2 cells secrete a set of cytokines, including interleukin (IL)-4 and IL-5, and their primary function is to stimulate IgE and eosinophil / mast cell-mediated immune responses and downregulate Th1 responses.

[0165] Therapeutic agents: Used in a general sense, this includes therapeutic agents, preventative agents, and replacement agents. Therapeutic agents are used to improve the condition of a specific group of subjects suffering from a disease or ailment (such as cancer).

[0166] Toxins: Effector molecules that induce cytotoxicity upon contact with cells. Specific, non-limiting examples of toxins include, but are not limited to, abrin, ricin, and auristatins (e.g., monomethylauribatin E (MMAE)). See For example, Francisco et al., Blood, 102: 1458-1465, 2003) and monomethylaurestatin F (MMAF); See For example, Doronina et al. , BioConjugate Chem., 17: 114-124, 2006), maytansinoids (e.g., DM1; See For example, Phillips et al., Cancer Res., 68:9280-9290, 2008), Pseudomonas ( Pseudomonas Exotoxins (PEs, such as PE35, PE37, PE38, and PE40), diphtheria toxin (DT), botulinum toxin, saporin, restrictocin, or gelonin, or modified toxins thereof, or other toxins that directly or indirectly inhibit cell growth or kill cells. For example, PEs and DTs are highly toxic compounds that typically cause death through hepatotoxicity. However, by removing the natural targeting components of the toxins (e.g., the Ia domain of PE and the B chain of DT) and replacing them with different targeting moieties (e.g., antibodies), PEs and DTs can be modified to function as immunotoxins.

[0167] Transformed: Transformed cells are cells into which nucleic acid molecules have been introduced using molecular biology techniques. As used herein, the term “transformed” and other terms (e.g., transformation, transfection, transduction, etc.) encompass all techniques that can introduce nucleic acid molecules into such cells, including transduction with viral vectors, transformation with plasmid vectors, and accelerated introduction of DNA via electroporation, liposomes, and particle guns.

[0168] Treatment or prevention of disease: Suppressing the full development of a disease or condition, for example, in subjects at risk of developing the disease or who already have the disease (e.g., a tumor). "Treatment" refers to a therapeutic intervention that improves the signs or symptoms of a disease or pathological condition after it has begun to develop. For a disease or pathological condition, the term "improvement" refers to any observable beneficial effect of the treatment. Such a beneficial effect can be demonstrated, for example, by delaying the onset of clinical symptoms of the disease in susceptible subjects, reducing the severity of some or all of the clinical symptoms of the disease, slowing disease progression, improving the overall health or well-being of the subject, or by other parameters specific to the disease. "Preventative" treatment is administered to subjects who do not exhibit signs of the disease or only exhibit early signs, with the aim of reducing the risk of developing the disease.

[0169] Vector: A nucleic acid molecule (e.g., DNA or RNA molecule) carrying a promoter that is operatively linked to and can express the coding sequence of a protein of interest. Non-limiting examples include naked DNA or packaged DNA (lipids and / or proteins), naked RNA or packaged RNA, viruses or bacteria or other microorganisms that may not be capable of replication, or subcomponents of viruses or bacteria or other microorganisms that may be capable of replication. Vectors are sometimes also referred to as constructs. A recombinant DNA vector is a vector containing recombinant DNA. A vector may include nucleic acid sequences that allow it to replicate in a host cell, such as an origin of replication. A vector may also include one or more optional marker genes and other genetic elements. A viral vector is a recombinant nucleic acid vector that has at least some nucleic acid sequences derived from one or more viruses. In some instances, a viral vector includes a nucleic acid molecule encoding a publicly disclosed humanized monoclonal antibody or antigen-binding fragment.

[0170] Under conditions sufficient to…: a phrase used to describe any environment that allows the desired activity to occur.

[0171] III. Overview of Several Public Aspects Isolated humanized monoclonal antibodies and their antigen-binding fragments are provided, which specifically bind to mutated / variant EGFRs (e.g., EGFRvIII and / or gene-amplified EGFR) expressed by cancer cells / tumor cells. In some aspects, the humanized monoclonal antibodies or antigen-binding fragments disclosed herein specifically bind to EGFRvIII. In some aspects, the humanized monoclonal antibodies or antigen-binding fragments disclosed herein specifically bind to gene-amplified EGFR. In some aspects, the humanized monoclonal antibodies or antigen-binding fragments disclosed herein specifically bind to EGFRvIII but not gene-amplified EGFR. In some aspects, the humanized monoclonal antibodies or antigen-binding fragments disclosed herein specifically bind to gene-amplified EGFR but not EGFRvIII. In some aspects, the humanized monoclonal antibodies or antigen-binding fragments disclosed herein specifically bind to cells expressing EGFRvIII and / or gene-amplified EGFR but not wild-type EGFR. In some aspects, the antibodies disclosed herein bind to EGFR cells exposed on the cell surface. 287-302 Cells in a ring (e.g., tumor cells).

[0172] In some respects, the antibodies disclosed herein bind to mammalian EGFRvIII and / or mammalian gene-amplified EGFR, for example, humans, non-human primates (e.g., apes (great apes)). Simiiformes ), chimpanzee (chimpanzee ( Pan troglodytes Bonobos (Bonobos ( Pan paniscus ), canines (e.g., domestic dogs) Canis lupus familiaris )) or felines (e.g., domestic cats) Felis catus EGFRvIII and / or amplified EGFR. In a non-limiting example, the antibodies disclosed herein bind to human EGFRvIII and / or amplified human EGFR.

[0173] In several respects, the monoclonal antibodies and antigen-binding fragments provided herein can be used to treat tumors expressing EGFRvIII, such as, but not limited to, head and neck cancer, breast cancer, pancreatic cancer, colon or rectal cancer, CNS cancer, or bladder cancer. In several respects, the monoclonal antibodies and antigen-binding fragments can be used to treat tumors expressing amplified EGFR genes, such as, but not limited to, head and neck cancer, breast cancer, pancreatic cancer, colon or rectal cancer, CNS cancer, or bladder cancer. These monoclonal antibodies and antigen-binding fragments can be used to treat tumors in human or animal subjects (e.g., non-human primates, dogs, cats).

[0174] Compositions including the antibodies or antigen-binding fragments disclosed herein and pharmaceutically acceptable vectors are also disclosed. Nucleic acids encoding antibody or antigen-binding fragments, expression vectors including these nucleic acids, and isolated host cells expressing these nucleic acids are also provided.

[0175] The compositions disclosed herein, comprising monoclonal antibodies or antigen-binding fragments, can be used for, for example, research, diagnostic, and / or therapeutic purposes. For instance, monoclonal antibodies can be used to diagnose and / or treat subjects with tumors expressing EGFRvIII and / or EGFR gene amplification. Subjects can be human or animal subjects (e.g., non-human primates, dogs, cats).

[0176] A. Antibody-antigen binding fragments Isolated humanized monoclonal antibodies and antigen-binding fragments are provided, which specifically bind to EGFRvIII and / or epitopes of EGFR gene amplification. These antibodies are parental monoclonal antibodies 40H3 ( See The disclosed humanized forms and / or variants of the monoclonal antibody (US Patent Publication No. 2022-0380474) are described herein. In some aspects, the isolated humanized monoclonal antibody or antigen-binding fragment specifically binds to the epitope of EGFRvIII. In some aspects, the isolated humanized monoclonal antibody or antigen-binding fragment specifically binds to the epitope of gene-amplified EGFR. The disclosed antibody includes one or more humanized residues, and therefore its frame region includes one or more alterations compared to the parental mouse monoclonal antibody 40H3. In some aspects, the disclosed humanized monoclonal antibody is less immunogenic than the parental monoclonal antibody 40H3. The disclosed monoclonal antibody unexpectedly exhibits increased binding to EGFRvIII compared to the parental monoclonal antibody 40H3.

[0177] In several respects, the disclosed humanized antibody and antigen-binding fragment can inhibit the biological function or properties of EGFRvIII protein in vivo, including but not limited to reducing and / or inhibiting tumor growth, or reducing and / or inhibiting tumor metastasis. The disclosed humanized monoclonal antibody and antigen-binding fragment preferentially bind to EGFRvIII expressed on tumor cells and overexpressed EGFR (e.g., gene-amplified EGFR), but substantially does not bind to EGFR expressed on wild-type (non-cancer cells). In some respects, the disclosed humanized monoclonal antibody and antigen-binding fragment bind to EGFR. 287-302 ring.

[0178] Under normal conditions (e.g., when expressed on healthy, non-cancer cells), a spatially inaccessible structural element of EGFR is the disulfide-restricted loop at residues 287-302 (numbered for the mature receptor; or residues 301-326 of the full-length receptor). 287-302The ring is exposed in EGFRvIII. In some respects, publicly available humanized monoclonal antibodies bind to EGFR. 287-302 Ring. In some instances, EGFR 287-302 The ring includes R300. The disclosed antibody is a humanized antibody, and therefore includes one or more variations in its frame region compared to the parental mouse monoclonal antibody 40H3.

[0179] The following discussion of monoclonal antibodies refers to antibodies including V H and V L The isolated monoclonal antibodies comprise HCDRs and LCDRs, respectively. Those skilled in the art will understand that various CDR numbering schemes (e.g., Kabat, Chothia, or IMGT numbering schemes) can be used to determine CDR positions. This document uses Kabat, IGMT, or Chothia numbering to display the amino acid sequences of the heavy and light chains and CDR positions of the disclosed monoclonal antibodies. However, those skilled in the art will readily understand the use of various CDR numbering schemes when referring to the specific amino acids of the antibodies disclosed herein. Procedures for identifying CDRs using Chothia, IGMT, or Kabat are publicly available.

[0180] The antibodies disclosed in this article include the heavy chain variable region (V). H ) and light chain variable region (V L In some instances, the antibodies disclosed herein include V as described in Table 1. H and V L Combinations (excluding A1).

[0181] Table 1: Exemplary antibodies, corresponding VH and VL, and corresponding cell binding.

[0182]

[0183] 1 MDA-MB-468 Chimera: Mouse VH and VL derived from 40H3 antibody + human IgG1CH / human IgκCL In some embodiments, the antibodies disclosed herein include a heavy chain variable region (V... H ) and light chain variable region (V L ), the V H and V L : a) Includes SEQ ID NO: 5 and SEQ ID NO: 8 (A10, VH3+VL3), respectively; b) Includes SEQ ID NO: 3 and SEQ ID NO: 6 (A2, VH1+VL1), respectively; c) Includes SEQ ID NO: 3 and SEQ ID NO: 7 (A3, VH1+VL2); d) Includes SEQ ID NO: 3 and SEQ ID NO: 8 (A4, VH1+VL3), respectively; e) includes SEQ ID NO: 4 and SEQ ID NO: 6 (A5, VH2+VL1), respectively; f) includes SEQ ID NO: 4 and SEQ ID NO: 7 (A6, VH2+VL2), respectively; g) includes SEQ ID NO: 4 and SEQ ID NO: 8 (A7, VH2+VL3), respectively; h) includes SEQ ID NO: 5 and SEQ ID NO: 6 (A8, VH3+VL1); i) Includes SEQ ID NO: 5 and SEQ ID NO: 7 (A9, VH3+VL2), respectively; j) includes SEQ ID NO: 1 and SEQ ID NO: 9 (B1, 40H3 VH+VL-EG); k) includes SEQ ID NO: 1 and SEQ ID NO: 10 (B2, 40H3 VH+VL-DA); l) includes SEQ ID NO: 4 and SEQ ID NO: 11 (B3, VH2+VL1-DA); m) respectively includes SEQ ID NO: 4 and SEQ ID NO: 12 (B4, VH2+VL2-DA); n) includes SEQ ID NO: 5 and SEQ ID NO: 11 (B5, VH3 and VL1-DA); o) respectively includes SEQ ID NO: 31 and SEQ ID NO: 32 (C10); p) includes SEQ ID NO: 5 and SEQ ID NO: 14 (D2) respectively; or q) includes SEQ ID NO: 5 and SEQ ID NO: 24 (D3), respectively.

[0184] Humanized monoclonal antibodies may include V H and V L These include the amino acids (A10, VH3+VL3) shown in SEQ ID NO: 5 and SEQ ID NO: 8, respectively. Humanized monoclonal antibodies may include V... H and VL These include the amino acids (VH1+VL1) shown in SEQ ID NO: 3 and SEQ ID NO: 6, respectively. Humanized monoclonal antibodies may include V... H and V L These include the amino acids (VH1+VL2) shown in SEQ ID NO: 3 and SEQ ID NO: 7, respectively. Humanized monoclonal antibodies may include V... H and V L These include the amino acids (VH1+VL3) shown in SEQ ID NO: 3 and SEQ ID NO: 8, respectively. Humanized monoclonal antibodies may include V... H and V L These include the amino acids (VH2+VL1) shown in SEQ ID NO: 4 and SEQ ID NO: 6, respectively. Humanized monoclonal antibodies may include V... H and V L These include the amino acids (VH2+VL2) shown in SEQ ID NO: 4 and SEQ ID NO: 7, respectively. Humanized monoclonal antibodies may include V... H and V L These include the amino acids (VH2+VL3) shown in SEQ ID NO: 4 and SEQ ID NO: 8, respectively. Humanized monoclonal antibodies may include V... H and V L These include the amino acids (VH3+VL1) shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively. Humanized monoclonal antibodies may include V... H and V L They respectively contain the amino acids (VH3+VL2) shown in SEQ ID NO: 5 and SEQ ID NO: 7. Humanized monoclonal antibodies may include V H and V L These include the amino acids (40H3 VH+VL-EG) shown in SEQ ID NO: 1 and SEQ ID NO: 9, respectively. Humanized monoclonal antibodies may include V... H and V L They respectively contain the amino acids shown in SEQ ID NO: 1 and SEQ ID NO: 10 (40H3 VH+VL-DA). Humanized monoclonal antibodies may include V H and V L These include the amino acids (VH2+VL1-DA) shown in SEQ ID NO:4 and SEQ ID NO:11, respectively. Humanized monoclonal antibodies may include V... H and V LThese include the amino acids (VH2+VL2-DA) shown in SEQ ID NO: 4 and SEQ ID NO: 12, respectively. Humanized monoclonal antibodies may include V... H and V L These include the amino acids (VH3 and VL1-DA) shown in SEQ ID NO: 5 and SEQ ID NO: 11, respectively. Humanized monoclonal antibodies may include V... H and V L They respectively contain the amino acids (C10) shown in SEQ ID NO: 31 and SEQ ID NO: 32. Humanized monoclonal antibodies may include V H and V L These include the amino acids (D2) shown in SEQ ID NO: 5 and SEQ ID NO: 14, respectively. Humanized monoclonal antibodies may include V H and V L These include, respectively, the amino acids (D3) shown in SEQ ID NO: 5 and SEQ ID NO: 24. In several embodiments, the antibodies disclosed herein bind to EGFRvIII and / or gene-amplified EGFR. In some instances, the antibodies disclosed herein bind to EGFRvIII. In some instances, the antibodies disclosed herein bind to gene-amplified EGFR.

[0185] Currently, publicly available monoclonal antibodies and antigen-binding fragments are humanized. Mutations that can occur in the human frame region, and in the human antibody frame region, can be introduced into the antibody using various methods. See For example, U.S. Patent No. 5,585,089, 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).

[0186] 1. Further description of antibody-antigen binding fragments Humanized monoclonal antibodies can be of any isotype. Monoclonal antibodies can be, for example, IgM antibodies or IgG antibodies, such as IgG1, IgG2, IgG3, or IgG4. The categories of humanized monoclonal antibodies disclosed herein can be converted to other categories. In one aspect, the separation encoding V... L or V H The nucleic acid molecule is designed so that it does not contain any nucleic acid sequences encoding either the light chain or the heavy chain constant region, respectively. Then, the sequence encoding V... L or V H The nucleic acid molecules can be operatively linked to C-cells encoding different classes of immunoglobulin molecules. L Or C H The nucleic acid sequence. As is known in the art, this can be achieved using C... L Or C H This is achieved through the use of carriers or nucleic acid molecules. For example, a humanized monoclonal antibody that was originally IgG may undergo class conversion. Class conversion can be used to convert one IgG subclass to another, such as from IgG1 to IgG2, IgG3, or IgG4.

[0187] In some instances, publicly available humanized antibodies can be prepared as antibody oligomers, such as dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, etc.

[0188] (a) Combining affinity In several aspects of this disclosure, antibody or antigen-binding fragments, for example, are in a size not exceeding 1.0 × 10⁻⁶. -8 M, not exceeding 5.0 × 10 -8 M, not exceeding 1.0 × 10 -9 M, not exceeding 5.0 × 10 -9 M, not exceeding 1.0 × 10 -10 M, not exceeding 5.0 × 10 -10 M or not exceeding 1.0 × 10 -11 The affinity of M (e.g., through K) d (Measurement) specifically binds to EGFRvIII or gene-amplified EGFR. In non-limiting examples, the antibody or antigen-binding fragments disclosed herein have an affinity of less than 6 nm (K0.05). d ) specifically binds to EGFRvIII or gene-amplified EGFR. In non-limiting examples, the antibody or antigen-binding fragments disclosed herein have an affinity of less than 4 nm (K0.05). d) specifically binds to the EGFRvIII protein. In some aspects, the antibody or antigen-binding fragments disclosed herein have an affinity (K0.5) of 0.5 nm to 10 nm, for example, 0.5 nm to 9 nm, 1 nm to 9 nm, 1 nm to 8 nm, 1 nm to 7 nm, 1 nm to 6 nm, 1 nm to 5 nm, 1 nm to 4 nm, 1 nm to 3 nm, 2 nm to 9 nm, 2 nm to 8 nm, 2 nm to 7 nm, 2 nm to 6 nm, 2 nm to 5 nm, 2 nm to 4 nm, 2 nm to 3 nm, 3 nm to 9 nm, 3 nm to 8 nm, 3 nm to 7 nm, 3 nm to 6 nm, 3 nm to 5 nm, or 3 nm to 4 nm. d ) specifically binds to EGFRvIII or gene-amplified EGFR. In non-limiting examples, the antibody or antigen-binding fragments disclosed herein have an affinity (K0.05) of 1 nm to 6 nm. d ) specifically binds to EGFRvIII or gene-amplified EGFR. In non-limiting examples, the antibody or antigen-binding fragments disclosed herein have an affinity (K0.05) of 2 nm to 6 nm. d It specifically binds to EGFRvIII or gene-amplified EGFR. In some instances, affinity is measured using the Octet® Bio-Layer Interferometry (BLI) platform. In several aspects of this disclosure, the antibody or antigen-binding fragments disclosed herein, for example, at a concentration not exceeding 1.0 × 10⁻⁶. - 8 M, not exceeding 5.0 × 10 -8 M, not exceeding 1.0 × 10 -9 M, not exceeding 5.0 × 10 -9 M, not exceeding 1.0 × 10 -10 M, not exceeding 5.0 × 10 -10 M or not exceeding 1.0 × 10 -11 The affinity of M (e.g., through K) d (Measurement) The product of EGFR amplification specifically binds to the antibody or antigen-binding fragment disclosed herein. In some instances, the binding affinity of the antibody or antigen-binding fragment to EGFRvIII is 2 nm to 5 nm (e.g., 2 nm to 4 nm, 2 nm to 3 nm, 3 nm to 5 nm, or 3 nm to 4 nm).

[0189] In other instances, K dThis can be measured, for example, by a radiolabeled antigen binding assay (RIA), which uses known methods with a Fab version of the humanized monoclonal antibody of interest and its antigen. In one assay, the antibody is titrated in the presence of an unlabeled antigen, using the lowest concentration of ( 125 I) Labeled antigens were balanced with Fab, and then the bound antigens were captured by a plate coated with anti-Fab antibody to measure the solution binding affinity of Fab to the antigen. See For example, Chen et al ., J. Mol. Biol. 293:865-881 (1999)). To establish assay conditions, MICROTITER® multiwell plates (ThermoScientific) were coated overnight with 5 μg / ml 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 two to five hours at room temperature (approximately 23°C). In non-adsorption plates (Nunc #269620), 100 μM or 26 pM [ 125 I]-The antigen is mixed with a series of dilutions of the Fab of interest (e.g., with Presta et al., Cancer Res. (Consistent with the evaluation of the anti-VEGF antibody Fab-12 in 57:4593-4599 (1997)). The Fab of interest was then incubated overnight; however, incubation could be continued for a longer period (e.g., about 65 hours) to ensure equilibration. The mixture was then transferred to a capture plate and incubated at room temperature (e.g., for one hour). The solution was then removed, and the plate was washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. After the plate dried, 150 μl / well of scintillation buffer (MICROSCINT-20™; Packard) was added, and the plate was counted for several tens of minutes on a TOPCOUNT™ γ counter (Packard). A competitive binding assay was performed using the concentration of each Fab that produced a maximum binding of less than or equal to 20%.

[0190] In another assay, surface plasmon resonance assays can be used to measure K at ~10 response units (RU) using an immobilized antigen CM5 chip with a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C. dIn short, following the supplier's instructions, the carboxymethylated dextran biosensor chip (CM5, BIACORE®, Inc.) was activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The antigen was diluted to 5 μg / ml (~0.2 μM) with 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 conjugated protein. After antigen injection, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, serial dilutions of Fab (0.78 nM to 500 nM) were injected at 25 °C into PBS (PBST) containing 0.05% polysorbate 20 (TWEEN-20™) surfactant at a flow rate of approximately 25 l / min. The association rate (k) was calculated by simultaneously fitting association and dissociation sensor maps using a simple one-to-one Langmuir binding model (BIACORE® evaluation software version 3.2). on ) and dissociation rate (k off The equilibrium dissociation constant (Kd) is calculated as k. off / k on ratio. See For example, Chen et al., J. Mol. Biol. 293:865-881 (1999). If the binding rate (on-rate) measured by the above surface plasmon resonance method exceeds 10⁶ M⁻¹. -1 s -1 The binding rate can be determined by using fluorescence quenching, a technique that measures 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) at 25°C in the presence of an increased concentration of antigen, as measured in a spectrophotometer (e.g., an Aviv Instruments spectrophotometer equipped with a flow-stopped flowmeter or an 8000 Series SLM-AMINCO™ spectrophotometer with a stirring tank, ThermoSpectronic).

[0191] (b) Multispecific antibodies In some respects, multispecific antibodies (e.g., bispecific antibodies) include a publicly disclosed humanized monoclonal antibody or antigen-binding fragment. Such multispecific antibodies can be generated by known methods, such as crosslinking two or more antibodies of the same or different types, or antigen-binding fragments (e.g., scFv). Exemplary methods for preparing multispecific antibodies include those described in PCT Publication No. WO2013 / 163427. Suitable crosslinking agents include heterobifunctional crosslinking agents having two distinct reactive groups separated by a suitable spacer (e.g., m-maleimide benzoyl-N-hydroxysuccinimide ester), or homobifunctional crosslinking agents (e.g., disuccinimide octanoate). Such linkers are available from Pierce Chemical Company, Rockford, Illinois.

[0192] In some respects, the bispecific antibody includes a publicly disclosed humanized monoclonal antibody or antigen-binding fragment that specifically binds to EGFRvIII and / or gene-amplified EGFR, and further specifically binds to a second tumor antigen (e.g., Her-2) or a checkpoint inhibitor (e.g., programmed death (PD)-1, PD ligand (PD-L1), or PD-L2). In some instances, the bispecific antibody further binds to the MET proto-oncogene antigen (…). See For example, Comaglio et al., “Known and novel roles of the MET oncogene in cancer: a coherent approach to targeted therapy,” Nature Reviews Cancer , 18:341-358, 2018).

[0193] 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 have been described. 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, and 6,723,538). Further 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 (“dimorph”) molecules is described, for example, by 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 dimers, the scFv molecule can be fused to one of the VL-CL(L) or VH-CH1 chains, for example, to produce a dimer in which scFv is fused to the C-terminus of the Fab chain.

[0194] (c) Antigen-binding fragment The antigen-binding fragments (e.g., Fab or scFv) of any monoclonal antibodies (e.g., A10) disclosed herein are also disclosed. The antigen-binding fragments disclosed herein include heavy and light chain variable regions and specifically bind to EGFRvIII and / or amplified EGFR. The antibody fragments retain the ability to selectively bind antigens (e.g., EGFRvIII and / or amplified EGFR) and include, for example: (1) Fab, which is a fragment containing a monovalent antigen-binding fragment of an antibody molecule, can be produced by digesting the whole antibody with papain to obtain a complete light chain and a part of a heavy chain. (2) Fab', which is a fragment of the antibody molecule: it can be obtained by treating the whole antibody with pepsin and then reducing it to obtain a portion of the complete light chain and heavy chain; each antibody molecule yields two Fab' fragments; (3) (Fab')2, namely the following antibody fragment: it can be obtained by treating the whole antibody with pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments linked together by two disulfide bonds; (4) Fv, namely the following genetically engineered fragment: containing a light chain variable region and a heavy chain variable region, expressed as two chains, and its disulfide linkage form (dsFV); and (5) Single-chain antibodies (e.g., scFv) are defined as genetically engineered molecules containing light chain variable regions and heavy chain variable regions, linked together by suitable polypeptide linkers to form gene-fused single-chain molecules. scFv is a fusion protein in which the light chain variable regions and heavy chain variable regions of immunoglobulins are linked by linkers ( See For example, Ahmad et al ., Clin. Dev. Immunol., 2012, doi:10.1155 / 2012 / 980250; Marbry, IDrugs, 13:543-549, 2010). scFv in V H - Domains and V L - The intramolecular orientation of the domain is not decisive for the provided antibody (e.g., for the provided multispecific antibody). Therefore, two possible arrangements (V) can be used. H -Structural Domain-Joint Structural Domain-V L - Domain; V L -Structural Domain-Joint Structural Domain-V H The scFv (within a structuring domain). Other forms are also used, such as ds-scFv.

[0195] (6) Single-chain antibody dimers (scFV2), defined as dimers of scFV. These are also known as "miniantibodies." Methods for preparing these fragments have been described. See For example, Harlow and Lane, Antibodies: A Laboratory Manual , 2 nd , Cold Spring Harbor Laboratory, New York, 2013).

[0196] In a further aspect, the antibody-binding fragment can be an Fv antibody, which is typically about 25 kDa and contains a complete antigen-binding site, with three CDRs on each heavy chain and each light chain. To generate F... V Antibody, V H and V L It can be expressed by two separate nucleic acid constructs in the host cell. If V H and V L In discontinuous expression, the chains of Fv antibodies are typically held together by non-covalent interactions. However, these chains tend to dissociate upon dilution, and therefore methods have been developed to crosslink these chains via glutaraldehyde, intermolecular disulfides, or peptide linkers. Thus, in one instance, Fv can be a disulfide-stabilized Fv (dsFv), where the heavy chain variable region and the light chain variable region are chemically linked by disulfide bonds.

[0197] In another instance, the Fv fragment includes a V linked by a peptide linker. H and V L These single-chain antigen-binding proteins (scFv) construct the V-chain. H and V L The domains are prepared by nucleic acid molecules linked by oligonucleotides. 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 with a linker peptide bridging two V domains. Methods for generating scFv have been described ( See For example, 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). A dimer of the single-chain antibody (scFV2) was also envisioned.

[0198] Antigen-binding fragments can be obtained through antibody proteolysis or through host cells (e.g., E. coli). E. coli The fragment is prepared by expressing DNA encoding the fragment in cells. Alternatively, the antigen-binding fragment can be obtained by conventional methods, such as digesting the intact antibody with pepsin or papain. For example, the antigen-binding fragment can be generated by enzymatic digestion of the antibody with pepsin to provide a 5S fragment called F(ab')2. This fragment can be further cleaved with a thiol reducing agent, and optionally with a blocking group targeting the thiol group generated by disulfide bond cleavage, to generate a 3.5S Fab' monovalent fragment. Alternatively, enzymatic digestion with pepsin can directly generate two monovalent Fab' fragments and an Fc fragment (see, for example, U.S. Patent Nos. 4,036,945 and 4,331,647; 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 .atsections 2.8.1-2.8.10 and 2.10.1-2.10.4).

[0199] Other methods for cleaving antibodies can also be used, such as separating the heavy chain to form a monovalent light chain-heavy chain fragment, further cleaving the fragment, or other enzymatic, chemical, or genetic techniques, as long as the fragment binds to the antigen recognized by the intact antibody.

[0200] It has also been derived from mouse V, which was amplified from the genomic DNA of immunized mice. H Antigen-binding monoclonal antibodies were identified in the gene library. H The domain is called a domain antibody (dAb). et al. Nature 341 :544-546, 1989). Human monoimmunoglobulin variable domain peptides capable of binding antigens with high affinity have also been described. See For example, PCT publications WO 2005 / 035572 and WO 2003 / 002609). The CDRs disclosed herein may also be included in dAbs.

[0201] In some respects, one or more heavy chain and / or light chain complementarity-determining regions (CDRs) from publicly available humanized monoclonal antibodies are expressed on the surface of another protein, such as a scaffold protein. The expression of antibody domains on scaffold protein surfaces has been described (see, e.g., Liu...). et al., J. Virology 85(17): 8467-8476, 2011). This expression creates a chimeric protein that retains binding to EGFRvIII and / or amplified EGFR. In certain non-limiting instances, one or more heavy chain CDRs are grafted onto a scaffold protein, such as one or more heavy chain CDR1, CDR2, and / or CDR3. One or more CDRs may also be included in a dimer or another type of single-chain antibody molecule.

[0202] In some aspects, the antigen-binding fragment disclosed herein is scFv. In some instances, the antigen-binding fragment comprises an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 100%) sequence identity with SEQ ID NO: 43 or 45. In some instances, the antigen-binding fragment comprises or consists of SEQ ID NO: 43 or 45. In some aspects, the antigen-binding fragment disclosed herein is Fab. In some instances, the antigen-binding fragment comprises an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 100%) sequence identity with SEQ ID NO: 47 and / or 49. In some instances, the antigen-binding fragment comprises or consists of SEQ ID NO: 47 and / or 49.

[0203] (d) variant This disclosure includes amino acid sequence variants of the humanized antibodies provided herein. For example, further enhancements in antibody binding affinity and / or other biological properties may be desired. Amino acid sequence variants of humanized monoclonal antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the antibody amino acid sequence. The final construct can be obtained by any combination of deletions, insertions, and substitutions, provided that the final construct possesses the desired properties, such as specific binding to EGFRvIII and / or EGFR-amplified genes.

[0204] In some respects, antibody variants with one or more amino acid substitutions are provided. Sites of interest for substitution mutagenesis include the CDR and frame regions. Amino acid substitutions can be introduced into humanized monoclonal antibodies of interest, and the desired activity of the product can be screened, such as maintained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0205] Variant usually retains V H and V L The correct folding and stabilization of the amino acid residues between regions, while preserving the charge properties of these residues, maintains the molecule's low pI and low toxicity. This can be achieved in V... H and V L The region undergoes amino acid substitution to increase yield. Conserved amino acids that provide functionally similar amino acids are known to be representative; for example, the following six groups are examples of amino acids considered to be conserved substitutes for each other: 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).

[0206] In some aspects, compared with the amino acid sequence shown in one of SEQ ID NO: 3, 4, 5 and / or 31, the heavy chain of the antibody includes 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 (e.g., conserved amino acid substitutions). In some aspects, compared with the amino acid sequence shown in one of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 14, 24 or 32, the light chain of the antibody includes 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 (e.g., conserved amino acid substitutions).

[0207] In some respects, compared to known frame regions or antibody frame regions disclosed herein, antibody or antigen-binding fragments may include up to 10 (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, or 9) amino acid substitutions (e.g., conserved amino acid substitutions) in the frame regions of the antibody heavy chain and / or antibody light chain, and maintain specific binding activity to products of EGFRvIII protein and / or gene amplification of EGFR.

[0208] In some respects, substitutions, insertions, or deletions may occur within one or more CDRs, as long as such changes do not significantly reduce the antibody's ability to bind to target antigens (e.g., EGFRvIII and / or amplified EGFR). For example, conserved changes that do not significantly reduce binding affinity can be made in CDRs (e.g., conserved substitutions as described herein). In some instances of the VH and VL sequence variants provided above, each CDR is either unchanged or contains no more than one, two, or three amino acid substitutions. In some instances, L-CDR2 may be modified with amino acids.

[0209] To enhance antibody binding affinity, a process similar to somatic cell mutation in vivo can be employed to target V... L and V H Random mutations are performed on segments, such as within the H-CDR3 or L-CDR3 regions. This in vivo somatic mutation process is responsible for antibody affinity maturation during innate immune responses. Therefore, V can be amplified using PCR primers complementary to H-CDR3 or L-CDR3, respectively.H and V L The primers are spiked with a random mixture of four nucleotide bases at specific positions to achieve in vitro affinity maturation. H and V L The segment in which random mutations have been introduced into V H and / or V L The CDR3 region. This can be used to detect these randomly mutated V... H and V L The segment was tested to determine its binding affinity to EGFRvIII and / or the product of EGFR gene amplification. Methods for in vitro affinity maturation have been described. 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)).

[0210] A useful method for identifying antibody residues or regions that may serve as mutagenic targets is called "alanine scanning mutagenesis," as exemplified by Cunningham and Wells (1989). Science As described in 244:1081-1085. In this method, a residue or a group of target residues (e.g., charged residues, such as arg, asp, his, lys, and glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction between the antibody and the antigen is affected. Further substitutions may be introduced at amino acid positions to elucidate the functional sensitivity to the initial substitution. Alternatively, or additionally, the crystal structure of the antigen-antibody complex may be used to identify contact points between the antibody and the antigen. Such contact residues and adjacent residues may be targeted or eliminated as substitution candidates. Variants may be screened to determine whether they contain the desired properties.

[0211] In some respects, humanized monoclonal antibodies or antigen-binding fragments are altered to increase or decrease the degree of glycosylation of the antibody or antigen-binding fragment. The addition or deletion of glycosylation sites can be conveniently accomplished by altering the amino acid sequence, thereby creating or removing one or more glycosylation sites.

[0212] In the case of antibodies containing an Fc region, the carbohydrates attached to it may be altered. Naturally occurring antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides, which are usually attached to Asn297 of the CH2 domain of the Fc region via N-bonds. See For example, Wright et al . TIBTECH 15:26-32 (1997). Oligosaccharides may include a variety of carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose, which is attached to the "backbone" of the biantennary oligosaccharide structure. In some respects, oligosaccharides in humanized monoclonal antibodies can be modified to create variants with certain improved properties.

[0213] On the one hand, antibody variants are provided that have carbohydrate structures lacking (directly or indirectly) fucose attached 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 within the glycan chain at Asn297 relative to the total amount of all sugar structures attached to Asn297 (e.g., complexes, mixtures, 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 approximately at position 297 in the Fc region; however, due to minor sequence variations in the antibody, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucoidylated variants may possess improved ADCC function. See For example, US Patent Publication Nos. 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa HakkoKogyo Co., Ltd.). Publicly disclosed examples associated with “defucosylated” or “fucose-deficient” antibody variants include: US2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US2004 / 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 defucosylation antibodies include Lec 13 CHO cells (Ripka) with protein fucosylation defects. et al . Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application Nos. US 2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al (especially Example 11), and knockout cell lines, such as CHO cells with α-1,6-fucosyltransferase gene FUT8 knocked out (…). See For example, Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al ., Biotechnol. Bioeng. 94(4):680-688 (2006); and WO2003 / 085107).

[0214] The antibody variant is further provided with a bimeric oligosaccharide, for example, wherein the biantennary oligosaccharide attached to the Fc region of the antibody is bimeric by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana). et al .); and US 2005 / 0123546 (Umana et al Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. These antibody variants are described, for example, in WO 1997 / 30087 (Patel et al.). al .), WO 1998 / 58964 (Raju, S.) and WO 1999 / 22764 (Raju, S.).

[0215] In several respects, the constant region of an antibody includes one or more amino acid substitutions to optimize the antibody's in vivo half-life. The serum half-life of IgG antibodies is regulated by the neonatal Fc receptor (FcRn). Therefore, in several respects, antibodies include amino acid substitutions that increase their binding to the FcRn. Several such substitutions are known, such as the substitutions in the IgG constant region T250Q and M428L (…). See For example, Hinton et al., J Immunol., 176:346-356, 2006); Substitution of M428L and N434S (“LS” mutation, SeeFor example, Zalevsky, et al. , Nature Biotechnology, 28:157-159, 2010); Replacement of N434A ( See For example, Petkova et al., Int. Immunol ., 18:1759-1769, 2006); Replacement of T307A, E380A and N434A ( See For example, Petkova et al., Int. Immunol ., 18:1759-1769, 2006); and the replacement of M252Y, S254T and T256E ( See For example, Dall'Acqua et al., J. Biol. Chem ., 281:23514-23524, 2006).

[0216] In some respects, the constant region of an antibody includes one or more amino acid substitutions to optimize antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC is primarily mediated through a closely related group of Fcγ receptors. In some respects, antibodies include one or more amino acid substitutions that increase their binding to FcγRIIIa. Several such substitutions are known, such as the substitutions at S239D and I332E in the IgG constant region (…). See For example, Lazar et al., Proc. Natl., Acad. Sci. U.S.A ., 103:4005-4010, 2006); and the replacement of S239D, A330L and I332E ( See For example, Lazar et al., Proc. Natl., Acad. Sci. U.S.A ., 103:4005-4010, 2006).

[0217] This also includes combinations of the above substitutions to generate an IgG constant region with increased binding to FcRn and FcγRIIIa. These combinations increase antibody half-life and ADCC. For example, such combinations may 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 / T256E; (6) (7) S239D / A330L / I332E and T250Q / M428L; (8) S239D / A330L / I332E and M428L / N434S; (9) S239D / A330L / I332E and N434A; or (10) S239D / A330L / I332E and M252Y / S254T / T256E.

[0218] In some instances, antibodies or their antigen-binding fragments are modified to make them directly cytotoxic to infected cells, or utilize natural defenses such as complement, antibody-dependent cell cytotoxicity (ADCC), or macrophage phagocytosis.

[0219] The humanized monoclonal antibodies described herein can be further modified to contain additional non-protein moieties. Suitable moieties for antibody derivatization 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, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in production due to its stability in water. The polymer can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they can be the same or different molecules. Typically, the quantity and / or type of polymer used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be improved, and whether the antibody derivative will be used for treatment under specific conditions.

[0220] Humanized antibodies or antigen-binding fragments can be derivatized or linked to another molecule, such as another peptide or protein. Typically, antibody or antigen-binding fragments are derivatized so that binding to the desired target is not adversely affected by derivatization or labeling. For example, humanized antibodies or antigen-binding fragments can be functionally linked (through chemical conjugation, gene fusion, non-covalent association, or other means) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or dimer), a detectable biomarker, an effector molecule, or a protein or peptide that can mediate association between the humanized antibody or antibody portion and other molecules, such as a streptavidin core region or a polyhistidine tag).

[0221] It also includes humanized antibodies that bind to the same epitopes on EGFRvIII as the humanized monoclonal antibodies disclosed herein. 287-302 Humanized monoclonal antibodies that bind to such epitopes on the ring can be identified based on their ability to cross-compete with specific antibodies (e.g., those described in the examples) in binding assays (e.g., competitively inhibiting their binding in a statistically significant manner). This is achieved when the concentration of the competing antibody is higher than 10... 6 ×K D In cases where the competitive antibody inhibits the humanized monoclonal antibody of this disclosure in relation to EGFR 287-302 When the binding of the ring exceeds 50%, the humanized antibody "competitively" binds. In a non-limiting example, the antibody of this disclosure binds to EGFR. 287-302 Humanized antibodies with identical epitopes on the ring are human monoclonal antibodies. These humanized monoclonal antibodies can be prepared and isolated as described herein.

[0222] B. Conjugate The humanized monoclonal antibodies or antigen-binding fragments disclosed herein can be conjugated with reagents (e.g., effector molecules or detectable biomarkers) using a variety of methods known in the art. Covalent or non-covalent attachment can be used. Conjugates include, but are not limited to, molecules in which effector molecules or detectable biomarkers are covalently linked to the humanized monoclonal antibodies or antigen-binding fragments disclosed herein. Those skilled in the art will understand that a wide variety of effector molecules and detectable biomarkers can be used, including (but not limited to) chemotherapeutic agents, anti-angiogenic agents, toxins, and radiation agents (e.g., [missing information]). 125 I, 32 P, 14 C 3 H and 35 S) and other markers, target parts and ligands, etc.

[0223] The selection of a specific effector molecule or detectable biomarker depends on the specific target molecule or cell and the desired biological effect. Thus, for example, an effector molecule could be a cytotoxin designed to cause the death of a specific target cell, such as a tumor cell expressing EGFRvIII and / or EGFR gene amplification.

[0224] The procedure for attaching an effector molecule or detectable biomarker to an antibody or antigen-binding fragment varies depending on the chemical structure of the effector. Peptides typically contain multiple functional groups, such as carboxylic acid (COOH), free amino (-NH2), or thiol (-SH) groups, which can react with suitable functional groups on the antibody to result in binding to the effector molecule or detectable biomarker. Alternatively, the antibody or antigen-binding fragment may be derivatized to expose or attach additional reactive functional groups. Derivatization may involve attaching known adapter molecules, such as those available from Pierce Chemical Company, Rockford, IL. The adapter can be any molecule used to link a humanized antibody or antigen-binding fragment to an effector molecule or detectable biomarker. The adapter is capable of forming a covalent bond with both the humanized antibody or antigen-binding fragment and the effector molecule or detectable biomarker. Suitable adapters are known and include, but are not limited to, straight-chain or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers. In the case of humanized antibodies or antigen-binding fragments and effector molecules or detectable markers being peptides, the linker can be connected to a constitutive amino acid (e.g., to cysteine ​​via a disulfide bond) or to the α-carbon amino and carboxyl groups of the terminal amino acid via its side chain groups.

[0225] Furthermore, the adapter may include a spacer element, the presence of which increases the size of the adapter, thereby increasing the distance between the effector molecule or detectable marker and the humanized antibody or antigen-binding fragment. Exemplary spacers are known and include those listed 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, and 5,554,720. 5, 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 U.S. Patent Publications 20110212088 and 20110070248.

[0226] In some instances, the conjugate includes a linker that links an effector molecule or detectable marker to the humanized monoclonal antibody or antigen-binding fragment disclosed herein. In some instances, the linker is cleavable under intracellular conditions, such that cleavage of the linker releases the effector molecule or detectable marker from the humanized antibody or antigen-binding fragment in the intracellular environment. In other instances, the linker is not cleavable, and the effector molecule or detectable marker is released, for example, through antibody degradation. In some instances, the linker is cleavable by a cleaving agent present in the intracellular environment (e.g., within lysosomes or endosomes or pits). The linker can be, for example, a peptide linker, cleaved by intracellular peptidases or proteases, including but not limited to lysosomal or endosomal proteases. In some aspects, the peptide linker is at least two amino acids long or at least three amino acids long. In some instances, the linker is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids long, for example, 1-2, 1-3, 2-5, 3-10, 3-15, 1-5, 1-10, or 1-15 amino acid lengths. Proteases can include cathepsins B and D, as well as plasmin, all of which are known to hydrolyze dipeptide drug derivatives, resulting in the release of the active drug within target cells. See For example, Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123. For instance, peptide linkers cleavable by the thiol-dependent protease cathepsin B (e.g., phenylalanine-leucine or glycine-phenylalanine-leucine-glycine linkers) can be used. Other examples of linkers are described, for example, in U.S. Patent No. 6,214,345. In certain instances, peptide linkers cleavable by intracellular proteases are valine-citrulline linkers or phenylalanine-lysine linkers (…). See For example, U.S. Patent No. 6,214,345 describes the synthesis of doxorubicin using a valine-citrulline linker.

[0227] In other respects, the severable connector is pH sensitive, meaning it is sensitive to hydrolysis at certain pH values. Typically, pH-sensitive connectors are hydrolyzable under acidic conditions. For example, acid-labile connectors that are hydrolyzable in lysosomes (e.g., hydrazones, ureas, thioureas, cis-aconitine, orthoesters, acetals, ketals, etc.) can be used. SeeFor 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 (e.g., in blood), but unstable below pH 5.5 or 5.0 (the approximate pH of lysosomes). In some instances, the hydrolyzable linker is a thioether linker (e.g., a thioether linker attached to a therapeutic agent via an acylhydrazone bond). See (e.g., U.S. Patent No. 5,622,929).

[0228] In other respects, the joint is cuttable under reducing conditions (e.g., disulfide joints). Various disulfide joints are known in the art, including, for example, those formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene), SPDB, and SMPT. See , for example, Thorpe et al., 1987, Cancer Res. 47:5924-5931;Wawrzynczak et al ., In Immunoconjugates: Antibody Conjugates in Radioimageryand Therapy of Cancer (CW Vogel ed., Oxford U. Press, 1987); Phillips et al ., Cancer Res. 68:92809290, 2008; See also U.S. Patent No. 4,880,935.

[0229] In other instances, the connector is a malonate connector (Johnson). et al ., 1995, Anticancer Res. 15:1387-93), maleimide benzoyl linker (Lau et al ., 1995, Bioorg-Med-Chem. 3(10):1299-1304) or 3'-N-amide analogs (Lauet al ., 1995, Bioorg-Med-Chem. 3(10):1305-12).

[0230] In several instances, the adapters are resistant to cleavage in the extracellular environment. For example, in the conjugate samples disclosed herein, when the conjugates are present in an extracellular environment (e.g., plasma), no more than about 20%, no more than about 15%, no more than about 10%, no more than about 5%, no more than about 3%, or no more than about 1% of the adapters are cleaved. For example, whether the adapters are resistant to cleavage in the extracellular environment can be determined by incubating the conjugates containing the adapters of interest with plasma for a predetermined period of time (e.g., 2 hours, 4 hours, 8 hours, 16 hours, or 24 hours) and then quantifying the amount of free effector molecules or detectable markers present in the plasma. Various exemplary adapters that can be used in conjugates are described in WO 2004-010957, U.S. Publication No. 2006 / 0074008, U.S. Publication No. 2005 / 0238649, and U.S. Publication No. 2006 / 0024317.

[0231] The humanized monoclonal antibodies or antigen-binding fragments disclosed herein can be derivatized, for example, by cross-linking two or more antibodies (of the same or different types, for example, for creating bispecific antibodies). Suitable cross-linking agents include heterobifunctional cross-linking agents having two distinct reactive groups separated by a suitable spacer (e.g., m-maleimide benzoyl-N-hydroxysuccinimide ester), or homobifunctional cross-linking agents (e.g., disuccinimide octanoate). Such linkers are commercially available.

[0232] The humanized antibodies or antigen-binding fragments disclosed herein can be conjugated with low molecular weight drugs, such as monomethylaurestatin E (MMAE), monomethylaurestatin F (MMAF), maytansine, maytansine derivatives (including maytansine derivatives called DM1 (also known as mertansine), delutecan, or other chemotherapeutic agents, to prepare antibody-drug conjugates (ADCs). The chemotherapeutic agents described herein can be conjugated with the provided antibodies to generate ADCs.

[0233] The humanized monoclonal antibodies or antigen-binding fragments disclosed herein can be conjugated to one or more small molecule toxins, such as calicheamicin, maytansines, dolalastatins, aurestatins, trichothecene, and CC1065, as well as derivatives of these toxins with toxic activity. Maytansine compounds suitable for use as the maytansine toxin moiety are available and can be isolated from natural sources using known methods and produced using genetic engineering techniques. See Yu et al(2002) PNAS 99:7968-7973), or maytanol and maytanol analogues can be synthesized by known methods. Maytansins are mitotic inhibitors that exert their effects by inhibiting tubulin polymerization. Maytansins were initially isolated from the East African shrub *Maytenus serrata* (US Patent No. 3,896,111). Subsequently, it was discovered that certain microorganisms also produce maytansins, such as maytanol and C-3 maytanol ester (US Patent No. 4,151,042). Synthetic maytanol and its derivatives and analogues are disclosed, for example, in U.S. Patent 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 maytansine, their preparation methods and therapeutic uses are disclosed, for example, in U.S. Patent Nos. 5,208,020; 5,416,064; 6,441,163 and European Patent EP 0425 235 B1.

[0234] Given the numerous methods already reported for attaching various radiodiagnostic compounds, radiotherapy compounds, labels (such as enzymes or fluorescent molecules), toxins, and other reagents to antibodies, those skilled in the art will be able to determine the appropriate method for attaching a given reagent to the humanized monoclonal antibody or antigen-binding fragment disclosed herein.

[0235] In specific, non-limiting instances, the conjugate includes the humanized monoclonal antibody that specifically binds to EGFRvIII or the gene-amplified EGFR product (or its antigen-binding fragment), an irreducible thioester linker, and maytansin toxin DM1 disclosed herein; for example, the conjugate may include the structure shown below (where “mAb” refers to the humanized monoclonal antibody or its antigen-binding fragment):

[0236] In some instances, the effector molecule is auristatin, such as auristatin E (also known as a derivative of saccharin-10) or a derivative thereof. For example, auristatin can be an ester formed from auristatin E and a keto acid. For example, auristatin E can be reacted with acetylbenzoic acid or benzoylvaleric acid, respectively, to produce AEB and AEVB. Other exemplary auristatins include AFP, MMAF, and MMAE. The synthesis and structure 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 02 / 088172 and 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; 5,554,725; 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. Further descriptions of antibody-drug conjugates, including auristatin MMAE, and methods for preparing such conjugates are provided, for example, in U.S. Publications 2011 / 0268751, 2008 / 0305044, and 2007 / 0258987. Auristatins have been shown to interfere with microtubule dynamics, as well as cell nucleus and cell division, and to possess anticancer activity. Auristatins bind to tubulin and can exert cytotoxic or inhibitory effects on cells. Various assays are known in the art for determining whether auristatin or its derivatives exert inhibitory or cytotoxic effects on desired cell lines.

[0237] In one example, the conjugate comprises a humanized monoclonal antibody (or its antigen-binding fragment) that specifically binds to EGFRvIII or a product of EGFR gene amplification disclosed herein, a cleavable linker including a valine-citrulline (Val-Cit) peptide cleavage site, a spacer, and a toxin MMAE; for example, the conjugate may comprise the structure shown below (where “mAb” refers to a monoclonal antibody or its antigen-binding fragment):

[0238] In another non-limiting instance, the conjugate is

[0239] Where n is an integer from 0 to 10 (e.g., 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 the humanized monoclonal antibody or its antigen-binding fragment disclosed herein, and S is a sulfur atom from the humanized antibody. In one instance, n is preferably an even number from 0 to 8, for example, an even number from 0 to 4. The S portion can be exposed by reducing or partially reducing the interchain disulfide of the humanized antibody (e.g., by treatment with a reducing agent such as DTT or TCEP).

[0240] In a non-limiting example, the conjugate is:

[0241] Where n is 4, and A is the monoclonal antibody or its antigen-binding fragment disclosed herein.

[0242] Other toxins may be employed in conjunction with humanized monoclonal antibodies that specifically bind to EGFRvIII and / or gene-amplified EGFR, as well as antigen-binding fragments of these antibodies. Exemplary toxins include Pseudomonas exotoxin (PE), ricin, abrin, diphtheria toxin and its subunits, ribosomal toxins, ribonucleases, saponins, and chalcogenide, and botulinum toxins A through F. These toxins are known, and many are readily available from commercial sources (e.g., Sigma Chemical Company, St. Louis, MO). Contemplated toxins also include variants of the toxins ( See (For example, see U.S. Patent Nos. 5,079,163 and 4,689,401). In some aspects, the conjugates disclosed herein are intended for the treatment of cancers such as head and neck cancer, breast cancer, pancreatic cancer, colon or rectal cancer, CNS cancer, or bladder cancer.

[0243] Saponins are derived from soapwort ( Saponaria officinalis The toxin interferes with protein synthesis by inactivating the 60S subunit of the ribosome complex. et al (Bio / Technology, 10:405-412, 1992). However, this toxin lacks a specific mechanism for entering cells and therefore requires conjugation with antibody or antigen-binding fragments that recognize internalized cell surface proteins in order to be effectively absorbed by the cell.

[0244] Diphtheria toxin is derived from Corynebacterium diphtheriae (Corynebacterium diphtheria Corynebacterium diphtheriae(Separation). Typically, diphtheria toxin used as an immunotoxin is mutated to reduce or eliminate its nonspecific toxicity. A mutant called CRM107, possessing 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.

[0245] Ricin is derived from castor beans ( Ricinus communis ricin (RCA) is the lectin from castor beans. For examples of ricin, see U.S. Patent Nos. 5,079,163 and 4,689,401. Ricin (RCA) exists in two forms, referred to as RCA according to their molecular weights (approximately 65 kD and 120 kD). 60 and RCA 120 (Nicholson & Blaustein, J. Biochim. Biophys. Acta 266:543, 1972). The A chain is responsible for inactivating protein synthesis and killing cells. The B chain binds ricin to galactose residues on the cell surface and promotes the transport of the A chain into the cytoplasm (Olsnes). et al (Nature 249:627-631, 1974 and U.S. Patent No. 3,060,165).

[0246] Ribonucleases are also conjugated to target molecules for use as immunotoxins. See Suzuki et al (Nat. Biotech. 17:265-70, 1999). Exemplary ribosomal toxins (e.g., α-sarcin and aspergillin) are discussed in, for example, Rathore. et al ., Gene 190:31-5, 1997; and Goyal and Batra, Biochem. 345 Pt 2:247-54, 2000. Kazimidycin was originally derived from Micromonospora echinococcosis ( Micromonospora echinospora It separates and is a member of the enediyne antitumor antibiotic family, which causes DNA double-strand breaks, leading to apoptosis. See For example, Lee et al., J. Antibiot. 42:1070-87, 1989. This drug is the toxicity component of immunotoxins in clinical trials ( See For example, Gillespieet al ., Ann. Oncol. 11:735-41, 2000).

[0247] Abrus toxins include those derived from arisaema (Abrus precatorius) Abrus precatorius The toxic lectins of abrinogen. The toxic components abrinogen a, b, c, and d have molecular weights of approximately 63 kDa to 67 kDa and are composed of polypeptide chains A and B linked by two disulfide bonds. Chain A inhibits protein synthesis; chain B (abrusinogen-b) binds to D-galactose residues (…). See ,Funatsu et al ., Agr. Biol. Chem. 52:1095, 1988; and Olsnes, Methods Enzymol. 50:330-335, 1978).

[0248] In one instance, the toxin is a pseudomonad exotoxin (PE) (US Patent No. 5,602,095). As used herein, PE includes full-length natural (naturally occurring) PE or modified PE. Such modifications may include, but are not limited to, the removal of various amino acid deletions in domain Ia, domains Ib, II, and III, single amino acid substitutions, and the addition of one or more sequences (e.g., at the carboxyl terminus) to the carboxyl terminus. See Siegall et al (J. Biol. Chem. 264:14256-14261, 1989). The PE used with the provided antibody may include the natural sequence, cytotoxic fragments of the natural sequence, and conserved modified variants of the natural PE and its cytotoxic fragments. Cytotoxic fragments of the PE include those that are cytotoxic in target cells, whether or not they have undergone subsequent proteolysis or other processing. Cytotoxic fragments of the PE include PE25, PE40, PE38, and PE35. For further descriptions of PE and its variants, see, for example, 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.

[0249] Protease-resistant PE variants and immunogenic PE variants were also envisioned, such as, but not limited to, PE-LR, PE-6X, PE-8X, PE-LR / 6X, and PE-LR / 8X. See For example, Weldon et al ., Blood 113(16):3792-3800,2009; Onda et al ., Proc. Natl. Acad. Sci. USA, 105(32):11311-11316, 2008; and PCT Publications WO 2007 / 016150, WO 2009 / 032954 and WO 2011 / 032022). The PE variant can be PE25 ( See See Weldon et al ., Blood 2009;113:3792–3800).

[0250] In some instances, PE is a variant 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, PE is a variant known as PE-LR / 6X (PCT Publication No. WO 2011 / 032022). In a further instance, PE is a variant known as PE-LR / 8M (PCT Publication No. WO 2011 / 032022).

[0251] Humanized monoclonal antibodies (or their antigen-binding fragments) that specifically bind to EGFRvIII and / or amplified EGFR genes can also be conjugated to detectable biomarkers, such as those that can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or diagnostic imaging techniques (e.g., computed tomography (CT), computed axial computed tomography (CAT), magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance imaging (MTR), ultrasound, fiberoptic endoscopy, and laparoscopy). Specific non-limiting examples of detectable biomarkers include fluorophores, chemiluminescent agents, enzyme-linked compounds, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for MRI detection). For example, useful detectable biomarkers include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, etc. Bioluminescent biomarkers can also be used, such as luciferase, green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), and cyan fluorescent protein (CFP). Humanized monoclonal antibodies or antigen-binding fragments can also be conjugated to enzymes used for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, and glucose oxidase. When humanized monoclonal antibodies or antigen-binding fragments are conjugated to detectable enzymes, detection can be achieved by adding another reagent to generate a recognizable reaction product using that enzyme. For example, in the presence of horseradish peroxidase reagent, the addition of hydrogen peroxide and diaminobenzidine produces a colored reaction product that is visually detectable. Humanized monoclonal antibodies or antigen-binding fragments can also be conjugated to biotin and detected by indirectly measuring the binding of avidin or streptavidin. Avidin itself can also be conjugated to enzymes or detectable biomarkers.

[0252] Humanized monoclonal antibodies or antigen-binding fragments can be conjugated to paramagnetic agents (e.g., gadolinium). Paramagnetic agents (e.g., superparamagnetic iron oxide) can also be used as labels. Antibodies can also be conjugated to lanthanides (e.g., europium and dysprosium) and manganese. Humanized monoclonal antibodies or antigen-binding fragments can also be labeled with predetermined peptide epitopes that can be recognized by secondary reporter sequences (e.g., leucine zipper pairs, binding sites of secondary antibodies, metal-binding domains, epitope tags).

[0253] Humanized monoclonal antibodies or antigen-binding fragments can also be conjugated to radiolabeled amino acids. Radiolabeling can be used for diagnostic and therapeutic purposes. For example, radiolabeling can be used to detect EGFRvIII and / or gene-amplified EGFR, and cells expressing EGFRvIII and / or gene-amplified EGFR, by X-ray, emission spectroscopy, or other diagnostic techniques. Furthermore, radiolabeling can be used therapeutically as a toxin to treat tumors in a subject, such as any tumor expressing EGFRvIII and / or gene-amplified EGFR, such as cancers like head and neck cancer, breast cancer, pancreatic cancer, colon or rectal cancer, CNS cancer, or bladder cancer. Examples of labeling for peptides include, but are not limited to, the following radioisotopes or radionuclides: 3 H, 14 C 15 N、 35 S, 90 Y、 99 Tc, 111 In、 125 I, 131 I.

[0254] Methods for detecting detectable markers have been described; for example, radioactive markers can be detected using photographic film or a scintillation counter, and fluorescent markers can be detected using a photodetector to detect emitted light. Enzyme markers are typically detected by providing a substrate to the enzyme and detecting the reaction products produced by the enzyme acting on the substrate, while colorimetric markers are detected by simply visualizing the colored marker.

[0255] The humanized monoclonal antibodies or antigen-binding fragments disclosed herein can also be derivatized with chemical groups, such as polyethylene glycol (PEG), methyl or ethyl groups, or carbohydrate groups. These groups can be used to improve the biological properties of humanized antibodies or antigen-binding fragments, such as prolonging serum half-life or increasing tissue binding.

[0256] In conjugates, the average number of effector molecules or detectable biomarker moieties per humanized monoclonal antibody or antigen-binding fragment can range from, for example, 1 to 20 moieties per antibody or antigen-binding fragment. For some conjugates, the average number of effector molecules or detectable biomarker moieties per antibody or antigen-binding fragment may be limited by the number of attachment sites on the humanized monoclonal antibody or antigen-binding fragment. For example, if the attachment is cysteine ​​thiol, the humanized monoclonal antibody or antigen-binding fragment may have only one or more cysteine ​​thiol groups, or may have only one or more sufficiently reactive thiol groups through which a linker can be attached. In some instances, the average number of effector molecules or detectable biomarker moieties in each humanized monoclonal antibody or antigen-binding fragment in the conjugate ranges from 1 to 10; for example, 2 to 6; 2 to 8; 3 to 5; 3 to 4; 3.1 to 3.9; 3.2 to 3.8; 3.2 to 3.7; 3.2 to 3.6; 3.3 to 3.8; or 3.3 to 3.7. In further instances, the average number of effector molecules or detectable biomarker moieties in each humanized monoclonal antibody or antigen-binding fragment is about 1, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, or about 9. In conjugate formulations, the average number of effector molecules or detectable biomarker moieties of each humanized monoclonal antibody or antigen-binding fragment can be characterized by conventional methods (e.g., mass spectrometry or ELISA assays). The loading of the conjugate (e.g., the effector molecule / antibody ratio) can be controlled in various ways, for example, by: (i) limiting the molar excess of the effector molecule-linker intermediate or linker reagent relative to the antibody; (ii) limiting the conjugation reaction time or temperature; (iii) partially or restrictively reducing conditions with cysteine ​​thiol modification; and (iv) engineering the amino acid sequence of the humanized antibody using recombinant techniques, such that the number and position of cysteine ​​residues are modified to control the number or position of linker-effect molecule attachments (e.g., thioMab or thioFab prepared as disclosed in WO2006 / 03448).

[0257] C. Chimeric Antigen Receptor (CAR) This document also discloses chimeric antigen receptors (CARs) comprising antibodies disclosed herein (e.g., humanized monoclonal antibodies or antigen-binding fragments thereof, specific for EGFRvIII and / or genetically amplified EGFR). A CAR is an artificially constructed chimeric receptor protein comprising an extracellular antigen-binding domain (e.g., a single-chain variable fragment (scFv)) that specifically binds to a target (e.g., EGFRvIII), which is linked to a transmembrane domain and to one or more intracellular T cell signaling domains. The disclosed CARs are characterized by their ability to specifically and reactivity redirect T cells to cells expressing EGFRvIII and / or genetically amplified EGFR in a non-MHC-restricted manner. Recognition of non-MHC-restricted EGFRvIII and / or genetically amplified EGFR enables immune cells (e.g., T cells) expressing the disclosed CARs to recognize antigens without antigen processing.

[0258] Intracellular T-cell signaling domains may include, for example, T-cell receptor signaling domains, T-cell co-stimulatory signaling domains, or both. T-cell receptor signaling domains refer to a portion of the CAR, including the intracellular domain of the T-cell receptor, such as the intracellular portion of the CD3ζ protein. Co-stimulatory signaling domains refer to a portion of the CAR, including the intracellular domain of the co-stimulatory molecule, which is a cell surface molecule, rather than an antigen receptor or its ligand required for an effective lymphocyte response to an antigen.

[0259] 1. Extracellular Region CARs include an antigen-binding domain that specifically binds to EGFRvIII and / or EGFR amplification. For example, the antigen-binding domain may be scFv, including the heavy chain variable region and light chain variable region of any humanized monoclonal antibody or its antigen-binding fragment disclosed herein.

[0260] In some respects, EGFRvIII and / or EGFR-specific antibodies that have undergone gene amplification include the variable heavy chain region (V... H ) and variable light chain region (V L ), and specifically binds to EGFRvIII and / or EGFR gene amplification. In some instances, the CAR includes both heavy and light chains: a) Each of the amino acid sequences shown in SEQ ID NO: 5 and SEQ ID NO: 8 (A10, VH3+VL3) is included. b) Each of the amino acid sequences shown in SEQ ID NO: 3 and SEQ ID NO: 6 (A2, VH1+VL1); c) Includes the amino acid sequences (A3, VH1+VL2) shown in SEQ ID NO: 3 and SEQ ID NO: 7, respectively. d) Includes the amino acid sequences shown in SEQ ID NO: 3 and SEQ ID NO: 8 (A4, VH1+VL3); e) Each of the amino acid sequences shown in SEQ ID NO: 4 and SEQ ID NO: 6 is an A5, VH2+VL1 sequence. f) includes the amino acid sequences (A6, VH2+VL2) shown in SEQ ID NO: 4 and SEQ ID NO: 7, respectively. g) includes the amino acid sequences (A7, VH2+VL3) shown in SEQ ID NO: 4 and SEQ ID NO: 8, respectively. h) includes the amino acid sequences (A8, VH3+VL1) shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively. i) Each of the amino acid sequences shown in SEQ ID NO: 5 and SEQ ID NO: 7 (A9, VH3+VL2); j) includes the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 9 (B1,40H3 VH+VL-EG); k) includes the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 10 (B2,40H3 VH+VL-DA); l) includes the amino acid sequences shown in SEQ ID NO: 4 and SEQ ID NO: 11 (B3, VH2+VL1-DA); m) includes the amino acid sequences shown in SEQ ID NO: 4 and SEQ ID NO: 12 (B4, VH2+VL2-DA); n) includes the amino acid sequences (B5, VH3 and VL1-DA) shown in SEQ ID NO: 5 and SEQ ID NO: 11, respectively. o) includes the amino acid sequences (C10) shown in SEQ ID NO: 31 and SEQ ID NO: 32, respectively; p) includes the amino acid sequences shown in SEQ ID NO: 5 and SEQ ID NO: 14, respectively (D2); or q) includes the amino acid sequences shown in SEQ ID NO: 5 and SEQ ID NO: 24, respectively (D3).

[0261] Any antibody or antigen-binding fragment disclosed herein can be used in a CAR. The disclosed antibody or antigen-binding fragment is humanized. In a non-limiting example, the heavy chain and light chain included in the CAR are SEQ ID NO: 5 and SEQ ID NO: 8, respectively.

[0262] In several respects, the antigen-binding fragment is an scFv. In some respects, the scFv includes a heavy chain variable region and a light chain variable region linked by a peptide linker (e.g., a linker comprising an amino acid sequence as shown in GGGGSGGGGSGGGGS (SEQ ID NO: 33)).

[0263] CARs may include a signal peptide sequence, such as a signal peptide sequence located at the N-terminus of the antigen-binding domain. The signal peptide sequence may include any suitable signal peptide sequence. In an example, the signal peptide sequence is a human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor sequence, such as LLVTSLLLCELPHPAFLLIPDT (SEQ ID NO: 34) or an amino acid sequence comprising it. While the signal peptide sequence may contribute to CAR expression on the cell surface, the presence of the signal peptide sequence in the expressed CAR is not essential for CAR function. Once the CAR is expressed on the cell surface, the signal peptide sequence may be cleaved from the CAR. Therefore, in some instances, the CAR lacks a signal peptide sequence.

[0264] 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 contain up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids. In some aspects, the spacer domain may include an immunoglobulin domain, such as a human immunoglobulin sequence. In an example, the immunoglobulin domain comprises the immunoglobulin CH2 and CH3 immunoglobulin G (IgG1) domain sequences (CH2CH3). In this respect, the spacer domain may include an immunoglobulin domain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 35: EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPK.

[0265] Unbound by any particular theory, it is believed that the CH2CH3 domain extends the antigen-binding domain of the CAR away from the membrane of the CAR-expressing cell and may more accurately mimic the size and domain structure of the native TCR.

[0266] 2. Transmembrane Domain Regarding transmembrane domains, CARs can be designed to include transmembrane domains fused with the extracellular domains of the CAR. In one example, a transmembrane domain naturally connected to one of the domains in the CAR is used.

[0267] The transmembrane domain can be derived from a natural or synthetic source. If the source is natural, the domain can be derived from any membrane-binding or transmembrane protein. Exemplary transmembrane domains for disclosed CARs may include at least the α, β, or ζ chain of the T cell receptor, and transmembrane regions of CD28, CD3ε, CD45, CD4, CD5, CDDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. Alternatively, the transmembrane domain may be synthetic, in which case it will primarily contain hydrophobic residues such as leucine and valine. In several instances, each end of the synthetic transmembrane domain contains a triplet of phenylalanine, tryptophan, and valine.

[0268] Optionally, short oligopeptides or polypeptide linkers (preferably 2 to 10 amino acids in length) can form a 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.

[0269] In some instances, the transmembrane domain comprises a transmembrane domain of a T-cell receptor, such as the CD8 transmembrane domain. Therefore, a CAR may include the CD8 transmembrane domain, which includes or comprises SEQ ID NO: 36: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC.

[0270] In other instances, the transmembrane domain comprises a transmembrane domain of a T-cell co-stimulatory molecule, such as CD137 or CD28. Therefore, a CAR may include a CD28 transmembrane domain comprising or consisting of SEQ ID NO: 37: IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVR.

[0271] 3. Intracellular Region The intracellular regions of a CAR include one or more intracellular T cell signaling domains that are responsible for activating at least one normal effector function of T cells to which the CAR is expressed or introduced. Exemplary T cell signaling domains are provided and described herein.

[0272] While the entire intracellular T cell signaling domain can be used in CARs, in many cases, the whole chain is not necessary. When using a truncated portion of the intracellular T cell signaling domain, it can replace the entire chain as long as that truncated portion transduces the relevant T cell effector signaling.

[0273] Examples of intracellular T-cell signaling domains used in CARs include cytoplasmic sequences of T-cell receptors (TCRs) and co-stimulatory molecules (which work together to initiate signal transduction upon antigen receptor binding), as well as any derivatives or variants of these sequences, and any synthetic sequences with the same functional capabilities.

[0274] T-cell receptor signaling domains regulate the initial activation of the T-cell receptor complex in a stimulatory or inhibitory manner. Disclosed CARs may include major cytoplasmic signaling sequences that function in a stimulatory manner, and these sequences may contain signaling motifs known as immune receptor tyrosine-based activation motifs or ITAMs. Examples of ITAMs containing major cytoplasmic signaling sequences that may be included in disclosed CARs include ITAMs derived from the proteins CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CDS, CD22, CD79a, CD79b, and CD66d. In some instances, the cytoplasmic signaling molecules in the CAR include intracellular T-cell signaling domains derived from CD3ζ.

[0275] The intracellular regions of a CAR may include a major cytoplasmic signaling domain (e.g., CD3ζ) containing an ITAM, either alone or in combination with any other desired cytoplasmic domains useful in the context of the CAR. For example, the cytoplasmic domains of a CAR may include a portion of the CD3ζ chain as well as an intracellular co-stimulatory signaling domain. A co-stimulatory signaling domain refers to an intracellular domain of the CAR that includes a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules, not antigen receptors or ligands required for an effective lymphocyte response to an antigen. 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. Another example of a signaling domain that may be included in a disclosed CAR is the tumor necrosis factor receptor superfamily member 18 (TNFRSF18; also known as glucocorticoid-induced TNFR-associated protein, GITR) signaling domain.

[0276] In some instances, the CAR includes a CD3ζ signaling domain, a CD8 signaling domain, a CD28 signaling domain, a CD137 signaling domain, or a combination of two or more of these. In another instance, the cytoplasmic domain includes a CD3ζ signaling domain and a CD28 signaling domain. In a further instance, the cytoplasmic domain includes a CD3ζ signaling domain and a CD137 signaling domain. In yet another instance, the cytoplasmic domain includes a CD3ζ signaling domain as well as CD28 and CD137 signaling domains. Those skilled in the art can change the order of one or more T cell signaling domains on the CAR as needed. Exemplary amino acid sequences of such T cell signaling domains are provided. For example, the CD3ζ signaling domain may include or consist of the amino acid sequence shown in SEQ ID NO: 38: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR. The CD8 signaling domain may include or consist of the amino acid sequence shown in SEQ ID NO: 39: FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNR. The CD28 signaling domain may include or consist of the amino acid sequence shown in SEQ ID NO: 40: SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS. The CD137 signal transduction domain may include or consist of the amino acid sequence shown in SEQ ID NO: 41 or SEQ ID NO: 42: SEQ ID NO: 41: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL, or SEQ ID NO: 42: RFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0277] The cytoplasmic signaling sequences within the cytoplasmic signaling region of the CAR disclosed herein can be linked together in a random or specified order. Optionally, short polypeptide linkers (preferably 2 to 10 amino acids in length) can form a linker. Glycine-serine dinucleotides provide particularly suitable linkers. Furthermore, 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 contain up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids.

[0278] 4. Additional Descriptions of CAR The functional portion of the CAR described herein is also provided. When referring to a CAR, the term "functional portion" means any part or fragment of the CAR that retains the biological activity of its parent CAR. For example, a functional portion encompasses those parts of the CAR that retain the ability to recognize target cells, or retain the ability to detect, treat, or prevent disease to a similar, equal, or greater degree than that of the parent CAR. Regarding the parent CAR, the functional portion may comprise, for example, approximately 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or higher of the parent CAR.

[0279] The CAR or its functional portion may include additional amino acids at the N-terminus, C-terminus, or both, which are not present in the amino acid sequence of the parent CAR. It is desirable that these additional amino acids do not interfere with the biological function of the CAR or its functional portion, such as recognizing target cells, detecting cancer, treating or preventing cancer. More desirablely, these additional amino acids should enhance the biological activity compared to that of the parent CAR.

[0280] Functional variants of the CAR described herein are also provided, which have substantial or significant sequence identity or similarity to the parental CAR and retain its biological activity as a variant CAR. For example, functional variants include variants of the CAR described herein (parental CAR) that retain the ability to recognize target cells to a degree, the same degree, or greater than that of the parental CAR. For the parental CAR, for example, the functional variants may be 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 higher, identical in amino acid sequence to the parental CAR.

[0281] For example, a functional variant may comprise the amino acid sequence of the parental CAR but have at least one conserved amino acid substitution. Alternatively, or additionally, a functional variant may comprise the amino acid sequence of the parental CAR but have at least one non-conserved amino acid substitution. In this case, preferably, the non-conserved amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. The non-conserved amino acid substitution may enhance the biological activity of the functional variant, resulting in increased biological activity of the functional variant compared to the parental CAR.

[0282] CARs (including functional parts and functional variants) can be of any length, meaning they can contain any number of amino acids, provided that the CAR (or its functional part or functional variant) retains its biological activity, such as the ability to specifically bind to antigens, the ability to detect diseased cells in mammals, or the ability to treat or prevent diseases in mammals. In some instances, CARs are approximately 50 to 5000 amino acids long, for example, 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more amino acids.

[0283] The CARs disclosed herein (including the functional portions and functional variants of the CARs disclosed herein) may include 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, leucine, α-aminodecanoic acid, homoserine, S-acetaminomethylcysteine, trans-3-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline- 2-Carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyllysine, N',N'-dibenzyllysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, oc-aminocycloheptanecarboxylic acid, -(2-amino-2-norbornene)-carboxylic acid, γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0284] The CARs disclosed herein (including functional moieties and functional variants) can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via, for example, disulfide bridging, or converted into acid addition salts, and / or optionally dimerized or polymerized, or conjugated.

[0285] Methods for generating chimeric antigen receptors, T cells including such receptors, and their uses (e.g., for cancer treatment) are known and further described, for example, by 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, 1 12:2261 -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 Oncol., 6:47, 2013; PCT Publications WO2012 / 079000, WO2013 / 126726, and U.S. Publication 2012 / 0213783).

[0286] In some instances, the nucleic acid molecule encoding the CAR disclosed herein is included in an expression vector (e.g., a lentiviral vector) for expression in host cells (e.g., T cells) to prepare the disclosed CAR. In some aspects, methods using chimeric antigen receptors include isolating immune cells (e.g., T cells) from a subject, transforming the isolated cells with an expression vector encoding the CAR disclosed herein (e.g., a lentiviral vector or an AAV vector), and administering the CAR-expressing engineered cells to the subject for treatment, e.g., treating a tumor in the subject.

[0287] D. Polynucleotides and Expression Nucleic acids encoding humanized monoclonal antibodies, antibody-binding fragments, conjugates, or CARs that specifically bind to EGFRvIII and / or EGFR gene amplification are provided. Using the amino acid sequences provided herein (e.g., CDR sequences, heavy and light chain sequences), sequences available in the prior art (e.g., framework sequences), and the genetic code, those skilled in the art can readily generate nucleic acids encoding these molecules. Those skilled in the art can readily use the genetic code to construct various functionally equivalent nucleic acids, such as nucleic acids with different sequences but encoding the same antibody sequence, or those encoding V... L and / or V H Conjugates or fusion proteins of nucleic acid sequences.

[0288] Nucleic acid sequences encoding publicly disclosed humanized monoclonal antibodies, antibody-binding fragments, conjugates, and CARs that specifically bind to EGFRvIII and / or gene amplification of EGFR can be prepared by any suitable method, such as cloning suitable sequences or by direct chemical synthesis, methods such as Narang et al., Meth. Enzymol. 68:90-99, the phosphate triester method of 1979; Brown et al. , Meth. Enzymol. Phosphodiester method, 68:109-151, 1979; Beaucage et al., Tetra. Lett. 22:1859-1862, 1981, diethylphosphoramide process; Beaucage & Caruthers, Tetra. Letts. Solid-phase phosphoramidite esterification, 22(20):1859-1862, 1981, for example, using automated synthesizers, as described in Needham-Van Devander. et al. , Nucl. Acids Res Solid-phase support method as described in .12:6159-6168, 1984; and US Patent No. 4,458,066. Chemical synthesis produces single-stranded oligonucleotides. These can be converted into double-stranded DNA by hybridization with complementary sequences or by polymerization with DNA polymerase using single strands as templates. Those skilled in the art will recognize that while the chemical synthesis of DNA is typically limited to sequences of about 100 bases, longer sequences can be obtained by joining shorter sequences.

[0289] The nucleic acids disclosed herein can be prepared using standard molecular biology techniques. Examples of suitable cloning and sequencing techniques are known ( See For example, Sambrook et al . (Molecular Cloning:A Laboratory Manual, 4 th ed, 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 provided by manufacturers of biological reagents and laboratory equipment also offers useful information. These manufacturers include SIGMAChemical Company (Saint Louis, MO), R&D Systems (Minneapolis, MN), PharmaciaAmersham (Piscataway, NJ), CLONTECH Laboratories, Inc. (Palo Alto, CA), ChemGenes Corp., Aldrich Chemical Company (Milwaukee, WI), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersburg, MD), Fluka Chemica-BiochemikaAnalytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen (Carlsbad, CA), and Applied Biosystems (Foster City, CA), as well as many other commercial sources.

[0290] Nucleic acids can also be prepared through amplification methods. These methods include polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification systems (TAS), and self-sustaining sequence replication systems (3SR). Various cloning methods, host cells, and in vitro amplification techniques are well-known.

[0291] In non-limiting examples, nucleic acid molecules are provided that encode the CAR disclosed herein for expression in immune cells, such as T cells, natural killer (NK) cells, natural killer T (NKT) cells, cytotoxic T lymphocytes (CTLs), or regulatory T cells. In some examples, the CAR disclosed herein is used for expression in T cells, for example, to generate chimeric antigen receptor T cells (CAR T cells). The nucleic acid molecule encoding the CAR may be included in a vector (e.g., a lentiviral vector). Methods for generating nucleic acid molecules encoding chimeric antigen receptors and cells comprising such receptors are known. See For example, 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 etal., 2008, Blood, 1 12:2261-2271; Park et al ., Trends Biotechnol ., 29:550-557,2011; Grupp et al., N Engl J Med., 368:1509-1518, 2013; Han et al., J. HematolOncol., 6:47, 2013; PCT Publications WO2012 / 079000, WO2013 / 126726; and U.S. Publication 2012 / 0213783).

[0292] Nucleic acid molecules can be expressed in recombinant engineered cells, such as bacterial, plant, yeast, insect, and mammalian cells. These antibodies, antigen-binding fragments, and conjugates can be expressed as individual V antibodies. H and / or V L The chain (linked to an effector molecule or detectable marker as needed) or can be expressed as a fusion protein. Methods for expressing and purifying antibodies and antigen-binding fragments are known and are further described herein. See For example, Al-Rubeai (ed). Antibody Expression and Production (Springer Press, 2011). Immunoadhesins can also be expressed. Therefore, in some instances, encoding V is provided. H and V L And the nucleic acid of immunoadhesins. The nucleic acid sequence may optionally encode a leader sequence.

[0293] To create an scFv, you can encode V. H and V L The DNA fragment is operatively linked to another fragment encoding a flexible linker (e.g., encoding the amino acid sequence (Gly4-Ser)3), enabling V H and V L The sequence can be expressed as a continuous single-chain protein, where V L and V H The structural domains are connected by flexible joints (see, for example, Bird). et al., Science 242:423-426, 1988; Huston et al., Proc. Natl. Acad. Sci. USA85:5879-5883, 1988; McCafferty et al., Nature 348:552-554, 1990; Kontermann and Dubel (Ed),Antibody Engineering, Vols. 1-2, 2 nd Ed., Springer Press, 2010; Harlow and Lane, Antibodies: A Laboratory Manual , 2 nd (Cold Spring Harbor Laboratory, New York, 2013). Optionally, the adapter may include a cleavage site, such as a furin cleavage site.

[0294] Encoding V H and / or V L The nucleic acid may optionally encode an Fc domain (immunoadhesin). The Fc domain may be an IgA, IgM, or IgG Fc domain. The Fc domain may be an optimized Fc domain, as described in U.S. Patent Application Publication No. 2010 / 093979. In one example, the immunoadhesin is IgG1Fc.

[0295] Single-chain antibodies can be monovalent (if only a single V is used). H and V L ), divalent (if two V's are used) H and V L ) or multi-valent (if more than two V's are used) H and V L This can generate bispecific or multivalent antibodies that can specifically bind to, for example, EGFRvIII and other antigens (e.g., but not limited to CD3). Encoding V H and V L V may be included optionally H and V L Frin protease cleavage sites between domains.

[0296] Many expression systems are known and can be used to express nucleic acids in hosts such as Escherichia coli, other bacterial hosts, yeast, or various higher eukaryotic cells, such as COS, CHO, HeLa, and myeloma cell lines.

[0297] One or more DNA sequences encoding a disclosed humanized monoclonal antibody, antibody-binding fragment, conjugate, or CAR can be expressed in vitro via DNA transfer to a suitable host cell. The cell can be a prokaryotic or eukaryotic cell. This term also includes any progeny of the subject's host cell. It should be understood that not all progeny cells are identical to the parent cell due to mutations that may occur during replication. Methods for stable transfer (i.e., the persistent maintenance of foreign DNA in the host) are known in the art. This disclosure also covers hybridomas expressing humanized monoclonal antibodies.

[0298] A polynucleotide sequence encoding a publicly disclosed humanized monoclonal antibody, antibody-binding fragment, conjugate, or CAR can be operatively linked to an expression control sequence. The operative linking of the expression control sequence to the coding sequence enables expression of the coding sequence under conditions compatible with the expression control sequence. Expression control sequences include, but are not limited to: promoters, enhancers, transcription terminators, start codons preceding protein-coding genes (e.g., ATG), intron splicing signals, spacers for maintaining the correct reading frame and allowing correct mRNA translation, and stop codons.

[0299] To achieve high-level expression of the cloned gene, it is desirable to construct an expression cassette containing at least the following: a strong promoter to guide transcription, a ribosome binding site (internal ribosome binding sequence) for translation initiation, and a transcription / translation terminator. For *E. coli*, this includes a promoter (e.g., T7, trp, lac, or lambda promoter), a ribosome binding site, and preferably a transcription termination signal. For eukaryotic cells, the control sequence may include a promoter and / or enhancer (e.g., derived from immunoglobulin genes, HTLV, SV40, or cytomegalovirus), a polyadenylated sequence, and may further include splice donor and / or acceptor sequences (e.g., CMV and / or HTLV splice acceptor and donor sequences). The expression cassette can be transferred to selected host cells by known methods (e.g., transformation or electroporation of *E. coli*, or calcium phosphate treatment, electroporation, or liposome transfection of mammalian cells). Cells transformed with the expression cassette or vector can be selected for antibiotic resistance conferred by the genes contained in these expression cassettes or vectors (e.g., amp, gpt, neo, and hyg genes).

[0300] Polynucleotide sequences encoding publicly available humanized monoclonal antibodies, antigen-binding fragments, conjugates, or CARs can be inserted into expression vectors, including but not limited to plasmids, viruses, or other solvents, which can be manipulated to allow sequence insertion or integration and can be expressed in prokaryotes or eukaryotes. Hosts can include microorganisms, yeast, insects, and mammalian organisms. Methods for expressing DNA sequences with eukaryotic or viral sequences in prokaryotes are known. Many viral and plasmid DNA vectors capable of expression and replication in host cells have been described, and many are commercially available.

[0301] When the host is a eukaryote, DNA transfection methods such as calcium phosphate coprecipitation can be used, along with conventional mechanical procedures such as microinjection, electroporation, liposome-encapsulated plasmid insertion, or viral vectors. Eukaryotic cells can also be co-transformed with polynucleotide sequences encoding, for example, humanized antibodies or antigen-binding fragments disclosed herein, and second foreign DNA molecules encoding, for example, selectable phenotypes (e.g., the herpes simplex virus thymidine kinase gene). Another approach is to use 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). Technicians can readily use expression systems, such as plasmids and vectors, to produce proteins in cells, including higher eukaryotic cells such as COS, CHO, HeLa, and myeloma cell lines.

[0302] To generate recombinant CARs, the host cell can be a mammalian cell. The host cell can be a human cell. In some instances, the host cell is a peripheral blood lymphocyte (PBL), a peripheral blood mononuclear cell (PBMC), or a T cell. The T cell can be any T cell, such as cultured T cells (e.g., primary T cells), or T cells derived from a cultured T cell line (e.g., Jurkat, SupTl, etc.), or T cells obtained from a mammal (e.g., T cells obtained from a human subject). If the T cells are obtained from a mammal, they can be obtained from a variety of sources (including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids). The T cells can also be enriched or purified. The T cells can be human T cells. The T cells can be T cells isolated from humans. The T cells can be any type of T cell and can be at any developmental stage, including, but not limited to, CD4. + / CD8 + Double-positive T cells, CD4 + Helper T cells (e.g., Th1 and Th2 cells), CD8 +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 + T cells.

[0303] A cell population is also provided, comprising at least one host cell as described herein. The cell population can be a heterogeneous population comprising host cells including any of the recombinant expression vectors described herein, and at least one other cell type, such as host cells not containing any recombinant expression vector (e.g., T cells), or cells other than T cells, such as B cells, macrophages, neutrophils, erythrocytes, hepatocytes, endothelial cells, epithelial cells, muscle cells, brain cells, etc. Alternatively, the cell population can be a substantially homogeneous population, wherein the population primarily comprises host cells containing the recombinant expression vector (e.g., substantially composed of host cells). The population can also be a clonal cell population, wherein all cells in the population are clones of a single host cell containing the recombinant expression vector, such that all cells in the population contain the recombinant expression vector. In one example, the cell population is a clonal population comprising host cells including the recombinant expression vectors described herein.

[0304] The nucleic acids encoding humanized monoclonal antibodies or antigen-binding fragments described herein can be modified without diminishing their biological activity. Modifications can be made to facilitate the cloning, expression, or integration of the target molecule into the fusion protein. Such modifications are known and include, for example, stop codons, the addition of a methionine at the amino terminus to provide a start site, additional amino acids at either end to create a positionally convenient restriction site, or additional amino acids (e.g., polyHis) to facilitate purification steps. In addition to recombinant methods, the immunoconjugates, effector moieties, and antibodies of this disclosure can also be constructed wholly or partially using standard peptide synthesis methods.

[0305] Once expressed, humanized monoclonal antibodies, antigen-binding fragments, and conjugates can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity column chromatography, column chromatography, etc. See Generally, antibodies, antigen-binding fragments, and conjugates do not need to be 100% pure. If intended for therapeutic use, these peptides should be substantially free of endotoxins after purification (partial purification or purification to homogenization, as needed).

[0306] Methods for expressing humanized monoclonal antibodies, antigen-binding fragments, and conjugates from mammalian cells and bacteria (e.g., Escherichia coli), and / or refolding them into suitable active forms, have been described, and these methods are applicable to the antibodies disclosed herein. See For example, Harlow and Lane, Antibodies: A Laboratory Manual , 2 nd , Cold Spring Harbor Laboratory, New York, 2013, Simpson ed., Basic methods inProtein Purification and Analysis: A laboratory Manual, Cold Harbor Press, 2008, and Ward et al. , Nature 341:544, 1989.

[0307] Typically, functional heterologous proteins from *E. coli* or other bacteria are isolated from inclusion bodies and require dissolution with a strong denaturing agent, followed by refolding. During the dissolution step, a reducing agent is present to separate disulfide bonds. An exemplary buffer containing the reducing agent is: 0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, and 0.3 M DTE (dithioerythritol). In the presence of low molecular weight thiols in both reduced and oxidized states, disulfide bonds can undergo reoxidation, such as in Saxena. et al. , Biochemistry As described in 9: 5015-5021, 1970, particularly as Buchner... et al. As stated above.

[0308] Besides recombinant methods, humanized monoclonal antibodies, antigen-binding fragments, and / or conjugates can also be synthesized, either fully or partially, using standard peptide synthesis. Solid-phase synthesis of peptides can be achieved by attaching the C-terminal amino acid of the sequence to an insoluble support, followed by the sequential addition of the remaining amino acids from the sequence. (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 describes solid-phase synthesis techniques. Longer proteins can be synthesized by condensation of the amino and carboxyl ends of shorter fragments. Methods for forming peptide bonds by activating the carboxyl end (e.g., using the coupling agent N,N'-dicyclohexylcarbodiimide) are known.

[0309] E. Detection Methods Methods are provided for detecting the presence of cells expressing EGFRvIII and / or amplified EGFR (e.g., tumor cells expressing EGFRvIII and / or amplified EGFR) in a subject. In some aspects, these methods include contacting cells (e.g., tumor cells) from a subject with one or more humanized monoclonal antibodies or conjugates thereof that specifically bind to EGFRvIII and / or amplified EGFR 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 and / or amplified EGFR in the subject.

[0310] In some instances, these methods involve contacting cells from a subject (e.g., tumor cells) with one or more humanized monoclonal 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, and the presence of the immune complex indicates the presence of EGFRvIII-expressing cells in the subject.

[0311] The detection methods may involve in vivo or in vitro detection of immune complexes. In several aspects, detecting cells expressing EGFRvIII includes detecting EGFRvIII expression on the cell surface of tumor cells. In several aspects of the provided methods, detecting cells expressing EGFRvIII and / or gene-amplified EGFR in a subject detects a tumor. In several non-limiting examples, the tumor is carcinoma, such as head and neck cancer, breast cancer, pancreatic cancer, colon or rectal cancer, CNS cancer, or bladder cancer. In some examples, these methods detect tumor cells overexpressing EGFRvIII (e.g., gene-amplified EGFR). In several examples, humanized monoclonal antibodies are used in conjunction with EGFR. 287-302 Ring combination.

[0312] Various methods can be used to detect cells (e.g., tumor cells) expressing EGFRvIII and / or amplified EGFR. In some aspects, subjects are selected who have a tumor (e.g., cancer), are suspected of having a tumor (e.g., cancer), or are at risk of having a tumor (e.g., cancer). For example, subjects may have, are suspected of having, or are at risk of having: head and neck cancer, breast cancer, pancreatic cancer, colon or rectal cancer, CNS cancer, or bladder cancer. In some instances, subjects may have, are suspected of having, or are at risk of having: head and neck cancer, breast cancer, or bladder cancer. Therefore, the presence of cells expressing EGFRvIII and / or amplified EGFR can be detected in these subjects.

[0313] The disclosed methods can also detect EGFR overexpression. Overexpression can be measured, enabling the detection of any 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 methods involve contacting tumor cells from a biological sample 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 complexes is then detected and / or quantified. The presence (or amount) of immune complexes on the subject's cells indicates the presence of tumor cells overexpressing EGFR in the subject.

[0314] In one non-limiting example, a sample is obtained from a subject, and the presence of tumor cells expressing EGFRvIII is assessed in vitro. For example, such a method involves contacting tumor cells from a biological sample from the subject with one or more of the conjugates or humanized monoclonal antibodies that specifically bind EGFRvIII or its antigen-binding fragments disclosed herein to form an immune complex. The presence (or absence) of the immune complex is then detected. The presence of immune complexes on the subject's cells indicates the presence of EGFRvIII-expressing tumor cells in the subject. For example, the increased presence of immune complexes in a sample compared to the formation of immune complexes in a control sample indicates the presence of EGFRvIII-expressing tumor cells in the subject. In some instances, a control is utilized. Similar methods can be used to detect the presence of tumor cells expressing EGFR-amplified genes using one or more of the humanized monoclonal antibodies or antigen-binding fragments or their conjugates disclosed herein that specifically bind EGFR gene amplification.

[0315] Biological samples are typically obtained from mammalian subjects of interest, such as humans. Samples can be any type of specimen, including but not limited to biopsy tissue, autopsy tissue, and pathological specimens. Biological samples also include tissue sections, such as frozen sections used for histological studies.

[0316] In some instances of the disclosed methods, the humanized monoclonal antibody or antigen-binding fragment disclosed herein is conjugated to a detectable biomarker. In some instances, these methods further include contacting a second antibody that specifically binds to the disclosed antibody, its antigen-binding fragment, or conjugate for a sufficient period to form an immune complex, and detecting the immune complex. The presence of such an immune complex in a biological sample from selected subjects (as described above) increases the detection of endothelial cells expressing EGFRvIII and / or amplified EGFR in that biological sample compared to the presence of the immune complex in a control sample or other standards. In some instances, the second antibody is conjugated to a detectable biomarker.

[0317] Detectable biomarkers suitable for humanized monoclonal antibodies or secondary antibodies have been described and are known to those skilled in the art. Examples include various enzymes, cofactors, fluorescent materials, luminescent materials, magnetic agents, and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Non-limiting examples of suitable cofactor complexes include streptavidin / biotin and avidin / biotin. Non-limiting examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazineamine fluorescein, dansyl chloride, or phycoerythrin. Non-limiting exemplary luminescent materials include luminol; non-limiting exemplary magnetic agents include gadolinium; and non-limiting exemplary radiolabels include… 125 I, 131 I, 35 S or 3 H.

[0318] The humanized monoclonal antibodies and their conjugates disclosed in this article can be used for immunohistochemical assays. These assays are known and have been described. See For example, Harlow & Lane, Antibodies, A Laboratory Manual ,2 nd ed., Cold Spring Harbor Publications, New York (2013)).

[0319] The humanized monoclonal antibody disclosed herein can also be used in vivo to detect tumor cells expressing EGFRvIII and / or amplified EGFR. In some instances, in vivo detection diagnoses the presence of tumors in a subject. Therefore, methods for detecting pathological conditions in a subject, such as tumors, such as cancers; for example, head and neck cancer, breast cancer, pancreatic cancer, colon or rectal cancer, CNS cancer, or bladder cancer, are disclosed. In one instance, an effective amount of the disclosed humanized monoclonal antibody (or its antigen-binding fragment) or its conjugate is administered to a subject for a sufficient duration to allow the humanized monoclonal antibody or antigen-binding fragment to form an immune complex, which can then be detected. Detection of the immune complex in the subject identifies the presence of tumor cells expressing EGFRvIII and / or amplified EGFR. In specific non-limiting instances, the detection of the immune complex is performed by immunoscintigraphy. Other specific non-limiting instances of immune complex detection include radiolocalization, radiographic imaging, magnetic resonance imaging (e.g., using biotinylated antibodies and anti-biotin-iron oxide), or fluorescence imaging (e.g., using antibodies labeled with luciferase or green fluorescent protein). See For example, Paty et al. , Transplantation. (77:1133-1137, 2004). In several instances, the disclosed methods detect, for example, head and neck cancer, breast cancer, pancreatic cancer, colon or rectal cancer, CNS cancer, or bladder cancer.

[0320] In magnetic resonance imaging (MRI) settings, contrast agent detection is significantly affected by the magnetic field strength of the MRI scanner. Increased magnetic field strength improves the detection capability of contrast agents by several orders of magnitude (Hu). et al. , Ann. Rev. Biomed. Eng ., 6:157-184, 2004; Wedeking et al. , Magn. Reson. Imaging. (17:569-575, 1999). For example, the detection limit for gadolinium at 2 Tesla (T) is ~30 μM. At 4T, the detection limit decreases to ~1 μM. Using newer 7 to 12T scanners, it is expected that low concentrations (10-100 nM) of this contrast agent can be detected. Similar sensitivity can also be identified using contrast agents (e.g., iron oxide). Once detected, the test results can be used to assist or guide surgical or other resection of tumors.

[0321] In one instance, an effective dose of the humanized monoclonal antibody or antigen-binding fragment or conjugate thereof that specifically binds to EGFRvIII, as disclosed herein, is administered to a subject with a tumor following anticancer treatment. The immune complex is detected after sufficient time has been allowed to form an immune complex with EGFRvIII on tumor cells. For example, the humanized monoclonal antibody or conjugate thereof that specifically binds to EGFRvIII may be administered to the subject before or after tumor treatment. The tumor may be (but is not limited to) cancer, such as head and neck cancer, breast cancer, pancreatic cancer, colon or rectal cancer, CNS cancer, or bladder cancer. The presence (or absence) of the immune complex indicates the effectiveness of the treatment. For example, an increase in immune complexes compared to a control collected before treatment indicates treatment ineffectiveness, while a decrease in immune complexes compared to a control collected before treatment indicates treatment effectiveness. Similar methods of use of the humanized monoclonal antibody or antigen-binding fragment or conjugate thereof that specifically binds to amplified EGFR, as disclosed herein, can be used to detect amplified EGFR, and the presence (or absence) of the immune complex indicates the effectiveness of the treatment.

[0322] F. Treatment methods A therapeutically effective dose of the disclosed EGFRvIII-specific humanized monoclonal antibody, antigen-binding fragment, conjugate thereof, or CAR-expressing immune cells (e.g., CAR T cells or CAR NK cells) can be administered to a subject to treat EGFRvIII-expressing tumors, such as cancers, including head and neck cancer, breast cancer, pancreatic cancer, colon or rectal cancer, CNS cancer, or bladder cancer. In some instances, administration of a therapeutically effective dose of the disclosed EGFRvIII-specific humanized monoclonal antibody, antigen-binding fragment, conjugate thereof, or CAR-expressing immune cells reduced the signs or symptoms of EGFRvIII-expressing tumors. Tumors can be cancers, such as head and neck cancer, breast cancer, pancreatic cancer, colon or rectal cancer, CNS cancer, or bladder cancer. Therefore, subjects with EGFRvIII-expressing tumors, suspected of having EGFRvIII-expressing tumors, or at risk of having EGFRvIII-expressing tumors can be selected for treatment. Therapeuticly effective doses of the disclosed nucleic acid molecules and vectors can also be used. In other instances, tumors may overexpress EGFR and / or express misfolded EGFR. In some instances, subjects with tumors that overexpress EGFR and / or express misfolded EGFR are selected. The compositions disclosed herein can be used to treat these tumors in subjects.

[0323] In a further example, a therapeutically effective amount of the humanized antibody or antigen-binding fragment disclosed herein can also be used in a method of inhibiting tumors in a subject that overexpress EGFR (e.g., gene-amplified EGFR). This method includes administering an effective amount of the humanized antibody, antigen-binding fragment, nucleic acid molecule, vector, T cell, or pharmaceutical composition disclosed herein to a subject with a tumor overexpressing EGFR. In a non-limiting example, the humanized monoclonal antibody or antigen-binding fragment includes V H and V L The V H and V L : a) Each of the amino acid sequences shown in SEQ ID NO: 5 and SEQ ID NO: 8 (A10, VH3+VL3) is included. b) Each of the amino acid sequences shown in SEQ ID NO: 3 and SEQ ID NO: 6 (A2, VH1+VL1); c) Includes the amino acid sequences (A3, VH1+VL2) shown in SEQ ID NO: 3 and SEQ ID NO: 7, respectively. d) Includes the amino acid sequences shown in SEQ ID NO: 3 and SEQ ID NO: 8 (A4, VH1+VL3); e) Each of the amino acid sequences shown in SEQ ID NO: 4 and SEQ ID NO: 6 is an A5, VH2+VL1 sequence. f) includes the amino acid sequences (A6, VH2+VL2) shown in SEQ ID NO: 4 and SEQ ID NO: 7, respectively. g) includes the amino acid sequences (A7, VH2+VL3) shown in SEQ ID NO: 4 and SEQ ID NO: 8, respectively. h) includes the amino acid sequences (A8, VH3+VL1) shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively. i) Each of the amino acid sequences shown in SEQ ID NO: 5 and SEQ ID NO: 7 (A9, VH3+VL2); j) includes the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 9 (B1,40H3 VH+VL-EG); k) includes the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 10 (B2,40H3 VH+VL-DA); l) includes the amino acid sequences shown in SEQ ID NO: 4 and SEQ ID NO: 11 (B3, VH2+VL1-DA); m) includes the amino acid sequences shown in SEQ ID NO: 4 and SEQ ID NO: 12 (B4, VH2+VL2-DA); n) includes the amino acid sequences (B5, VH3 and VL1-DA) shown in SEQ ID NO: 5 and SEQ ID NO: 11, respectively. o) includes the amino acid sequences (C10) shown in SEQ ID NO: 31 and SEQ ID NO: 32, respectively; p) includes the amino acid sequences shown in SEQ ID NO: 5 and SEQ ID NO: 14, respectively (D2); or q) includes the amino acid sequences shown in SEQ ID NO: 5 and SEQ ID NO: 24, respectively (D3).

[0324] Nucleic acid molecules, vectors, and CAR-expressing immune cells that include these antigen-binding fragments can also be used. Tumors can be cancers, such as head and neck cancer, breast cancer, pancreatic cancer, colon or rectal cancer, CNS cancer, or bladder cancer. Therefore, subjects with tumors that overexpress EGFR (e.g., tumors expressing amplified EGFR), suspected of having tumors that overexpress EGFR (e.g., tumors expressing amplified EGFR), or at risk of having tumors that overexpress EGFR (e.g., tumors expressing amplified EGFR) can be selected for treatment.

[0325] In some instances, subjects may be administered the humanized monoclonal antibodies, antigen-binding fragments, CAR-expressing immune cells (e.g., CAR T cells), compositions, and conjugates disclosed herein to slow or inhibit tumor growth or metastasis, reduce tumor volume, or reduce metastasis. In these applications, a therapeutically effective dose of the humanized monoclonal antibody, antigen-binding fragment, conjugate, CAR T cell, or composition disclosed herein that specifically binds to EGFRvIII and / or genetically amplified EGFR is administered to the subject in an amount and under conditions sufficient to form an immune complex with EGFRvIII and / or genetically amplified EGFR, thereby slowing or inhibiting tumor growth or metastasis, reducing tumor volume, or suppressing signs or symptoms of cancer. Examples of suitable subjects include subjects diagnosed with or suspected of having tumors expressing EGFRvIII and / or genetically amplified EGFR, such as subjects with cancer (e.g., head and neck cancer, breast cancer, pancreatic cancer, colon or rectal cancer, CNS cancer, or bladder cancer).

[0326] The therapeutically effective dose will depend on the severity of the disease and the patient's overall health. A therapeutically effective dose is the amount that provides subjective relief of symptoms or an improvement objectively identified by a clinician or other qualified observer. In one instance, a therapeutically effective dose is the amount required to inhibit tumor growth (e.g., cancer growth, such as head and neck cancer, breast cancer, pancreatic cancer, colon or rectal cancer, CNS cancer, or bladder cancer), inhibit metastasis, reduce tumor volume, or effectively alleviate the signs or symptoms of the tumor. The therapeutically effective dose of the administered agent can vary depending on the desired effect and the subject being treated. In some instances, a therapeutic dose is the amount that eliminates or reduces the patient's tumor burden, or inhibits or reduces the proliferation of metastatic cells, or alleviates tumor symptoms.

[0327] Subjects who can benefit from the publicly available method include human and animal subjects (e.g., cats, dogs, non-human primates, etc.). In some instances, subjects are mammalian subjects, such as humans, non-human primates, canines, or felines. In some instances, subjects are humans, non-human primates, domestic dogs (domestic dogs ( Canis lupus familiaris )) or domestic cat (e.g., domestic cat ( Felis catus In a non-limiting instance, the subject is a human being. Subjects may be screened, for example, to determine if they have a tumor, prior to initiating the publicly available therapy. The presence of a tumor expressing EGFRvIII and / or amplified EGFR indicates that the composition can be treated using the methods described herein.

[0328] Any method of administration can be used, including local or systemic administration. For example, topical, oral, intravascular (e.g., intravenous, intramuscular, intraperitoneal), intranasal, intradermal, intrathecal, intratumoral, and subcutaneous administration can be used. The attending physician can choose the specific administration method and dosage regimen based on the specific case circumstances (e.g., the subject, the disease, the disease state involved, and whether the treatment is prophylactic). When administering more than one agent or composition, one or more routes of administration may be used; for example, chemotherapeutic agents can be administered orally, and humanized monoclonal antibodies or antigen-binding fragments or conjugates or compositions can be administered intravenously. Administration methods include injection, in which the conjugates, humanized monoclonal antibodies, antigen-binding fragments, CAR-containing immune cells, nucleic acid molecules, or compositions provided herein are in a non-toxic, pharmaceutically acceptable carrier (e.g., water, saline, Ringer's solution, glucose solution, 5% human serum albumin, fixation oil, ethyl oleate, or liposomes). In some instances, local application may be used, such as by applying the humanized antibodies or antigen-binding fragments disclosed herein to a tissue area where a tumor has been removed, or to an area suspected of being prone to tumor formation. In some instances, sustained release of a pharmaceutical formulation comprising a therapeutically effective amount of a humanized antibody or antigen-binding fragment (or a conjugate thereof) within (or near) a tumor may be beneficial.

[0329] The compositions disclosed herein, comprising humanized monoclonal antibodies or antigen-binding fragments or conjugates thereof, or immune cells containing CARs, can be formulated into unit dosage forms suitable for precise individual dosing. Furthermore, these compositions can be administered in single-dose or multi-dose regimens. A multi-dose regimen is one in which the primary course of treatment may involve more than one single dose (e.g., 1-10 doses), followed by additional doses at subsequent time intervals as needed to maintain or enhance the effect of the composition. Treatment may involve administering the compound once daily or multiple times daily over periods ranging from days to months or even years. Therefore, the dosing regimen will also be at least in part based on the specific needs of the subject to be treated and will depend on the judgment of the administering technician.

[0330] Exemplary doses of humanized monoclonal antibodies, antigen-binding fragments, conjugates, compositions, or other reagents may range from about 0.01 mg / kg to about 30 mg / kg (subject weight), for example, from about 0.1 mg / kg to 10 mg / kg, or from about 5 mg / kg to 15 mg / kg. In some instances, the dosage is 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, 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. In some instances, the dose does not exceed about 1 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, or 30 mg / kg. In some instances, the dosage of the humanized monoclonal antibodies, antigen-binding fragments, conjugates, compositions, or other reagents disclosed herein is from 1 mg / kg to 20 mg / kg, for example, 2 mg / kg to 20 mg / kg, 2 mg / kg to 15 mg / kg, 2 mg / kg to 10 mg / kg, 2 mg / kg to 5 mg / kg, 5 mg / kg to 20 mg / kg, 5 mg / kg to 15 mg / kg, 5 mg / kg to 10 mg / kg, 8 mg / kg to 20 mg / kg, 8 mg / kg to 15 mg / kg, 8 mg / kg to 12 mg / kg, or 8 mg / kg to 10 mg / kg. In a non-limiting instance, the dosage of the humanized monoclonal antibodies or antigen-binding fragments disclosed herein is from 5 mg / kg to 15 mg / kg. In another non-limiting instance, the dosage of the humanized monoclonal antibodies or antigen-binding fragments disclosed herein is from 8 mg / kg to 12 mg / kg.

[0331] In some instances, the dosage range of the humanized monoclonal antibodies, antigen-binding fragments, conjugates, compositions, or other reagents disclosed herein is from 100 mg / m² to 700 mg / m², for example, 200 mg / m². 2 Up to 700 mg / m 2 200 mg / m 2 Up to 600 mg / m 2 200 mg / m 2 Up to 500 mg / m 2 200 mg / m 2 Up to 400 mg / m 2 300 mg / m 2 Up to 700 mg / m 2 300mg / m 2 Up to 600 mg / m 2 300 mg / m 2 Up to 500 mg / m 2 300 mg / m 2 Up to 400 mg / m 2 350 mg / m 2 Up to 700 mg / m 2 350 mg / m 2 Up to 600 mg / m 2 350 mg / m 2 Up to 500 mg / m 2 350 mg / m 2 Up to 400 mg / m 2 In a non-limiting example, the dose of the humanized monoclonal antibody or antigen-binding fragment disclosed herein is 200 mg / m². 2 Up to 500 mg / m 2 In another non-limiting example, the dosage of the humanized monoclonal antibody or antigen-binding fragment disclosed herein is 300 mg / m². 2 Up to 400 mg / m 2 .

[0332] In some aspects, the subject was given 10 kg of the subject's body weight per kg of medication. 4 -10¹² immune cells containing CAR (e.g., CAR T cells), for example, 10 4 -10 8 cells / kg, 10 6 -10 7 cells / kg, 10 6 -10 8 cells / kg, 10 6 -10¹ 0cells / kg, 10 6 -10¹² cells / kg, 10 8 -10¹ 0 cells / kg or 10 8 -10¹² cells / kg. In some instances, subjects were administered 5 × 10¹² cells / kg of body weight. 6 Up to 5 × 10 8 One CAR-containing immune cell (e.g., CAR T cells). In some instances, 1 × 10⁻⁶ cells per kg of subject body weight are administered to the subject. 7 Up to 5 × 10 7 One CAR-containing immune cell (e.g., CAR T cell). In some instances, approximately 10 [units of something] are administered to the subject. 6 cells / kg to 10 8 Cells / kg, for example, 0.2 × 10⁻⁶ 6 / kg to 5.0 × 10 6 / kg, or 0.1 × 10 8 / kg to 2.5 × 10 8 / kg. In some instances, at least 10 g was administered to the subject. 4 cells / kg, 10 5 cells / kg, 10 6 cells / kg, 10 7 cells / kg or 10 8 Cells / kg. In a further example, no more than 10 cells / kg were administered to the subject. 4 cells / kg, 10 5 cells / kg, 10 6 cells / kg, 10 7 cells / kg, 10 8 cells / kg, 10 9 cells / kg or 10 10 Cells / kg. Multiple doses can be administered; for example, CAR-containing immune cells (e.g., CAR T cells) can be administered daily, every other day, twice a week, weekly, every two weeks, every three weeks, monthly, or less frequently. The dosage and frequency of administration can be influenced by a variety of factors, such as the condition being treated, the severity of symptoms, the age of the subject, or their overall health. Typically, treatment regimens are determined through clinical trials.

[0333] CAR-containing immune cells (e.g., CAR T cells) can be administered using any suitable route of administration. In some instances, CAR-containing immune cells are administered via a parenteral route (e.g., intravenous); however, injection or infusion near the target tumor (e.g., local administration) or intraperitoneal administration may also be used. In some instances, the CAR-containing immune cells disclosed herein are delivered locally to the tumor via injection or catheter. Technicians can determine the appropriate route of administration based on a variety of factors, such as the patient's condition, the severity of symptoms, the patient's age or overall health, or other factors.

[0334] In a specific instance, a therapeutic composition comprising one or more of a conjugate, a humanized monoclonal antibody, an antigen-binding fragment, a composition, CAR T cells, or other reagents is administered to a subject according to a multiple-day dosing regimen, such as for at least two consecutive days, for at least 10 consecutive days, or for a period of time such as weeks, months, or years. In one instance, a conjugate, antibody, composition, or other reagent is administered to a subject for a period of time of at least 30 days, such as 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.

[0335] In some instances, the disclosed therapeutic agent is administered intravenously, subcutaneously, or otherwise for a period of time, either daily or multiple times weekly, followed by a period of no treatment, and then the cycle is repeated. In some instances, the initial treatment period (e.g., daily or multiple times 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 some instances, the no-treatment period lasts for 3 days, 1 week, 2 weeks, 3 weeks, or 4 weeks. In some instances, the therapeutic agent is administered once daily for 3 days, followed by a 3-day break; or once daily or multiple times weekly for 1 week, followed by a 3-day or 1-week break; or once daily or multiple times weekly for 2 weeks, followed by a 1-week or 2-week break; or once daily or multiple times weekly for 3 weeks, followed by a 1-week, 2-week, or 3-week break; or once daily or multiple times weekly for 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks, followed by a 1-week, 2-week, 3-week, or 4-week break.

[0336] Administration of humanized monoclonal antibodies, antigen-binding fragments, conjugates, CAR-containing immune cells, or compositions may be concurrent with the administration of other anticancer or anti-angiogenic agents or therapeutic treatments (e.g., surgical resection of tumors or radiotherapy). For example, a subject may receive one or more additional therapies before, during, or after administration of a therapeutic amount of the antibody or conjugate. In one instance, a subject receives one or more treatments to remove or reduce a tumor before administration of a therapeutic amount of one or more agents for treating tumors. For example, additional agents may include, but are not limited to, chemotherapeutic agents, anti-angiogenic agents, or combinations thereof. In another instance, at least part of the tumor is surgically or otherwise removed or reduced in size or volume before administration of a therapeutically effective amount of a humanized antibody or antigen-binding fragment or conjugate.

[0337] Specific examples of other therapeutic agents that may be used include microtubule binders, DNA intercalators or cross-linkers, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors. These agents (administered at therapeutically effective amounts) and treatments may be used alone or in combination. For example, any suitable anticancer agent or antiangiogenic agent may be administered in combination with the antibodies or conjugates disclosed herein. Methods and therapeutic dosages for such agents are known and can be determined by those skilled in the art. In one instance, the chemotherapeutic agent includes 5-FU or IRT, or both.

[0338] Microtubule binders interact with tubulin to stabilize or disrupt microtubule formation, thereby inhibiting cell division. Examples of microtubule binders that can be used in combination with publicly disclosed therapies include, but are not limited to, paclitaxel, docetaxel, vinblastine, vinorelbine (novibenten), epothilone, colchicine, dolastatin 15, nocodazole, podophyllotoxin, and rhizomycin. Analogs and derivatives of such compounds may also be used. For example, suitable epothilones and epothilone analogs are described in International Publication No. WO 2004 / 018478. Taxanes, such as paclitaxel and docetaxel, and paclitaxel analogs taught in U.S. Patent Nos. 6,610,860, 5,530,020, and 5,912,264, may be used.

[0339] Suitable DNA and RNA transcription regulators include, but are not limited to, actinomycin D, daunorubicin, doxorubicin, and their derivatives and analogues, and are also suitable for use in combination with publicly available therapies. DNA intercalators and cross-linkers that can be administered to subjects include, but are not limited to, cisplatin, carboplatin, oxaliplatin, mitomycin (e.g., mitomycin C), bleomycin, chlorambucil, cyclophosphamide, and their derivatives and analogues. Suitable DNA synthesis inhibitors for use as therapeutic agents include, but are not limited to, methotrexate, 5-fluoro-5'-deoxyuridine, 5-FU, and their analogues. Examples of suitable enzyme inhibitors include, but are not limited to, camptothecin, etoposide, formetanan, trichostatin, and their derivatives and analogues. Suitable compounds that affect gene regulation include agents that cause an increase or decrease in the expression of one or more genes, such as raloxifene, 5-azacytidine, 5-aza-2'-deoxycytidine, tamoxifen, 4-hydroxytamoxifen, mifepristone and their derivatives and analogs.

[0340] Examples of commonly used chemotherapy drugs include doxorubicin, Alkeran, Ara-C, BiCNU, busulfan, CCNU, carboplatinum, cisplatinum, cytoxan, daunorubicin, DTIC, 5-FU, fludarabine, hydroxyurea, idarubicin, ifosfamide, methotrexate, mithramycin, mitomycin, mitoxantrone, nitrogen mustard, paclitaxel (or other taxanes, such as docetaxel), velban, vincristine, and vinblastine. Some newer drugs include gemcitabine, Herceptin, IRT (Camptosar, CPT-11), leustatin, novibenten, Rituxan STI-571, Taxotere, topotecan (Hycamtin), Xeloda (capecitabine), Zevelin, and calcitriol.

[0341] Non-limiting examples of immunomodulators 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 (CutterBiological), IMREG (from Imreg of New Orleans, La.), SK&F 106528, and TNF (tumor necrosis factor; Genentech).

[0342] Therefore, non-limiting examples of chemotherapeutic agents used in combination with the antibodies, antigen-binding fragments, conjugates thereof, CAR-containing immune cells, and nucleic acid molecules disclosed herein include chemotherapeutic agents such as erlotinib (TARCEVA®, Genentech / OSI Pharm.), bortezomib (VELCADE®, Millenium Pharm.), fulvestrant (FASLODEX®, AstraZeneca), sunitinib (SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis), and 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.), gefitinib (IRESSA®, AstraZeneca), AG1478, AG1571 (SU 5271; Sugen); alkylating agents, such as thiotepa and CYTOXAN® cyclophosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; antifolate antitumor drugs, such as pemetrexed (ALIMTA® Eli Lilly); aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylene imines and methyl melamine, including hexamethylmelamine, triethylenemelamine, triethylenephosphamide, triethylenethiophosphamide, and trimethylamine; acetogenin (especially bulbatacin and bulbatacinone); camptothecin (including the synthetic analogue topotecan); lichen inhibitors Brystatin; Callystatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin, and bizelesin); Cryptophycin (especially Cryptophycin 1 and Cryptophycin 8); Dolastatin; Duocarmycin (including synthetic analogues KW-2189 and CB1-TM1);Soft coral alcohol (eleutherobin); pancratistatin; sarcodictyin; spongistatin; nitrogen mustard, such as chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novombhichin, phenesterine, prednimustine, trofosfamide, uracil Mustard; nitrosoureas, such as carmustine, chlorozotocin, formustine, lomustine, nimustine, and ranimnustine; antibiotics, such as enediyne antibiotics, calicheamicin, calicheamicin γ1I, and calicheamicin ωI1; dynemicin, including dynemicin A; bisphosphonates, such as clophosphonate; esperamicin.and neocarzinostatin Chromophore and related chromophores of ethynylene antibiotics, aclacinomysin, neofulvicin, antramycin, azaserine, bleomycin, cactinomycin C, carabicin, carminomycin, carzinophilin, chromomycinis, actinomycin D, daunorubicin, detorubicin, 6-diazo-5-oxo-L-leucine, ADRIAMYCIN® doxorubicin (including morpholino-dxorubicin, cyanomorpholino-dxorubicin, 2-pyrrolino-dxorubicin, and deoxydxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin C, mycophenolic acid (acid), nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenmethoxazole, zinostatin, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); Folic acid analogs, such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; and pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmoflurane, cytarabine, dideoxyuridine, deoxyfluorouridine, enocitabine, and fluxuridine.Androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; antiadrenergic agents, such as aminoglutethimide, mitotan, and trilostane; folic acid supplements, such as frolinic acid; aceglucanolactone; aldehydephosphatidylglycoside; aminolevulinic acid; eniluracil; acridine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate); epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansine, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazine; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); raszoxin; rhizomycin; sizofiran; spirogermanium; tenuazonic acid acid); triaziquone; 2,2',2''-trichlorotriethylamine; trichothecene (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethan; vindesine; dacarbazine; mannomustine; dibromomannitol; mitolactone; pipebroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa;Taxanes, such as paclitaxel (TAXOL®, Bristol-MyersSquibb Oncology, Princeton, NJ), ABRAXANE™ Cremophor-free, albumin-free, paclitaxel nanoparticle formulation (American Pharmaceutical Partners, Schaumberg, Ill.), and AXOTERE® docetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vincristine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoroformylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; and any pharmaceutically acceptable salts, acids, or derivatives thereof.

[0343] Non-limiting examples of anti-angiogenic agents include molecules such as proteins, enzymes, polysaccharides, oligonucleotides, DNA, RNA, and recombinant vectors, as well as small molecules that reduce or even inhibit angiogenesis. Examples of suitable angiogenesis inhibitors include, but are not limited to: angiostatin K1-3, staurosporine, genistein, fumagillin, medroxyprogesterone, suramin, interferon-α, inhibitors of metalloproteinases, platelet factor 4, somatostatin, thromobospondin, endostatin, thalidomide, and their derivatives and analogues. For example, in some instances, the anti-angiogenic agent is an antibody that specifically binds to VEGF (e.g., AVASTIN®, Roche) or the VEGF receptor (e.g., a VEGFR2 antibody). In one instance, 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.

[0344] In some instances, additional reagents are monoclonal antibodies, such as 3F8, abagovomab, adecatumumab, atoruzumab, alacizumab, alemtuzumab, atumomab pentetate, anatumomab mafenatox, apolizumab, arcitumomab, bavituximab, beectumomab, belimumab, besilesomab, bevacizumab, bivatuzumab mertansine, blinatumomab, brentuximab, and cantuzumab. mertansine, capromab pendetide, catutoxomab, CC49, cetuximab, and citatuzumab bogatox, cixutumumab, clivatuzumabtetraxetan, conatumumab, dacetuzumab, detumomab, ecromeximab, eculizumab, edrecolomab, eprecolomab, epratuzumab, ertumaxomab, edaracizumab, farletuzumab, figitumumab, galiximab, gemtuzumab ozogamicin, girentuximab, glembatumumab vedotin, teimozumab (Ibritumomab)tiuxetan, Igovomab, Imciromab, Intetumumab, Oga-Itozumab, Iratumumab, Labetuzumab, Lexatumumab, Lintuzumab, Lorvotuzumab mertansine, Lucatumumab, Lumiliximab, Mapatumumab, Matuzumab, Metelimumab, Milatuzumab, Mitumomab, Morolimumab, Nacolomab Tafenatox, Naptumomab estafenatox, Necitumumab, Nofetumomab merpentan, Olaratumab, Oportuzumab monatox, Oregovomab, Pertumumab, Pemtumomab, Pintumomab, Pritumumab, Ramosimumab, Rilotumumab, Rituximab, Robatumumab, Satumomab / Bendetide, Sibrotuzumab, Sonepizumab, Sorafenib, Sunitinib, Tacatuzumab Tetraxetan, Tapipramomab paptox, Tenatumomab, TGN1412, Ticilimumab (tremelimumab), Tigatuzumab, TNX-650, Trastuzumab, Ticilimumab, Tucotuzumab celmoleukin, Veltuzumab, Volociximab, Votumumab, and Zalutumumab.

[0345] Another common treatment for some types of cancer is surgery, such as surgically removing the cancer or a portion thereof. Another example of treatment is radiation therapy, which involves applying radioactive materials or energy (such as external beam radiation therapy) to the tumor site to help eradicate the tumor or shrink it before surgical removal.

[0346] Other therapeutic agents, such as antitumor agents, whether or not they fall into one or more of the above categories, are also suitable for use in combination with publicly available therapies. Examples of such agents include doxorubicin, apigenin, rapamycin, zebularine, cimetidine, and their derivatives and analogues.

[0347] Other reagents may be prepared and administered according to the manufacturer's instructions or determined by the physician based on experience. Preparation and administration regimens for such chemotherapy are also described, for example, in Chemotherapy Service, (1992) Ed., MC Perry, Williams & Wilkins, Baltimore, Md.

[0348] Combination therapies can provide synergistic effects and have been demonstrated to have synergistic effects, meaning that the effect achieved when the active ingredients are used together is greater than the sum of the effects produced by using these compounds alone. Synergistic effects can be achieved when the active ingredients are: (1) co-formulated as combined unit-dose formulations and administered or delivered simultaneously; (2) delivered as individual formulations in an alternating or parallel manner; or (3) by other means. In alternating delivery, synergistic effects can be achieved when the compounds are administered or delivered sequentially, for example by different injections in separate syringes. Typically, in alternating delivery, each active ingredient is administered in an effective dose sequentially, for example, consecutively, while in combination therapy, two or more active ingredients are administered in effective doses together.

[0349] G. Composition The provided compositions comprise one or more humanized monoclonal antibodies, antigen-binding fragments, conjugates, CAR-containing immune cells (e.g., CAR T cells), or nucleic acid molecules disclosed herein, contained in a carrier (e.g., a pharmaceutically acceptable carrier). In some instances, the composition is a pharmaceutical composition. These compositions may be prepared in unit dosage forms for administration to a subject. The amount and timing of administration are determined by the attending physician on a case-by-case basis to achieve the desired outcome. These compositions may be formulated for systemic (e.g., intravenous) or local (e.g., intratumoral) administration. In one instance, the humanized monoclonal antibody disclosed herein, or its antigen-binding fragment, conjugate, nucleic acid molecule, or CAR-containing immune cell, is formulated for parenteral administration, such as intravenous administration. The disclosed compositions may be used, for example, to treat and / or detect tumors, such as those occurring in the head and / or neck, breast, pancreas, colon or rectum, central nervous system (CNS), or bladder. In some instances, these compositions may be used to treat or detect cancer.

[0350] Compositions intended for administration to subjects may include a therapeutic solution (or cells suspended therein) dissolved in a pharmaceutically acceptable carrier (e.g., an aqueous carrier). Various aqueous carriers may be used, such as buffered salt solutions. These solutions are sterile and generally free of unwanted substances. These compositions may be sterilized using conventional sterilization techniques. These compositions may contain pharmaceutically acceptable excipients as needed to mimic physiological conditions, such as pH adjusters and buffers, toxicity modifiers, etc., for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentrations of humanized monoclonal antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, or CAR-containing immune cells in these formulations can vary considerably and are selected primarily based on liquid volume, viscosity, body weight, etc., depending on the specific route of administration chosen and the needs of the subject. Practical methods for preparing such dosage forms are known or obvious to those skilled in the art.

[0351] Exemplary compositions for intravenous administration include, for example, doses of about 0.01 mg / kg to about 30 mg / kg per subject per day of antibody or antigen-binding fragment or conjugate (or corresponding doses of conjugates comprising humanized antibody or antigen-binding fragments). Practical methods for preparing administerable compositions are known or obvious to those skilled in the art and are described in more detail in the following publications, for example, Remington's Pharmaceutical Science, 19th ed.Mack Publishing Company, Easton, PA (1995). In some instances, the composition is a liquid formulation comprising one or more antibodies, antigen-binding fragments (e.g., humanized monoclonal antibodies or antigen-binding fragments that specifically bind to EGFRvIII), in concentrations ranging from 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.

[0352] The compositions disclosed herein may be provided in lyophilized form and reconstituted with sterile water prior to administration, although they may also be provided as sterile solutions of known concentrations. In one example, a lyophilized humanized monoclonal antibody or antigen-binding fragment or conjugate is suspended and added to an infusion bag containing 0.9% sodium chloride (USP), and in some cases, administered at a dose of 0.5 mg / kg to 15 mg / kg body weight. There is considerable experience in the art regarding the administration of antibodies or antigen-binding fragments and conjugates; for example, antibody drugs have been marketed 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 by intravenous bolus or bolus injection. In one example, a higher loading dose may be administered first, followed by a maintenance dose at a lower level. For example, an initial loading dose of 4 mg / kg of antibody or antigen-binding fragment (or a corresponding dose of a conjugate containing humanized monoclonal antibody or antigen-binding fragment) can be infused over a 90-minute period. If the initial dose is well tolerated, a maintenance dose of 2 mg / kg can then be infused weekly over a 30-minute period for 4–8 weeks.

[0353] Any of the compositions disclosed herein can be formulated into controlled-release formulations. Controlled-release parenteral formulations can be formulated as implants, oil-based injectables, or granule systems. A comprehensive overview of protein delivery systems is provided. See For example, 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 (e.g., 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 generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of about 5 μm, therefore only nanoparticles are administered intravenously. Microparticles typically have a diameter of about 100 μm and are administered via subcutaneous injection or intramuscular injection. 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).

[0354] Polymers can be used for ion-controlled release of the compositions disclosed herein. Various degradable and non-degradable polymer matrices for controlled drug delivery are known and described. See For example, Langer, Accounts Chem. Res. 26:537-542, 1993). For example, the block copolymer poloxamer 407 is a viscous but fluid liquid at low temperatures, but forms a semi-solid gel at body temperature. It has been shown to be an effective solvent for the formulation and sustained-release 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). Optionally, hydroxyapatite has been used as a microcarrier for controlled protein release (Ijntema). et al. , Int. J. Pharm. 112:215-224, 1994). On the other hand, liposomes are used for the controlled release of drugs encapsulated in liposomes and for targeted drug delivery (Betageri). et al., Liposome Drug Delivery Systems(Technomic Publishing Co., Inc., Lancaster, PA (1993)). Many other systems for the 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).

[0355] In some instances, DNA encoding humanized monoclonal antibodies, their antigen-binding fragments, or conjugates (e.g., conjugated with toxins) disclosed herein is administered to a subject to provide in vivo antibody production, for example, using the subject's cellular mechanisms. Immunization via nucleic acid constructs is known and is taught, for example, in U.S. Patent Nos. 5,643,578 and 5,593,972, 5,817,637, and 5,880,103. These methods may include liposome delivery of nucleic acids. Such methods can be applied by those skilled in the art to the production of humanized monoclonal antibodies or their antibody-binding fragments.

[0356] One method for administering nucleic acids is by directly administering a vector, such as a mammalian expression plasmid (or viral vector). The nucleotide sequence encoding a publicly disclosed humanized monoclonal antibody or its antibody-binding fragment can be included in the vector and placed under the control of a promoter to increase expression.

[0357] In another method using nucleic acids, the disclosed humanized monoclonal antibody or antibody-binding fragment, or conjugate thereof, can also be expressed via an attenuated viral host or vector or bacterial vector. Recombinant vaccinia virus, adeno-associated virus (AAV), herpesvirus, retrovirus, cytomegalovirus, or other viral vectors can be used to express humanized antibodies. For example, a useful scheme for a vaccinia virus vector and method is described in U.S. Patent No. 4,722,848. BCG (Bacillus Calmette-Guérin) provides another vector for expressing disclosed humanized monoclonal antibodies (see Stover, ...). Nature 351:456-460, 1991).

[0358] In one instance, nucleic acids encoding a publicly disclosed humanized monoclonal antibody or its antibody-binding fragment are directly introduced into cells. For example, nucleic acids can be loaded onto gold microspheres using standard methods and introduced into the skin using a device such as Bio-Rad's Helios™ gene gun. The nucleic acids can be "naked" nucleic acids, consisting of plasmids controlled by a strong promoter. Typically, the DNA is injected into muscle, although it can also be injected directly into other sites. Injection doses are typically from about 0.5 μg / kg to about 50 mg / kg, and usually from about 0.005 mg / kg to 5 mg / kg. See For example, U.S. Patent No. 5,589,466.

[0359] H. Reagent kit Kits are provided. For example, kits are provided for detecting tumor cells expressing EGFRvIII and / or amplified EGFR in subjects, and / or for treating tumors in subjects. These kits will typically include, as disclosed herein, humanized monoclonal antibodies or antigen-binding fragments that specifically bind to EGFRvIII and / or amplified EGFR; and / or conjugates thereof. In some instances, components in the kits may be used for detecting and / or treating tumors.

[0360] The kit may include more than one of the conjugates, humanized antibodies, antigen-binding fragments, CAR-containing immune cells, nucleic acids, or compositions disclosed herein. Therefore, the kit may include two or more humanized monoclonal antibodies that specifically bind to EGFRvIII and / or amplified EGFR. The kit may include at least two of the following: humanized monoclonal antibodies or antigen-binding fragments, conjugates thereof, or combinations thereof. In some instances, the kit includes an antigen-binding fragment or a conjugate comprising an antigen-binding fragment, such as an Fv fragment. In one instance, for example, for in vivo use, the fragment may be an scFv fragment.

[0361] The kit may include a container and a label or packaging instructions on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. Containers can be made of various materials, such as glass or plastic. Containers typically contain a composition comprising one or more of the disclosed antibodies, antigen-binding fragments, or conjugates. In several instances, the container may have a sterile opening (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be penetrated by a hypodermic needle). The label or packaging instructions indicate that the composition is intended to treat a specific condition.

[0362] Labels or package inserts typically include further instructions for use of the kit components (e.g., antibodies or fragments thereof, conjugates, nucleic acids, CAR-containing immune cells, or compositions disclosed herein), for example, methods of treating, preventing, or detecting tumors. Package inserts typically include instructions common in commercial packaging of therapeutic products, containing information on indications, usage, dosage, administration, contraindications, and / or precautions for such therapeutic products. Instructional materials can be in written, electronic (e.g., computer floppy disks or CDs), or visual (e.g., video files). Kits may also include additional components to facilitate specific applications for which the kit is designed. Thus, for example, a kit may additionally contain devices for detecting labeled substances (e.g., enzyme substrates for enzyme labeling, filter sets for detecting fluorescent labels, suitable secondary labels (e.g., secondary antibodies), etc.). Kits may also additionally include buffers and other reagents for practicing specific experimental methods.

[0363] Example The mouse monoclonal antibody 40H3 binds to human amplified EGFR when the receptor is overexpressed on cancer cells. 40H3 also binds to immobilized EGFRvIII or EGFRvIII when expressed on transfected cells. To use 40H3 as a clinical antibody, variable portions of the heavy and light chains of 40H3 are humanized.

[0364] Each CDR was initially identified by comparison with KABAT or other similar databases. The heavy and light chain variable regions of 40H3 were compared with the closest human immunoglobulin family, and residues were then altered to generate candidate humanized antibodies. The candidates contained varying numbers of human-substituted amino acids, and these amino acids were located in slightly different positions. Not only were several heavy and light chain candidates generated, but combinations were also generated in which a given humanized heavy chain was matched with several light chains—and vice versa. Thus, optimal pairings were established.

[0365] Humanization of mouse antibodies is a modifiable process. Replacing multiple human residues into the mouse framework generates proteins with lower immunogenicity, but carries the risk of loss of binding activity. The binding activity of all candidates against immobilized EGFRvIII (by ELISA) or against cells expressing high levels of EGFR or EGFRvIII (by flow cytometry) was determined.

[0366] To evaluate the binding activity of humanized candidates, immobilized EGFRvIII extracellular domains (measured by ELISA or Octet), various EGFR-expressing cancer cells (measured by flow cytometry), or cancer cells expressing transfected EGFRvIII (also measured by flow cytometry) were used. All published humanized sequences retained binding activity with immobilized EGFRvIII, but only a subset bound to cells overexpressing EGFR or transfected with EGFRvIII. Antibodies with excellent cell-binding activity were identified.

[0367] Example 1 Materials and Methods ELISA Binding of candidate-derived antibodies was assessed using an ELISA assay. Human EGFRvIII extracellular domain (ECD) with a C-terminal His tag (EGI-H52H4, ACROBiosystems, Newark, DE, USA) was bound at 3.5 μg / ml to nickel-coated clear 96-well plates (Thermo Fisher Scientific) for 1 h at room temperature. The plates were washed three times with PBS-Tween 20 (PBST) containing 0.05% TWEEN®. Different concentrations of primary antibody were added to the immobilized EGFRvIII on a shaker for 1 h at room temperature. The antibody was diluted to the desired concentration with PBS containing 5% BSA (Sigma-Aldrich). After adding the primary antibody, the plates were washed three times with PBST. Peroxidase-labeled AffiniPure donkey anti-human IgG (H+L) (catalog number 709035149; Jackson ImmunoResearch, ME, USA) was diluted 1:20,000 and incubated with shaking at room temperature for 1 h. The plate was washed three times with PBST, and the horseradish peroxidase (HRP)-labeled secondary antibody was quantified by adding 3,3',5,5'-tetramethylbenzidine substrate solution (Thermo Fisher Scientific). The reaction was terminated by adding 1 M sulfuric acid (Sigma-Aldrich). Horseradish peroxidase activity was analyzed using a VersaMax microplate reader with SoftMax Pro software (Molecular Devices, CA, USA).

[0368] Flow cytometry Mix the antibody with 500 μl of FACS buffer containing suspended cells (2.5 × 10⁶ cells per tube). 5Cells were incubated on ice for 30 min in FACS buffer composed 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). Antibody binding was detected on ice for 30 min using R-phycoerythrin AffiniPure F(ab')2 fragment goat anti-human IgG (H+L) (catalog number 109116088; Jackson Immuno Research, ME, USA) at a 1:250 dilution. Afterward, the viability dye eFluor R780 (catalog number 65086514, Invitrogen) was added to each tube and incubated on ice for 15 min (1 / 700 dilution). Antibody binding was characterized using the BD FACSCanto™ II system (BD Bioscience, San Jose, CA, USA), and the data were analyzed using FlowJo (Tree Star, Inc., Ashland, OR, USA) and displayed as histograms, plotting the median fluorescence intensity. EC50% binding to cells was calculated using GraphPad Prism version 9.4. 50 value.

[0369] Example 2 result When evaluating binding to cells or immobilized EGFRvIII, the A10 antibody was the best binding agent (among all candidates) and superior to chimeric antibodies with original mouse VH and mouse VL (as determined by assay results). See For example, Figure 1A-1B , Figure 2A-2B and Figures 4A-4B A10 exhibits enhanced binding affinity (i.e., tighter binding) to both cells and immobilized antigens. The A10 antibody is a combination of candidate heavy chain “VH3” and candidate light chain “VL3”. Other combinations yield candidates that retain binding activity, albeit with lower binding affinity. The superior binding activity of all candidates could not be predicted by sequence analysis alone.

[0370] Figures 4A-4B The data summarizes all antibody binding data, while results for specific cell lines are presented in separate graphs. Figure 1A-1B The binding of EGFRvIII to transfected F98 cells was shown when the cells were analyzed by flow cytometry with various antibodies at increasing concentrations. Figure 2A-2B The antibody was shown to interact with the EGFR-amplified cell line MDA-MB-468. Figure 2A ) and A431 ( Figure 2B The binding of 40H3 to WT EGFR was observed. 40H3 does not bind to WT EGFR. To examine any enhancement in binding activity to WT EGFR, WI-38 and U87MG cells (which express only WT EGFR) were incubated with several humanized candidates. Figures 3A-3B The results showed that binding to WT EGFR was not enhanced by any of the humanized candidates. However, the presence of WT EGFR was confirmed by cetuximab binding.

[0371] Figures 4A-4B Four series of humanized antibodies are provided. Series A consists of VH1-3 candidates paired with VL1-3. Except for A1, all candidates have amino acid substitutions in the framework portions of the variable heavy and light chains. Candidate A1 is an original mouse VH1-3 fused with the human CH1-3 constant sequence and constant κ chain. H and V L (Invivogen's pFUSE vectors (pFUSE2ss-CLIg-hK and pFUSEss-hchg1)).

[0372] The B series includes two light chain variants (DA and EG – see sequence information) that alter the last residue of the light chain CDR2. These alterations are intended to “protect” the antibody from potential post-translational modifications. These variants did not outperform the A10 candidate and were not further characterized.

[0373] The C series includes sequences generated using the BioPhi platform (Prihoda). et al (MABs, 2022). Surprisingly, only C10 exhibited cell-binding beneficial properties. C1-C9 did not show cell binding but bound to the antigen. The D series included the variable light chain of antibody C1 paired with VH3 of the A series, or the light chain of antibody C6 paired with VH3. D1 (VH and VL3 from C1) did not bind to cells, while D2 and D3 did.

[0374] For cell binding assays, cetuximab was used as a positive control to detect all surface-expressed EGFR. The 40H3 antibody (and all humanized versions of 40H3) binds to EGFR subsets expressed only on cancer cells, while cetuximab binds to all cells expressing EGFR on their surface. See ,For example, Figures 5A-5B Using 40H3 and its humanized version allows for specific binding to cancer cells in vivo, while avoiding binding to normal cells, thus reducing potential side effects.

[0375] Example 3 A10 crystal structure Determine the crystal structure of EGFR ring-A10 fab ( See , Figures 6A-6C The crystallization conditions are as follows: 1357 Wizard G11 NaH2PO4 (0.8M) KH2PO4 (1.2M) Sodium acetate (0.1M, pH 4.5) No cryoprotectant Table 2: Data Collection.

[0376]

[0377] Table 3: Contact residues between EGFR target peptide and A10 antibody.

[0378]

[0379] Example 4 A10 CAR-T cells like Figures 8A-8B The CAR vector was prepared as shown. CAR T cells were transduced via lentivirus and T cell generation was activated using anti-CD3 and IL-2. Figure 9 The cell viability of the A431-luc cell line is shown at 24 or 48 hours after receiving the indicated treatment. "CAR-1" and "CAR-2" refer to cells containing / expressing [CAR-1 and CAR-2, respectively]. Figure 8A or Figure 8B T cells of the A10 scFv-CAR construct.

[0380] For in vivo administration, cells are transformed and allowed to grow for one week before being administered to the tumor site via IV, IP, or local infusion. T-cell transduction is monitored by introducing surface markers (e.g., truncated EGFR), which can be detected using antibodies (e.g., cetuximab). For cetuximab, 35%–50% of T-cell preparations are suitable for infusion.

[0381] Example 5 A10 antibody-drug conjugate Purified A10 antibodies were conjugated to either drutecan (A10-Dxd) or monomethylaurestatin E (A10-MMAE) payloads. The conjugation reaction was commercially performed by NJBio®, Princeton, NJ. Briefly, to achieve conjugation via internal reducing disulfide, the antibody formulation was subjected to mild reduction (4.5 moles of excess TCEP relative to the antibody in PBS). Samples were desalted using a Zeba® column (ThermoFisher® Scientific, NY, USA) according to the manufacturer's instructions, followed by conjugation via maleimide groups on the linker-payload composition. Following the conjugation reaction, the ADCs were fractionated to remove under- and over-modified antibodies. The drug-antibody ratio (DAR) for each ADC was determined. ADCs carrying 2–8 drugs per antibody (without aggregation) were used. Figure 10 The cell viability of MDA-MB-468 cells after treatment with A10-Dxd or A10-MMAE is shown.

[0382] Example 6 Additional research The antibodies or fragments thereof disclosed in this article ( See For example, Table 1) can be further modified by attaching a drug-based cytotoxic payload, constructing a CAR construct containing an antibody or a fragment thereof, or combining it with a T-cell conjugate to produce a novel antitumor agent. In each case, cancer cells carrying amplified EGFR or EGFRvIII are killed. Furthermore, cells expressing low or normal levels of WT EGFR (non-cancer cells) are not killed.

[0383] It is evident that changes or modifications may be made to the specific details of the methods or compositions described herein without departing from the spirit of this disclosure. We claim protection for all such modifications and changes falling within the scope and spirit of the following claims.

Claims

1. An isolated humanized monoclonal antibody or its antigen-binding fragment, which contains a heavy chain variable region (V... H ) and light chain variable region (V L ), the V H and V L : a) Contains SEQ ID NO: 5 and SEQ ID NO: 8 (A10, VH3+VL3), respectively; b) Contains SEQ ID NO: 3 and SEQ ID NO: 6 (A2, VH1+VL1), respectively; c) Contains SEQ ID NO: 3 and SEQ ID NO: 7 (A3, VH1+VL2), respectively; d) Contains SEQ ID NO: 3 and SEQ ID NO: 8 (A4, VH1+VL3), respectively; e) Contains SEQ ID NO: 4 and SEQ ID NO: 6 (A5, VH2+VL1), respectively; f) Contains SEQ ID NO: 4 and SEQ ID NO: 7 (A6, VH2+VL2), respectively; g) contains SEQ ID NO: 4 and SEQ ID NO: 8 (A7, VH2+VL3), respectively; h) contains SEQ ID NO: 5 and SEQ ID NO: 6 (A8, VH3+VL1), respectively. i) Contains SEQ ID NO: 5 and SEQ ID NO: 7 (A9, VH3+VL2), respectively; j) Contains SEQ ID NO: 1 and SEQ ID NO: 9 (B1, 40H3 VH+VL-EG); k) respectively contains SEQ ID NO: 1 and SEQ ID NO: 10 (B2, 40H3 VH+VL-DA); l) respectively contain SEQ ID NO: 4 and SEQ ID NO: 11 (B3, VH2+VL1-DA); m) respectively contains SEQ ID NO: 4 and SEQ ID NO: 12 (B4, VH2+VL2-DA); n) respectively contain SEQ ID NO: 5 and SEQ ID NO: 11 (B5, VH3 and VL1-DA); o) respectively contains SEQ ID NO: 31 and SEQ ID NO: 32 (C10); p) contains SEQ ID NO: 5 and SEQ ID NO: 14 (D2) respectively; or q) contains SEQ ID NO: 5 and SEQ ID NO: 24 (D3) respectively.

2. The isolated humanized monoclonal antibody of claim 1, wherein the antibody comprises a human constant domain.

3. The isolated humanized monoclonal antibody according to claim 1 or claim 2, wherein the antibody is IgG.

4. The isolated humanized monoclonal antibody according to any one of the preceding claims, comprising a recombinant constant domain, said recombinant constant domain comprising a modification to extend the half-life of said antibody.

5. The isolated humanized monoclonal antibody or antigen-binding fragment of any of the preceding claims, conjugated to a toxin or chemotherapeutic agent.

6. The isolated humanized monoclonal antibody or antigen-binding fragment of claim 5, wherein the toxin is Pseudomonas aeruginosa (…). Pseudononas Exotoxins (PE), ricin, absinthecin, diphtheria toxin, ribosomal toxin, ribonuclease, saponins, cazithromycin, or botulinum toxin.

7. The isolated humanized monoclonal antibody or antigen-binding fragment of claim 5 or 6, wherein the toxin is PE, and wherein the PE is PE25, PE38 or PE40.

8. The isolated humanized monoclonal antibody or antigen-binding fragment of claim 5, wherein the chemotherapeutic agent is monomethylaurestatin E or a maytansine.

9. The antigen-binding fragment according to any one of the preceding claims.

10. The antigen-binding fragment of any one of the preceding claims, wherein the antigen-binding fragment is an Fv, dsFV, ds-scvFV, Fab, F(ab')2, scFV, or scFV2 ​​fragment.

11. The antigen-binding fragment of any one of the preceding claims, wherein the antigen-binding fragment is Fab or scFV.

12. The isolated humanized monoclonal antibody or antigen-binding fragment of any of the preceding claims, conjugated to a detectable biomarker.

13. A chimeric antigen receptor (CAR) comprising the antigen-binding fragment of any one of the preceding claims.

14. A bispecific antibody comprising any one of claims 1-11, a humanized monoclonal antibody or an antigen-binding fragment.

15. An isolated nucleic acid molecule encoding a humanized antibody or antigen-binding fragment as described in any one of claims 1-12, or a V-type of said antibody or antigen-binding fragment. H or V L The CAR of claim 13, or the bispecific antibody of claim 14.

16. The isolated nucleic acid molecule of claim 15, wherein the nucleic acid molecule is a cDNA sequence.

17. The isolated nucleic acid molecule of claim 15 or claim 16, operatively linked to a promoter.

18. A vector comprising the nucleic acid molecule of any one of claims 15-17.

19. The vector of claim 18, wherein the vector is a viral vector.

20. An isolated host cell comprising the nucleic acid molecule of any one of claims 15-17, or the vector of claim 18 or 19.

21. Isolated T cells expressing the CAR of claim 13.

22. A pharmaceutical composition for treating cancers expressing EGFRvIII, comprising an effective amount of any one of claims 1-12, a humanized monoclonal antibody or antigen-binding fragment, a bispecific antibody as claimed in claim 14, a nucleic acid molecule as claimed in any one of claims 15-17, a vector as claimed in claim 18 or 19, a host cell as claimed in claim 20 or a T cell as claimed in claim 21; and a pharmaceutically acceptable carrier.

23. A method for generating a monoclonal antibody or antigen-binding fragment that specifically binds to EGFRvIII, or a bispecific antibody comprising said monoclonal antibody or antigen-binding fragment, said method comprising: Expressing one or more nucleic acid molecules as described in claims 15-17 in host cells; and Purify the antibody, antigen-binding fragment, or bispecific antibody.

24. A method for detecting the presence of EGFRvIII in a biological sample from a subject, comprising: Under conditions sufficient to form immune complexes, the biological sample is contacted with an effective amount of any one of claims 1-12, the humanized monoclonal antibody or antigen-binding fragment, or the bispecific antibody of claim 14; and The presence of the immune complex in the biological sample is detected, wherein the presence of the immune complex in the biological sample indicates the presence of EGFRvIII in the sample.

25. The method of claim 24, wherein the biological sample is a biopsy sample from glioma, head and neck cancer, breast cancer, pancreatic cancer, colorectal cancer, or bladder cancer.

26. A method for inhibiting tumors expressing EGFRvIII and / or expressing amplified EGFR in a subject, comprising administering to the subject an effective amount of any humanized monoclonal antibody or antigen-binding fragment of any one of claims 1-12, a bispecific antibody of claim 14, a nucleic acid molecule of any one of claims 15-17, a vector of claim 18 or 19, a host cell of claim 20, a T cell of claim 21, or a pharmaceutical composition of claim 22, wherein the subject has a tumor expressing EGFRvIII or amplified EGFR, respectively.

27. The method of claim 26, wherein the method inhibits tumors expressing EGFRvIII, and subjects suffering from tumors expressing EGFRvIII are selected for treatment.

28. A method for inhibiting tumors that overexpress EGFR in a subject, comprising: The administration of an effective amount of any one of claims 1-12, the humanized monoclonal antibody or antigen-binding fragment of claim 14, the bispecific antibody of claim 14, the nucleic acid molecule of any one of claims 15-17, the vector of claim 18 or 19, the T cell of claim 21, or the pharmaceutical composition of claim 22 to a subject with a tumor that overexpresses EGFR, wherein the VH of the monoclonal antibody or the antigen-binding fragment thereof comprises SEQ ID NO: 5, and the VL of the monoclonal antibody or the antigen-binding fragment thereof comprises SEQ ID NO:

8.

29. The method of any one of claims 24 to 28, wherein the tumor is a glioma, head and neck cancer, breast cancer, pancreatic cancer, colorectal cancer, or bladder cancer.

30. The method of any one of claims 24 to 29, wherein the subject is a human.

31. The method of any one of claims 26-33, wherein inhibiting the tumor comprises reducing the growth, size, or metastasis of the tumor.

32. Use of the humanized monoclonal antibody or antigen-binding fragment of any one of claims 1-12, the bispecific antibody of claim 14, the nucleic acid molecule of any one of claims 15-17, the vector of claim 18 or claim 19, the T cell of claim 21, or the pharmaceutical composition of claim 22 for inhibiting tumors expressing EGFRvIII in a subject or for detecting the presence of EGFRvIII in a biological sample.

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