Anti-EGFRVIII antibodies and antigen binding fragments thereof

Abstract

The present invention provides antigen binding agents that specifically bind to epidermal growth factor receptor variant III (EGFRvIII), such as antibodies or antigen binding fragments thereof, chimeric antigen receptors (CAR), bispecific T-cell adapters (BiTE), and the like. EGFRvIII-specific antibodies or antigen binding fragments thereof, CARs, and BiTE may be used to treat cancer. The present invention encompasses, inter alia, antibody drug conjugates that target cells expressing EGFRvIII.

Description

[0001] This application is a divisional application. The corresponding parent application has application number 202080037602.8, filing date March 23, 2020, and the name of the invention is “Anti-EGFRVIII antibodies and antigen-binding fragments thereof”. Technical Field

[0002] The present disclosure provides antigen binding agents specifically binding to epidermal growth factor receptor variant III (EGFRvIII), such as antibodies or their antigen binding fragments, chimeric antigen receptors (CAR), bispecific T-cell adapters (BiTE) etc. The EGFRvIII-specific antibodies or their antigen binding fragments, CAR or BiTE of the present disclosure can be used, for example, to treat cancer. The present disclosure particularly encompasses antibody drug conjugates targeting cells expressing EGFRvIII. Background Art

[0003] Epidermal growth factor receptor variant III (EGFRvIII) is amplified, highly expressed, and present in 25-64% of glioblastoma multiforme (GBM). It should be noted that different detection methods can produce inconsistent results, but EGFRvIII mRNA and protein expression have been detected in a subset of breast cancer and head and neck squamous cell carcinoma (HNSCC) using a variety of complementary technologies (reviewed in Gan et al. 2013). Unlike the wild-type (wt) epidermal growth factor receptor (EGFR), which is expressed in tissues of epithelial, mesenchymal, and neuronal origin and plays a major role in normal cellular processes such as proliferation, differentiation, and development, EGFRvIII is not expressed on normal tissues.

[0004] The EGFRvIII variant is derived from the in-frame deletion of exon 2-7 of the EGFR gene, which results in the removal of a sequence of 267 amino acid residues encoding the extracellular domain. The newly formed splice junction encodes a glycine residue that has no counterpart in wild-type human EGFR and thus forms a new epitope. In addition, numerous studies have shown that normal tissues lack EGFRvIII. Therefore, EGFRvIII contains new tumor-specific cell surface epitopes that can be used for antibody-targeted therapy. However, compared with the rest of the human sequence, the immunogenicity of the EGFRvIII new epitope is not very strong, and many antibodies produced so far have not yet been confirmed to specifically recognize EGFRvIII (reviewed in Gan et al. 2013).

[0005] In rare cases, monoclonal antibodies (mAbs) against EGFRvIII neo-epitopes have been described, including antibody 13.1.2 (U.S. Pat. No. 7,736,644), which is also developed by Amgen as an antibody drug conjugate (ADC) (AMG 595: Hamblett KJ, et al., Molecular Cancer Therapeutics, Vol. 14(7), pp. 1614-24, 2015). U.S. Pat. No. 9,562,102 also describes the 806 antibody (ABT-806, ABT-414) developed by Abbvie, which binds to EGFRvIII and a subset of amplified EGFR on tumor cells overexpressing EGFR (Cleary, JM et al., Invest New Drugs, 33(3), pp. 671-8, 2015; Reilly, EB., Molecular Cancer Therapeutics, Vol. 14(5), pp. 1141-51, 2015). Although the antibody has been shown to preferentially bind to tumor EGFR in preclinical models, binding of the antibody to wild-type EGFR present in human skin has been shown to be responsible for the skin toxicity exhibited by ABT-806 in some patients (Cleary et al. 2015).

[0006] Antibodies or antigen-binding fragments thereof that specifically target epitopes of EGFRvIII that are absent or inaccessible in cells expressing EGFR would be beneficial in the treatment of cancer patients. Summary of the invention

[0007] The present invention provides antigen binding agents that specifically bind to epidermal growth factor receptor variant III (EGFRvIII), such as antibodies or antigen binding fragments thereof, chimeric antigen receptors (CARs), bispecific T-cell engagers (BiTEs), and the like.

[0008] As described in more detail below, some anti-EGFRvIII antibodies or its Fab can be combined with the EGFRvIII at the surface of cancer cell (for example, glioblastoma cell).In certain embodiments, described antibody or its Fab can not significantly combine with the EGFR expressed on cancer cell (for example, U87MG-EGFR WT).

[0009] Antibody or its Fab of the present disclosure can be internalized by cancer cell, and therefore can be used for delivering cargo molecule in one aspect thereof.Present disclosure especially encompasses anti-EGFRvIII antibody or its Fab that are conjugated to treatment part.Anti-EGFRvIII antibody described herein can be used for suppressing the growth of tumor cells expressing EGFRvIII.

[0010] In certain embodiments of the present disclosure, the anti-EGFRvIII antibody or antigen-binding fragment thereof may be capable of binding to an epitope present in native EGFRvIII (e.g., native recombinant EGFRvIII) and denatured EGFRvIII (e.g., denatured recombinant EGFRvIII).

[0011] Generally, the antibody of the present disclosure or its Fab can be in conjunction with peptide, and the peptide comprises the EGFRvIII fragment consisting of the amino-acid residue 1-76 (SEQ ID NO:119) of EGFRvIII.The subset of the antibody or its Fab can be in conjunction with the amino-acid residue 1-18 (SEQ ID NO:125) of EGFRvIII, and another subset of the antibody can be in conjunction with the amino-acid residue 15-37 (SEQ ID NO:6) of EGFRvIII.

[0012] More specifically, the present disclosure provides anti-EGFRvIII antibodies or antigen-binding fragments thereof that may be capable of binding to one or more of the EGFRvIII fragments illustrated in Figures 4a and / or 4b.

[0013] Embodiments of anti-EGFRvIII antibodies or antigen-binding fragments thereof encompassed by the present disclosure include, for example:

[0014] - an antibody capable of binding to a peptide comprising an EGFRvIII fragment consisting of amino acid residues 1-18 of EGFRvIII (SEQ ID NO: 125);

[0015] - an antibody capable of binding to a peptide comprising an EGFRvIII fragment consisting of amino acid residues 3-18 of EGFRvIII (SEQ ID NO: 129);

[0016] - an antibody capable of binding to a peptide comprising an EGFRvIII fragment consisting of amino acid residues 15-37 of EGFRvIII (SEQ ID NO: 6); or

[0017] - an antibody capable of binding to a peptide comprising an EGFRvIII fragment consisting of amino acid residues 19-37 of EGFRvIII (SEQ ID NO: 142).

[0018] Some specific anti-EFGRvIII antibodies or antigen-binding fragments thereof encompassed by the present disclosure include those that do not require the presence of amino acid residues 1-2 of EGFRvIII for binding. Specifically contemplated are anti-EFGRvIII antibodies or antigen-binding fragments thereof that are capable of binding to one or more of the following EGFRvIII fragments: fragment 19-76 (SEQ ID NO: 138), fragment 19-62 (SEQ ID NO: 139), fragment 19-49 (SEQ ID NO: 140), fragment 19-45 (SEQ ID NO: 141), fragment 28-45 (SEQ ID NO: 143), fragment 28-37 (SEQ ID NO: 144), fragment 19-37 (SEQ ID NO: 142), fragment 3-45 (SEQ ID NO: 127), fragment 3-49 (SEQ ID NO: 126), fragment 3-37 (SEQ ID NO: 128), fragment 6-49 (SEQ ID NO: 130), fragment 6-45 (SEQ ID NO: 131), fragment 6-37 (SEQ ID NO: 132), fragment 10-49 (SEQ ID NO: 133), fragment 10-45 (SEQ ID NO: 134), fragment 10-49 (SEQ ID NO: 135), fragment 10-45 (SEQ ID NO: 136), fragment 10-49 (SEQ ID NO: 137), fragment 10-45 (SEQ ID NO: 138), fragment 10-49 (SEQ ID NO: 139), fragment 10-45 (SEQ ID NO: 141), fragment 10-45 (SEQ ID NO: 139), fragment 10-49 (SEQ ID NO: 142), fragment 10-45 (SEQ ID NO: 133), fragment 10-45 (SEQ ID NO: 134), fragment 10-45 (SEQ ID NO: 135), fragment 10-45 (SEQ ID NO: 136), fragment 10-45 (SEQ ID NO: 137), fragment NO: 134), fragment 10-37 (SEQ ID NO: 135), fragment 15-49 (SEQ ID NO: 136), fragment 15-45 (SEQ ID NO: 137) or fragment 15-37 (SEQ ID NO: 6).

[0019] The anti-EGFRvIII antibodies or antigen-binding fragments provided herein include antibodies or antigen-binding fragments capable of binding to a peptide comprising an EGFRvIII fragment consisting of amino acid residues 3-37 of EGFRvIII (SEQ ID NO: 128), for example, the antibodies are F260-5G6 (also referred to herein as 5G6), F263-1A8 (also referred to herein as 1A8), F263-4B3 (also referred to herein as 4B3), F263-4E11 (also referred to herein as 4E11), F263-5D8 (also referred to herein as 5D8) and F265-9C9 (also referred to herein as 9C9) antibodies.

[0020] Other anti-EGFRvIII antibodies or antigen-binding fragments provided herein include antibodies or antigen-binding fragments capable of binding to a peptide comprising an EGFRvIII fragment consisting of amino acid residues 1-33 of EGFRvIII (SEQ ID NO: 124).

[0021] Exemplary antibodies or antigen-binding fragments thereof of the present disclosure include antibodies or antigen-binding fragments thereof that specifically bind to EGFRvIII (SEQ ID NO: 5) and are capable of binding to an EGFRvIII fragment selected from the group consisting of:

[0022] a. A fragment consisting of amino acid residues 15-37 of EGFRvIII (SEQ ID NO: 6);

[0023] b. a fragment consisting of amino acid residues 1-76 of EGFRvIII (SEQ ID NO: 119);

[0024] c. a fragment consisting of amino acid residues 1-62 of EGFRvIII (SEQ ID NO: 120);

[0025] d. a fragment consisting of amino acid residues 1-49 of EGFRvIII (SEQ ID NO: 121);

[0026] e. a fragment consisting of amino acid residues 1-45 of EGFRvIII (SEQ ID NO: 122);

[0027] f. a fragment consisting of amino acid residues 1-37 of EGFRvIII (SEQ ID NO: 123);

[0028] g. a fragment consisting of amino acid residues 3-49 of EGFRvIII (SEQ ID NO: 126);

[0029] h. a fragment consisting of amino acid residues 3-45 of EGFRvIII (SEQ ID NO: 127);

[0030] i. a fragment consisting of amino acid residues 3-37 of EGFRvIII (SEQ ID NO: 128);

[0031] j. a fragment consisting of amino acid residues 6-49 of EGFRvIII (SEQ ID NO: 130);

[0032] k. a fragment consisting of amino acid residues 6-45 of EGFRvIII (SEQ ID NO: 131);

[0033] 1. A fragment consisting of amino acid residues 6-37 of EGFRvIII (SEQ ID NO: 132);

[0034] m. a fragment consisting of amino acid residues 10-49 of EGFRvIII (SEQ ID NO: 133);

[0035] n. a fragment consisting of amino acid residues 10-45 of EGFRvIII (SEQ ID NO: 134);

[0036] o. a fragment consisting of amino acid residues 10-37 of EGFRvIII (SEQ ID NO: 135);

[0037] p. a fragment consisting of amino acid residues 15-49 of EGFRvIII (SEQ ID NO: 136);

[0038] q. a fragment consisting of amino acid residues 15-45 of EGFRvIII (SEQ ID NO: 137);

[0039] r. a fragment consisting of amino acid residues 19-76 of EGFRvIII (SEQ ID NO: 138);

[0040] s. A fragment consisting of amino acid residues 19-62 of EGFRvIII (SEQ ID NO: 139);

[0041] t. A fragment consisting of amino acid residues 19-49 of EGFRvIII (SEQ ID NO: 140);

[0042] u. a fragment consisting of amino acid residues 19-45 of EGFRvIII (SEQ ID NO: 141);

[0043] v. a fragment consisting of amino acid residues 19-37 (SEQ ID NO: 142) of EGFRvIII; and

[0044] w. Any combination of the above fragments,

[0045] The antibody or antigen-binding fragment thereof cannot bind to a peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 149.

[0046] The antibodies or antigen-binding fragments thereof of the present disclosure may be capable of binding to a peptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 165, and combinations thereof.

[0047] The antibody or antigen-binding fragment thereof may also be capable of binding to a peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:160.

[0048] Other exemplary embodiments of the present disclosure include antibodies or antigen-binding fragments thereof that specifically bind to EGFRvIII (SEQ ID NO: 5) and are capable of binding to an EGFRvIII fragment selected from the group consisting of:

[0049] a. A fragment consisting of amino acid residues 15-37 of EGFRvIII (SEQ ID NO: 6);

[0050] b. a fragment consisting of amino acid residues 1-76 of EGFRvIII (SEQ ID NO: 119);

[0051] c. a fragment consisting of amino acid residues 1-49 of EGFRvIII (SEQ ID NO: 121);

[0052] d. a fragment consisting of amino acid residues 1-37 of EGFRvIII (SEQ ID NO: 123);

[0053] e. a fragment consisting of amino acid residues 3-37 of EGFRvIII (SEQ ID NO: 128); and

[0054] f. Any combination of the above or its fragments,

[0055] The antibody or antigen-binding fragment thereof cannot bind to a peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 149.

[0056] The antibodies or antigen-binding fragments thereof of the present disclosure may be capable of binding to a peptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 165, and combinations thereof. The antibodies or antigen-binding fragments thereof may also be capable of binding to a peptide comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 154. The antibodies or antigen-binding fragments thereof may also be capable of binding to a peptide comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 159.

[0057] Other exemplary embodiments of the present disclosure include antibodies or antigen-binding fragments thereof that specifically bind to EGFRvIII (SEQ ID NO: 5) and are capable of binding to an EGFRvIII fragment selected from the group consisting of:

[0058] a. A fragment consisting of amino acid residues 15-37 of EGFRvIII (SEQ ID NO: 6);

[0059] b. a fragment consisting of amino acid residues 1-76 of EGFRvIII (SEQ ID NO: 119);

[0060] c. a fragment consisting of amino acid residues 1-62 of EGFRvIII (SEQ ID NO: 120);

[0061] d. a fragment consisting of amino acid residues 1-49 of EGFRvIII (SEQ ID NO: 121);

[0062] e. a fragment consisting of amino acid residues 1-45 of EGFRvIII (SEQ ID NO: 122);

[0063] f. a fragment consisting of amino acid residues 1-37 of EGFRvIII (SEQ ID NO: 123);

[0064] g. a fragment consisting of amino acid residues 3-49 of EGFRvIII (SEQ ID NO: 126);

[0065] h. a fragment consisting of amino acid residues 3-45 of EGFRvIII (SEQ ID NO: 127);

[0066] i. a fragment consisting of amino acid residues 3-37 of EGFRvIII (SEQ ID NO: 128);

[0067] j. a fragment consisting of amino acid residues 6-49 of EGFRvIII (SEQ ID NO: 130);

[0068] k. a fragment consisting of amino acid residues 6-45 of EGFRvIII (SEQ ID NO: 131);

[0069] 1. A fragment consisting of amino acid residues 6-37 of EGFRvIII (SEQ ID NO: 132);

[0070] m. a fragment consisting of amino acid residues 10-49 of EGFRvIII (SEQ ID NO: 133);

[0071] n. a fragment consisting of amino acid residues 10-45 of EGFRvIII (SEQ ID NO: 134);

[0072] o. a fragment consisting of amino acid residues 10-37 of EGFRvIII (SEQ ID NO: 135);

[0073] p. a fragment consisting of amino acid residues 15-49 of EGFRvIII (SEQ ID NO: 136);

[0074] q. a fragment consisting of amino acid residues 15-45 of EGFRvIII (SEQ ID NO: 137); and

[0075] r. Any combination of the above fragments.

[0076] The antibodies or antigen-binding fragments thereof may also bind:

[0077] a. A fragment consisting of amino acid residues 19-49 of EGFRvIII (SEQ ID NO: 140);

[0078] b. a fragment consisting of amino acid residues 19-37 of EGFRvIII (SEQ ID NO: 142);

[0079] c. a fragment consisting of amino acid residues 28-45 of EGFRvIII (SEQ ID NO: 143);

[0080] d. a fragment consisting of amino acid residues 28-37 of EGFRvIII (SEQ ID NO: 144); or

[0081] Any combination of the above fragments.

[0082] The antibody or antigen-binding fragment thereof may also be capable of binding to a peptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 165, and combinations thereof. The antibody or antigen-binding fragment thereof may also be capable of binding to a peptide comprising or consisting of an amino acid sequence shown in SEQ ID NO: 149.

[0083] Also provided is an anti-EGFRvIII antibody or its Fab that can bind epi-position, and the epi-position comprises or relates to the amino acid residue Cys20 in the peptide.These include, for example, binding EGFRvIII and / or comprising the peptide of EGFRvIII fragment (this fragment is made up of the amino acid sequence shown in SEQ ID NO:6), but can not bind the anti-EGFRvIII antibody or Fab of the peptide that comprises amino acid sequence SCVRAAGADSYEMEEDGVRKCKK (SEQ ID NO:149) or is made up of it.Such antibody or Fab encompass, for example, 4B3, 5D8 and 4E11 antibodies.

[0084] The disclosure also specifically encompasses anti-EGFRvIII antibodies or their Fabs that can bind epitopes, and the epitopes include or relate to the amino acid residue Cys35 in the peptide. These include, for example, binding to EGFRvIII and / or comprising a peptide of an EGFRvIII fragment (the fragment is composed of the amino acid sequence shown in SEQ ID NO:6), but can not bind to an anti-EGFRvIII antibody or Fab of a peptide comprising an amino acid sequence SCVRACGADSYEMEEDGVRKAKK (SEQ ID NO:163) or consisting thereof. Such antibodies or Fabs encompass, for example, 4B3, 5D8, 9C9 and 4E11 antibodies.

[0085] The disclosure further encompasses anti-EGFRvIII antibodies or antigen-binding fragments thereof that can bind to an epitope in a peptide, the epitope comprising or relating to amino acid residues Cys20 and Cys35 in the peptide. These include, for example, binding to EGFRvIII and / or comprising the EGFRvIII fragment shown in SEQ ID NO:6 or a peptide consisting thereof, but cannot bind to an amino acid sequence selected from SCVRAAGADSYEMEEDGVRKCKK (SEQ ID NO:149) or SCVRACGADSYEMEEDGVRKAKK (SEQ ID NO:163) or an anti-EGFRvIII antibody or antigen-binding fragment of a peptide consisting thereof. Such an antibody or antigen-binding fragment encompasses, for example, 4B3, 5D8 and 4E11 antibodies.

[0086] In addition to amino acid residues Cys20 and / or Cys35, the epitope to which the anti-EGFRvIII antibody or its antigen-binding fragment binds or participates in its binding may further include Glu26, Asp30, Gly31 and / or Arg33. The epitope may further include Asp23 and / or Val32.

[0087] For example, anti-EGFRvIII antibodies or antigen-binding fragments thereof encompassed by the present disclosure include those that bind to an epitope that comprises or is related to:

[0088] -Cys20, Glu26, Asp30, Gly31, Arg33, and Cys35, or

[0089] -Cys20, Asp23, Glu26, Asp30, Gly31, Val32, Arg33 and Cys35.

[0090] In addition to amino acid residues Cys20 and / or Cys35, the epitope to which the anti-EGFRvIII antibody or antigen-binding fragment thereof binds or participates in its binding may further include Arg18 and / or Gly21. The epitope may further include Glu26 and / or Gly31.

[0091] For example, anti-EGFRvIII antibodies or antigen-binding fragments thereof encompassed by the present disclosure include those that bind to an epitope that comprises or is related to:

[0092] -Arg18, Cys20, Gly21 and Cys35, or

[0093] -Arg18, Cys20, Gly21, Glu26, Gly31 and Cys35.

[0094] Additional anti-EGFRvIII antibodies or antigen-binding fragments thereof encompassed by the present disclosure include those that bind to an epitope comprising or involving:

[0095] - Cys16, Glu26, Gly31, Val32, Arg33, Lys34, Cys35 and Lys36, or

[0096] -Cys16, Cys20, Glu26, Asp30, Gly31, Val32, Arg33, Lys34, Cys35 and Lys36.

[0097] Embodiments of the present disclosure specifically include anti-EGFRvIII antibodies or antigen-binding fragments thereof that are capable of competing with the 4E11 antibody or antigen-binding fragment thereof, or with antibodies or antigen-binding fragments thereof that comprise the CDRs of the 4E11 antibody.

[0098] Other embodiments of the present disclosure include anti-EGFRvIII antibodies or antigen-binding fragments thereof that are capable of competing with the 5G6 antibody or antigen-binding fragment thereof, or with antibodies or antigen-binding fragments thereof that comprise the CDRs of the 5G6 antibody.

[0099] Other embodiments of the present disclosure include anti-EGFRvIII antibodies or antigen-binding fragments thereof that are capable of competing with the 9C9 antibody or antigen-binding fragment thereof, or with antibodies or antigen-binding fragments thereof that comprise the CDRs of the 9C9 antibody.

[0100] One specific embodiment contemplated by the present disclosure relates to an anti-EGFRvIII antibody or antigen-binding fragment thereof comprising the CDRs of the 4E11 antibody.

[0101] The present disclosure provides an anti-EGFRvIII antibody or antigen-binding fragment comprising a sequence selected from the group consisting of:

[0102] - an antibody or fragment thereof comprising CDRL1 (SEQ ID NO:38), CDRL2 (SEQ ID NO:39), CDRL3 (SEQ ID NO:40), CDRH1 (SEQ ID NO:43), CDRH2 (SEQ ID NO:44) and CDRH3 (SEQ ID NO:45) of the 4E11 antibody;

[0103] - an antibody or fragment thereof comprising CDRL1 (SEQ ID NO: 8), CDRL2 (SEQ ID NO: 9), CDR L3 (SEQ ID NO: 10), CDRH1 (SEQ ID NO: 13), CDRH2 (SEQ ID NO: 14) and CDRH3 (SEQ ID NO: 15) of the 5G6 antibody;

[0104] - an antibody or fragment thereof comprising CDRL1 (SEQ ID NO: 18), CDRL2 (SEQ ID NO: 19), CDRL3 (SEQ ID NO: 20), CDRH1 (SEQ ID NO: 23), CDRH2 (SEQ ID NO: 24) and CDRH3 (SEQ ID NO: 25) of the 1A8 antibody;

[0105] - an antibody or fragment thereof comprising CDRL1 (SEQ ID NO:28), CDRL2 (SEQ ID NO:29), CDRL3 (SEQ ID NO:30), CDRH1 (SEQ ID NO:33), CDRH2 (SEQ ID NO:34) and CDRH3 (SEQ ID NO:35) of the 4B3 antibody;

[0106] - an antibody or fragment thereof comprising CDRL1 (SEQ ID NO:48), CDRL2 (SEQ ID NO:49), CDRL3 (SEQ ID NO:50), CDRH1 (SEQ ID NO:53), CDRH2 (SEQ ID NO:54) and CDRH3 (SEQ ID NO:55) of the 5D8 antibody;

[0107] - an antibody or a fragment thereof comprising CDRL1 (SEQ ID NO: 58), CDRL2 (SEQ ID NO: 59), CDRL3 (SEQ ID NO: 60), CDRH1 (SEQ ID NO: 63), CDRH2 (SEQ ID NO: 64), CDRH3 (SEQ ID NO: 65) of the 9C9 antibody; and

[0108] -Antibodies or fragments thereof comprising CDRL1 (SEQ ID NO: 68 or 73), CDRL2 (SEQ ID NO: 69 or 74), CDRL3 (SEQ ID NO: 70 or 75), CDRH1 (SEQ ID NO: 78), CDRH2 (SEQ ID NO: 79) and CDRH3 (SEQ ID NO: 80) of the F266-11B1 (referred to herein as 11B1), F266-11C8 (referred to herein as 11C8), F266-11H5 (referred to herein as 11H5) and / or F266-11H3 (referred to herein as 11H3) antibodies.

[0109] Embodiments encompassed by the present disclosure relate to anti-EGFRvIII antibodies or antigen-binding fragments thereof comprising the CDRs of the 5G6 antibody.

[0110] Another embodiment encompassed by the present disclosure relates to an anti-EGFRvIII antibody or antigen-binding fragment thereof comprising the CDRs of the 1A8 antibody.

[0111] Another embodiment encompassed by the present disclosure relates to an anti-EGFRvIII antibody or antigen-binding fragment thereof comprising the CDRs of the 4B3 antibody.

[0112] Another embodiment contemplated by the present disclosure relates to an anti-EGFRvIII antibody or antigen-binding fragment thereof comprising the CDRs of the 5D8 antibody.

[0113] Another embodiment encompassed by the present disclosure relates to an anti-EGFRvIII antibody or antigen-binding fragment thereof comprising the CDRs of the 9C9 antibody.

[0114] Another embodiment encompassed by the present disclosure relates to an anti-EGFRvIII antibody or antigen-binding fragment thereof comprising the CDRs of the 11B1 or 11C8 antibody.

[0115] The present disclosure provides an anti-EGFRvIII antibody or antigen-binding fragment selected from the group consisting of:

[0116] - an antibody or antigen-binding fragment thereof comprising a CDR sequence consisting of CDRL1 (SEQ ID NO: 8), CDRL2 (SEQ ID NO: 9), CDRL3 (SEQ ID NO: 10), CDRH1 (SEQ ID NO: 13), CDRH2 (SEQ ID NO: 14), CDRH3 (SEQ ID NO: 15);

[0117] - an antibody or antigen-binding fragment thereof comprising a CDR sequence consisting of CDRL1 (SEQ ID NO: 18), CDRL2 (SEQ ID NO: 19), CDRL3 (SEQ ID NO: 20), CDRH1 (SEQ ID NO: 23), CDRH2 (SEQ ID NO: 24), CDRH3 (SEQ ID NO: 25);

[0118] - an antibody or antigen-binding fragment thereof comprising a CDR sequence consisting of CDRL1 (SEQ ID NO: 28), CDRL2 (SEQ ID NO: 29), CDRL3 (SEQ ID NO: 30), CDRH1 (SEQ ID NO: 33), CDRH2 (SEQ ID NO: 34), CDRH3 (SEQ ID NO: 35);

[0119] - an antibody or antigen-binding fragment thereof comprising a CDR sequence consisting of CDRL1 (SEQ ID NO: 38), CDRL2 (SEQ ID NO: 39), CDRL3 (SEQ ID NO: 40), CDRH1 (SEQ ID NO: 43), CDRH2 (SEQ ID NO: 44), CDRH3 (SEQ ID NO: 45);

[0120] - an antibody or antigen-binding fragment thereof comprising a CDR sequence consisting of CDRL1 (SEQ ID NO: 48), CDRL2 (SEQ ID NO: 49), CDRL3 (SEQ ID NO: 50), CDRH1 (SEQ ID NO: 53), CDRH2 (SEQ ID NO: 54), CDRH3 (SEQ ID NO: 55);

[0121] - an antibody or antigen-binding fragment thereof comprising a CDR sequence consisting of CDRL1 (SEQ ID NO: 58), CDRL2 (SEQ ID NO: 59), CDRL3 (SEQ ID NO: 60), CDRH1 (SEQ ID NO: 63), CDRH2 (SEQ ID NO: 64), CDRH3 (SEQ ID NO: 65);

[0122] - an antibody or fragment thereof comprising a CDR sequence consisting of CDRL1 (SEQ ID NO: 68), CDRL2 (SEQ ID NO: 69), CDRL3 (SEQ ID NO: 70), CDRH1 (SEQ ID NO: 78), CDRH2 (SEQ ID NO: 79), CDRH3 (SEQ ID NO: 80); and

[0123] -An antibody or antigen-binding fragment thereof comprising a CDR sequence consisting of CDRL1 (SEQ ID NO: 73), CDRL2 (SEQ ID NO: 74), CDRL3 (SEQ ID NO: 75), CDRH1 (SEQ ID NO: 78), CDRH2 (SEQ ID NO: 79), and CDRH3 (SEQ ID NO: 80).

[0124] In certain embodiments, the present disclosure provides antigen binding agents, such as antibodies or antigen binding fragments thereof, CARs, BiTEs, etc., which specifically bind to EGFRvIII and may comprise, for example:

[0125] a. a light chain variable region and a heavy chain variable region, wherein the light chain variable region may comprise CDRL1 having the amino acid sequence shown in SEQ ID NO: 8, CDRL2 having the amino acid sequence shown in SEQ ID NO: 9, and CDRL3 having the amino acid sequence shown in SEQ ID NO: 10; the heavy chain variable region may comprise CDRH1 having the amino acid sequence shown in SEQ ID NO: 13, CDRH2 having the amino acid sequence shown in SEQ ID NO: 14, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 15;

[0126] b. a light chain variable region and a heavy chain variable region, wherein the light chain variable region may comprise CDRL1 having the amino acid sequence shown in SEQ ID NO: 18, CDRL2 having the amino acid sequence shown in SEQ ID NO: 19, and CDRL3 having the amino acid sequence shown in SEQ ID NO: 20; the heavy chain variable region may comprise CDRH1 having the amino acid sequence shown in SEQ ID NO: 23, CDRH2 having the amino acid sequence shown in SEQ ID NO: 24, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 25;

[0127] c. a light chain variable region and a heavy chain variable region, wherein the light chain variable region may comprise CDRL1 having the amino acid sequence shown in SEQ ID NO: 28, CDRL2 having the amino acid sequence shown in SEQ ID NO: 29, and CDRL3 having the amino acid sequence shown in SEQ ID NO: 30; and the heavy chain variable region may comprise CDRH1 having the amino acid sequence shown in SEQ ID NO: 33, CDRH2 having the amino acid sequence shown in SEQ ID NO: 34, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 35;

[0128] d. a light chain variable region and a heavy chain variable region, wherein the light chain variable region may comprise CDRL1 having the amino acid sequence shown in SEQ ID NO: 38, CDRL2 having the amino acid sequence shown in SEQ ID NO: 39, and CDRL3 having the amino acid sequence shown in SEQ ID NO: 40; and the heavy chain variable region may comprise CDRH1 having the amino acid sequence shown in SEQ ID NO: 43, CDRH2 having the amino acid sequence shown in SEQ ID NO: 44, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 45;

[0129] e. a light chain variable region and a heavy chain variable region, wherein the light chain variable region may comprise CDRL1 having the amino acid sequence shown in SEQ ID NO: 48, CDRL2 having the amino acid sequence shown in SEQ ID NO: 49, and CDRL3 having the amino acid sequence shown in SEQ ID NO: 50; the heavy chain variable region may comprise CDRH1 having the amino acid sequence shown in SEQ ID NO: 53, CDRH2 having the amino acid sequence shown in SEQ ID NO: 54, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 55;

[0130] f. a light chain variable region and a heavy chain variable region, wherein the light chain variable region may comprise CDRL1 having the amino acid sequence shown in SEQ ID NO: 58, CDRL2 having the amino acid sequence shown in SEQ ID NO: 59, and CDRL3 having the amino acid sequence shown in SEQ ID NO: 60; the heavy chain variable region may comprise CDRH1 having the amino acid sequence shown in SEQ ID NO: 63, CDRH2 having the amino acid sequence shown in SEQ ID NO: 64, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 65;

[0131] g. a light chain variable region and a heavy chain variable region, wherein the light chain variable region may comprise CDRL1 having the amino acid sequence shown in SEQ ID NO: 68, CDRL2 having the amino acid sequence shown in SEQ ID NO: 69, and CDRL3 having the amino acid sequence shown in SEQ ID NO: 70; and the heavy chain variable region may comprise CDRH1 having the amino acid sequence shown in SEQ ID NO: 78, CDRH2 having the amino acid sequence shown in SEQ ID NO: 79, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 80; or

[0132] h. a light chain variable region and a heavy chain variable region, wherein the light chain variable region may comprise CDRL1 having the amino acid sequence shown in SEQ ID NO:73, CDRL2 having the amino acid sequence shown in SEQ ID NO:74, and CDRL3 having the amino acid sequence shown in SEQ ID NO:75; and the heavy chain variable region may comprise CDRH1 having the amino acid sequence shown in SEQ ID NO:78, CDRH2 having the amino acid sequence shown in SEQ ID NO:79, and CDRH3 having the amino acid sequence shown in SEQ ID NO:80.

[0133] The present disclosure provides, in further embodiments, antigen binding agents that specifically bind to EGFRvIII, such as antibodies or antigen binding fragments thereof, CARs, BiTEs, etc., which may comprise:

[0134] a. a light chain variable region and / or a heavy chain variable region, wherein the light chain variable region may comprise an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 118 or substantially the same as SEQ ID NO: 118; and the heavy chain variable region may comprise an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 116 or substantially the same as SEQ ID NO: 116;

[0135] b. a light chain variable region and a heavy chain variable region, wherein the light chain variable region may comprise an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 115 or substantially the same as SEQ ID NO: 115; and the heavy chain variable region may comprise an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 116 or substantially the same as SEQ ID NO: 116; or

[0136] c. a light chain variable region and a heavy chain variable region, wherein the light chain variable region may comprise an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 118 or substantially identical to SEQ ID NO: 118; and the heavy chain variable region may comprise an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 62 or substantially identical to SEQ ID NO: 62.

[0137] In one aspect thereof, an antigen-binding agent having a light chain that is at least 80% identical or substantially identical to the amino acid sequence shown in SEQ ID NO: 115 or SEQ ID NO: 118 may have CDRs that are identical to the CDRs of SEQ ID NO: 115 or SEQ ID NO: 118, respectively.

[0138] In one aspect thereof, an antigen-binding agent having a heavy chain that is at least 80% identical or substantially identical to the amino acid sequence shown in SEQ ID NO:62 or SEQ ID NO:116 may have CDRs that are identical to the CDRs of SEQ ID NO:62 or SEQ ID NO:116, respectively.

[0139] The present disclosure provides in other embodiments an antigen binding agent that specifically binds to EGFRvIII, such as an antibody or an antigen binding fragment thereof, CAR, BiTE, etc., which may comprise:

[0140] a. a light chain variable region and a heavy chain variable region, wherein the light chain variable region may comprise an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 7 or substantially the same as SEQ ID NO: 7; and the heavy chain variable region may comprise an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 12 or substantially the same as SEQ ID NO: 12;

[0141] b. a light chain variable region and a heavy chain variable region, wherein the light chain variable region may comprise an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 17 or substantially the same as SEQ ID NO: 17; and the heavy chain variable region may comprise an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 22 or substantially the same as SEQ ID NO: 22;

[0142] c. a light chain variable region and a heavy chain variable region, wherein the light chain variable region may comprise an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 27 or substantially the same as SEQ ID NO: 27; and the heavy chain variable region may comprise an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 32 or substantially the same as SEQ ID NO: 32;

[0143] d. a light chain variable region and a heavy chain variable region, wherein the light chain variable region may comprise an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 37 or substantially the same as SEQ ID NO: 37; and the heavy chain variable region may comprise an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 42 or substantially the same as SEQ ID NO: 42;

[0144] e. a light chain variable region and a heavy chain variable region, wherein the light chain variable region may comprise an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 47 or substantially the same as SEQ ID NO: 47; and the heavy chain variable region may comprise an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 52 or substantially the same as SEQ ID NO: 52;

[0145] f. a light chain variable region and a heavy chain variable region, wherein the light chain variable region may comprise an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 57 or substantially the same as SEQ ID NO: 57; and the heavy chain variable region may comprise an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 62 or substantially the same as SEQ ID NO: 62;

[0146] g. a light chain variable region and a heavy chain variable region, wherein the light chain variable region may comprise an amino acid sequence that is at least 80% identical to or substantially identical to the amino acid sequence of SEQ ID NO: 67; the heavy chain variable region may comprise an amino acid sequence that is at least 80% identical to or substantially identical to the amino acid sequence of SEQ ID NO: 77, at least 80% identical to or substantially identical to the amino acid sequence of SEQ ID NO: 92, or at least 80% identical to or substantially identical to the amino acid sequence of SEQ ID NO: 102; or

[0147] h. a light chain variable region and a heavy chain variable region, wherein the light chain variable region may comprise an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO:72 or substantially the same as SEQ ID NO:72; and the heavy chain variable region may comprise an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO:77 or substantially the same as SEQ ID NO:77, or an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO:92 or substantially the same as SEQ ID NO:92.

[0148] A light chain variable region, a light chain, a heavy chain variable region, or a heavy chain that may comprise an amino acid sequence that is at least 80% identical to the corresponding portion of a given antibody may have the same CDRs as that antibody. In one embodiment of the present disclosure, the VL and VH sequences of the antibodies and antigen-binding fragments provided in the present disclosure may comprise sequences that are substantially identical to the VL and VH sequences provided herein, or may comprise sequences that have at least 80%, at least 90%, or at least 95% sequence identity, wherein the sequence variations are preferably outside the CDRs of the provided VL and VH sequences.

[0149] In addition, the present disclosure particularly provides antigen binding agents, such as antibodies or antigen binding fragments thereof, CARs, BiTEs, etc., which specifically bind to EGFRvIII and which may comprise:

[0150] a. a light chain, which may comprise an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 108 or substantially the same as SEQ ID NO: 108; and a heavy chain, which may comprise an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 107 or substantially the same as SEQ ID NO: 107, or

[0151] b. a light chain, which may comprise an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 110 or substantially the same as SEQ ID NO: 110; and a heavy chain, which may comprise an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 109 or substantially the same as SEQ ID NO: 109.

[0152] The present disclosure particularly provides antigen binding agents, such as anti-EGFRvIII antibodies or antigen binding fragments thereof, CARs, BiTEs, etc., which may comprise:

[0153] a. CDRL1 having the amino acid sequence shown in SEQ ID NO: 38, CDRL2 having the amino acid sequence shown in SEQ ID NO: 39, CDRL3 having the amino acid sequence shown in SEQ ID NO: 40, CDRH1 having the amino acid sequence shown in SEQ ID NO: 43, CDRH2 having the amino acid sequence shown in SEQ ID NO: 44, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 45;

[0154] b. a light chain variable region and a heavy chain variable region, wherein the light chain variable region may comprise an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or identical to the amino acid sequence shown in SEQ ID NO: 37; and the heavy chain variable region may comprise an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or identical to the amino acid sequence shown in SEQ ID NO: 42; or

[0155] c. a light chain, which may comprise an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or identical to the amino acid sequence shown in SEQ ID NO: 108; and a heavy chain, which may comprise an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or identical to the amino acid sequence shown in SEQ ID NO: 107.

[0156] According to the present disclosure, the antibody or antigen-binding fragment thereof of item b or c may have CDRs that are identical or substantially identical to the CDRs shown in SEQ ID NOs: 38, 39, 40, 43, 44 and 45.

[0157] According to the present disclosure, the antibody or antigen-binding fragment thereof can have, for example, an affinity of less than 100 nM, for example, an affinity of 50 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, etc.

[0158] Exemplary embodiments of the present disclosure include antibodies or antigen-binding fragments thereof that may comprise a human IgG constant region.An antibody or antigen-binding fragment of the present disclosure may comprise, for example, but not limited to, a human IgG1 constant region or a human IgG2 constant region.

[0159] In an exemplary embodiment, an antigen binding agent disclosed herein may comprise a humanized framework region.

[0160] According to the present disclosure, the antibody or antigen-binding fragment thereof can be a monoclonal antibody, a polyclonal antibody, a humanized antibody, a chimeric antibody, a human antibody, a single-chain antibody, or a multispecific antibody (eg, a bispecific antibody).

[0161] Bispecific antibodies or antigen-binding fragments thereof of the present disclosure include those (eg, biparatopic antibodies) that may comprise a first arm that specifically binds a first human EGFRvIII epitope and a second arm that specifically binds a second (non-overlapping) human EGFRvIII epitope.

[0162] Additional embodiments of the bispecific antibodies or antigen-binding fragments thereof of the present disclosure include those that may comprise a first arm that specifically binds to a first human EGFRvIII epitope and a second arm that specifically binds to another antigen.

[0163] The bispecific antibodies or antigen-binding fragments thereof of the present disclosure include bispecific immune cell engagers, such as those comprising a first arm that specifically binds to human EGFRvIII and a second arm that specifically binds to CD3.

[0164] According to the present disclosure, the antigen binding fragment comprises, for example, scFv, Fab, Fab' or (Fab')2.

[0165] In another aspect, the present disclosure provides an anti-EGFRvIII antibody or antigen-binding fragment thereof that can be linked to a cargo molecule.

[0166] According to the present disclosure, the cargo molecule may comprise a therapeutic moiety, e.g., a cytotoxic agent, a cytostatic agent, an anticancer agent, or a radiotherapeutic agent. In a specific embodiment of the present disclosure, the antibody drug conjugate may comprise a cytotoxic agent. Another specific embodiment of the present disclosure relates to an antibody drug conjugate comprising a radiotherapeutic agent.

[0167] According to the present disclosure, the cargo molecule may comprise a detectable moiety.

[0168] The antibodies or antigen-binding fragments thereof of the present disclosure may be provided in the form of a pharmaceutical composition. The pharmaceutical composition may include, for example, a pharmaceutically acceptable carrier, diluent or excipient.

[0169] The present disclosure further provides nucleic acid molecules encoding the light chain variable region and / or the heavy chain variable region of the antibodies or antigen-binding fragments thereof disclosed herein.

[0170] Exemplary embodiments of nucleic acid molecules of the present disclosure include:

[0171] a. a nucleic acid molecule, which may comprise a sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 11; and / or a nucleic acid molecule, which may comprise a sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 16 or a sequence encoding an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO: 7 and / or SEQ ID NO: 12;

[0172] b. a nucleic acid molecule which may comprise a sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 21; and / or a nucleic acid molecule which may comprise a sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 26 or which encodes an amino acid sequence which is substantially identical to the amino acid sequence of SEQ ID NO: 17 and / or SEQ ID NO: 22;

[0173] c. a nucleic acid molecule comprising a sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO:31; and / or a nucleic acid molecule comprising a sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO:36 or encoding an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO:27 and / or SEQ ID NO:32;

[0174] d. a nucleic acid molecule which may comprise a sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO:41; and / or a nucleic acid molecule which may comprise a sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO:46 or which encodes an amino acid sequence which is substantially identical to the sequence of SEQ ID NO:37 and / or SEQ ID NO:42;

[0175] e. a nucleic acid molecule comprising a sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO:51; and / or a nucleic acid molecule comprising a sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO:56 or encoding an amino acid sequence substantially identical to the sequence of SEQ ID NO:47 and / or SEQ ID NO:52;

[0176] f. a nucleic acid molecule which may comprise a sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO:61; and / or a nucleic acid molecule which may comprise a sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO:66 or which encodes an amino acid sequence which is substantially identical to the sequence of SEQ ID NO:57 and / or SEQ ID NO:62;

[0177] g. a nucleic acid molecule which may comprise a sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 71; and / or a nucleic acid molecule which may comprise a sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 81 or which encodes an amino acid sequence which is substantially identical to the sequence of SEQ ID NO: 67 and / or SEQ ID NO: 77;

[0178] h. a nucleic acid molecule which may comprise a sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 76; and / or a nucleic acid molecule which may comprise a sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 81 or which encodes an amino acid sequence which is substantially identical to the sequence of SEQ ID NO: 72 and / or SEQ ID NO: 77;

[0179] i. a nucleic acid molecule which may comprise a sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 86; and / or a nucleic acid molecule which may comprise a sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 96 or which encodes an amino acid sequence which is substantially identical to the sequence of SEQ ID NO: 82 and / or SEQ ID NO: 92;

[0180] j. a nucleic acid molecule which may comprise a sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 91; and / or a nucleic acid molecule which may comprise a sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 96 or which encodes an amino acid sequence which is substantially identical to the sequence of SEQ ID NO: 87 and / or SEQ ID NO: 92; or

[0181] k. A nucleic acid molecule, which may comprise a sequence that is at least 80% identical to the nucleotide sequence shown in SEQ ID NO:101; and / or a nucleic acid molecule, which may comprise a sequence that is at least 80% identical to the nucleotide sequence shown in SEQ ID NO:106 or which encodes an amino acid sequence that is substantially identical to the sequence of SEQ ID NO:97 and / or SEQ ID NO:102.

[0182] In another aspect, the present disclosure provides a kit comprising at least one antibody or antigen-binding fragment thereof disclosed herein.

[0183] Another aspect of the present disclosure relates to a vector or vector set, which may contain nucleic acids encoding the light chain variable region and the heavy chain variable region of the antibody or antigen-binding fragment disclosed herein. The nucleic acids encoding the light chain variable region or light chain and the heavy chain variable region or heavy chain may be provided on the same vector or on separate vectors.

[0184] Other aspects of the present disclosure relate to isolated cells comprising a vector or vector collection described herein. The isolated cells may be capable of expressing, assembling and / or secreting the antibody or antigen-binding fragment thereof.

[0185] Other aspects of the present disclosure relate to a kit comprising a first bottle comprising a nucleic acid or vector encoding the light chain of an antibody or antigen-binding fragment thereof of the present disclosure and a second bottle comprising a nucleic acid or vector encoding the heavy chain of the antibody or antigen-binding fragment thereof.

[0186] Other aspects of the present disclosure relate to methods for treating cancer, and the cancer comprises cells (e.g., tumor cells) expressing EGFRvIII. The method may include administering antibody or its Fab as described herein to the subject in need. Antibodies or Fab (ADC) conjugated to the treatment portion are particularly contemplated in the therapeutic method.

[0187] The present disclosure further relates to the use of the antibodies or antigen-binding fragments thereof described herein in the treatment of cancer.

[0188] The present disclosure further relates to the use of the antibodies or antigen-binding fragments thereof described herein in the preparation of a medicament for treating cancer.

[0189] According to the present disclosure, the antibodies or antigen-binding fragments thereof can be used in combination with chemotherapeutic agents.

[0190] According to the present disclosure, a subject in need thereof suffers from or is suspected of suffering from glioblastoma multiforme.

[0191] According further to the present disclosure, a subject in need thereof has or is suspected of having cancer.

[0192] Other aspects of the disclosure relate to methods of detecting EGFRvIII. The methods may include contacting a sample containing or suspected of containing EGFRvIII with an antibody or antigen-binding fragment described herein.

[0193] Another aspect of the present disclosure relates to a method for preparing an antibody or antigen-binding fragment thereof of the present disclosure, comprising culturing a cell comprising a nucleic acid or vector encoding the antibody or antigen-binding fragment, thereby producing the antibody or antigen-binding fragment thereof. The antibody or antigen-binding fragment thereof can then be isolated and / or purified.

[0194] The method can further comprise conjugating the antibody or antigen-binding fragment thereof to a cargo molecule (eg, a therapeutic moiety).

[0195] Other scopes, applicability, and advantages of the present disclosure will be apparent from the non-limiting detailed description given below.It should be understood, however, that the detailed description, while indicating exemplary embodiments of the present disclosure, is given by way of example only with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0196] Figures 1A-1K represent histograms obtained using flow cytometry for supernatants of selected hybridomas for the U87MG cell line overexpressing wild-type human EGFR (U87WT) or EGFRvIII (U87vIII) as indicated.

[0197] Figures 2A-2D represent dose-response binding curves obtained from flow cytometric data of purified monoclonal antibodies against U87MG glioblastoma cell lines overexpressing EGFR vIII (U87MG EGFR vIII) or wild-type EGFR (U87MG EGFR wt) as indicated.

[0198] Figure 3 is an alignment between the amino acid sequences of the extracellular domains of wild-type human EGFR and EGFRvIII. Identical amino acids are underlined.

[0199] Figure 4A and 4B The results of the binding performance of anti-hEGFRvIII mAbs to various fragments of EGFRvIII displayed on yeast cells are shown (+++ represents 95% + / - 5% positive yeast cells, characterized by positive antibody binding with high affinity; ++ represents 70% + / - 20% positive yeast cells, characterized by positive antibody binding with medium affinity; + represents 30% + / - 20% positive yeast cells, characterized by positive antibody binding with low affinity; (+) represents 5-9% positive yeast cells, characterized by positive antibody binding with very low affinity; - / + represents less than 5% positive yeast cells, characterized by unclear antibody binding, - represents 0% positive yeast cells, characterized by no binding; nt = not tested.

[0200] Figure 5A is an alignment between the amino acid sequences of the heavy chain variable regions of 11B1 (SEQ ID NO: 77), 11C8 (SEQ ID NO: 92), and 11H3 (SEQ ID NO: 102), where "*" means that the residues in that column are identical in all sequences in the alignment, and ":" means that conservative substitutions have been observed (Sievers F. et al., Molecular Systems Biology, Oct 11, 2011, 7:539).

[0201] Figure 5B is an alignment between the amino acid sequences of the light chain variable regions of 4B3 (SEQ ID NO:27) and 5D8 (SEQ ID NO:47), where "*" means that the residues in that column are identical in all sequences in the alignment, ":" means that conservative substitutions have been observed, and "." means that semi-conservative substitutions have been observed.

[0202] Figure 5C is an alignment between the amino acid sequences of the heavy chain variable regions of 4B3 (SEQ ID NO:32) and 5D8 (SEQ ID NO:52).

[0203] Figure 5D is an alignment between the amino acid sequences of the light chain variable regions of 4B3 (SEQ ID: 27), 5D8 (SEQ ID: 47), and 9C9 (SEQ ID: 57), where "*" means that the residues in that column are identical in all sequences in the alignment, ":" means that conservative substitutions have been observed, and "." means that semi-conservative substitutions have been observed.

[0204] Figure 6A-F shows the results of the effect of anti-EGFRvIII antibodies as DM1-conjugates (A, B, and C) or MMAE-conjugates (D, E, and F) on cell viability in glioblastoma cells expressing wild-type EGFR (U87 wt) or EGFRvIII (U87EGFRvIII or DKMG EGFRvIII) as indicated.

[0205] Figure 7 The graph shows tumor growth curves in mice bearing U87MG EGFRvIII tumors treated twice (day 0 and day 4) with 5 mg / kg of selected ADCs based on DAR=3. Tumor volumes (mm 3 ). Each data point represents the mean ± SEM (n = 8).

[0206] FIG8A is a graph showing tumor growth inhibition in mice bearing U87MG EGFRvIII tumors treated twice (day 0 and day 4) with 3 mg / kg of selected DM1-ADCs based on DAR=3. Tumor volume (mm 3 ). Each data point represents the mean ± SEM (n = 12).

[0207] FIG8B is a graph showing tumor growth inhibition in mice bearing U87MG EGFRvIII tumors treated twice (day 0 and day 4) with 3 mg / kg of selected MMAE-ADCs based on DAR=3. Tumor volume (mm 3 ). Each data point represents the mean ± SEM (n = 12).

[0208] Figure 9: Schematic diagram illustrating the synthetic assembly and sequence of the 5G6 or 4E11 antigen binding domains in synthetic chimeric antigen receptor (CAR) constructs.

[0209] Fig.10:The figure of the in vitro function of EGFRvIIICAR-T construct is illustrated.The Jurkat cells electroporated with various CAR constructs (EGFRvIII5G6 CAR, EGFRvIII 4E11 CAR or the specific FMC63CAR of control CD19-) are exposed to the target cells 24 hours with (U87vIII) or without (U87, OVCAR3 and A20) EGFRvIII target expression.The surface expression of CD69 on Jurkat cells (CD45 positive) is quantified by flow cytometry to measure the cell activation level.

[0210] Fig.11 :This figure illustrates the inhibition of target cell growth in the T-cell / target cell co-culture of CAR transduction. Human primary peripheral blood-derived T cells are transduced with EGFRvIII-specific CAR lentivirus (4E11-CAR-T or 5G6-CAR-T), CD19-specific CAR lentivirus (FMC63), or similarly treated (simulated) in the absence of lentivirus, and grown in culture for several days. CAR-transduced or non-transduced T cells are then placed in co-culture with target cells expressing EGFRvIII antigen, which express nuclear-localized mKate2-fluorescent protein (U87vIII). In vivo fluorescence microscopy (Incucyte TM , Sartorius). The graph depicts relative target cell growth over 6 days as measured by automated counting of mKate2+ cells.

[0211] Figure 12: This figure illustrates tumor growth (left) and survival (right) of tumor-bearing NOD / SCID / IL-2Rγ-null (NSG) mice. NSG mice (Jackson Laboratory, Barr Harbor, ME) were injected subcutaneously with 1x10 6 U87vIII human glioblastoma cells. On day 7 after tumor cell injection, mice were administered primary human T cells transduced with EGFRvIII-4E11 CAR or left untreated. Tumor growth was then monitored by caliper measurement ( Fig. 12A The tumor size (length and width) was measured using a digital vernier caliper. The tumor volume was calculated using the following formula: tumor volume = (0.4)(ab 2 ), where a = large diameter and b = small diameter. Survival was defined as reaching a humane endpoint (defined as a tumor volume greater than 2000 mm) with and without CAR-T therapy. 3 ) time( Fig. 12B ).

[0212] Figure 13: Schematic diagram illustrating potential bispecific T-cell engagers and bispecific killer cell engagers.

[0213] Fig.14 :This figure illustrates the EGFRvIII specific construct screening bispecific T cell adapter activity with the 4E11-scFV sequence that contains the scFV that is connected with OKT3 people CD3-specific.The supernatant of human embryonic kidney (HEK293) cell from transient expression 4E11-OKT3 bispecific adapter construct is transferred in the hole containing Jurkat cell and the antigen expression target cell (U87vIII) of different doses.By using human CD69-specific antibody staining and flow cytometry to check CD69 expression level, measure the activation of target induction under having or not having bispecific T-cell adapter.Here shows the fold change of CD69 expression when using and not using bispecific T cell adapter in target cell of different doses.

[0214] Fig.15 :This figure illustrates the inhibition of target cell growth in T-cell / target cell co-culture in the presence of a bispecific T cell adapter construct containing a 4E11-scFV sequence connected to the specific scFV of OKT3 people CD3-.For comparison, a control bispecific T cell adapter consisting of human CD19-specific scFv connected to OKT3 is shown. The supernatant from human embryonic kidney cells of transiently expressing 4E11-OKT3 bispecific T cell adapter construct or CD19-OKT3 bispecific T cell adapter construct is transferred to a well containing primary blood-derived T cells and target cells expressing EGFRvIII antigen, which express nuclear localized mKate2-fluorescent protein (U87vIII). Then using in vivo fluorescence microscopy (Incucyte TM , Sartorius, USA). The graph depicts relative target cell growth over 72 hours as measured by automated counting of mKate2+ cells. DETAILED DESCRIPTION

[0215] The term "EGFRvIII" as used herein means epidermal growth factor receptor variant III. The terms "EGFRvIII" and "vIII" are used interchangeably.

[0216] As used herein, the term "EGFR" refers to human epidermal growth factor receptor. The terms "wt EGFR", "WT EGFR", "EGFR WT" or "EGFR wt" are used interchangeably to refer to wild-type EGFR.

[0217] The use of the terms “a” and “an” and “the” and similar referents in the context of describing the disclosure (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0218] Unless otherwise stated or obvious from the context, the term "or" as used herein is to be construed as inclusive and encompasses "or" as well as "and."

[0219] The term "and / or" as used herein shall be deemed as a specific disclosure of each particular feature or component with or without the other features or components.

[0220] The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (ie, meaning "including, but not limited to") unless otherwise noted. The term "consisting of" is to be construed as closed.

[0221] As used herein, the term "native" with respect to a protein such as EGFRvIII or EGFR refers to the native conformation of the protein, and includes a correctly folded and / or functional protein.

[0222] As used herein, the term "denatured" with respect to a protein such as EGFRvIII or EGFR refers to a protein that has lost its native conformation and may result in, for example, loss of tertiary and secondary structure.

[0223] The expression "a peptide comprising or consisting of an EGFRvIII fragment" used herein means that the peptide may comprise a portion other than the EGFRvIII fragment, or it consists of an EGFRvIII fragment.

[0224] As used herein, the term "binding to an epitope comprising an amino acid residue" means that the amino acid residue is a part of the epitope or it is necessary for the binding of the antibody.

[0225] As used herein, the term "failure to bind" a peptide or protein means that the antibody or antigen-binding fragment a) does not significantly bind to the peptide or protein when recombinantly expressed or expressed in a cell, b) no detectable binding is observed, c) has binding properties similar to a negative control antibody, d) does not specifically bind, or e) binds at a value of 0% to 15% as determined by flow cytometry experiments performed in Example 10.

[0226] The term "autologous" as used herein refers to material derived from the same individual.

[0227] The term "antigen binding domain" as used herein refers to the domain of an antibody or antigen binding fragment that allows specific binding to an antigen.

[0228] The term "antibody" as used herein encompasses monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies, human antibodies, single domain antibodies (such as VHH, VH, VL, nanobodies or any camelid or llama single domain antibodies), multispecific antibodies (e.g., bispecific antibodies), etc. The term "antibody" encompasses molecules having forms similar to those found in nature (e.g., human IgG, etc.). The term "antibody" as used herein is also referred to as "immunoglobulin" (Ig) in the art, meaning a protein constructed from paired heavy and light chain polypeptide chains; there are various Ig isotypes, including IgA, IgD, IgE, IgG, and IgM. When the antibody is correctly folded, each chain folds into many different globular domains, which are connected by a more linear polypeptide sequence. For example, an immunoglobulin light chain folds into a variable (VL) and a constant (CL) domain, while a heavy chain folds into a variable (VH) and three constant (CH1, CH2, CH3) domains. The interaction of the heavy and light chain variable domains (VH and VL) results in the formation of an antigen binding region (Fv). Each domain has a well-defined structure familiar to those skilled in the art.

[0229] Typically, antibodies are made up of a pairing of two light chains and two heavy chains. There are different antibody isotypes, including IgA, IgD, IgE, IgG, and IgM. Human IgG is further divided into four different subgroups, namely: IgG1, IgG2, IgG3, and IgG4. Therapeutic antibodies are usually developed as IgG1 or IgG2.

[0230] In one exemplary embodiment, the antibody or antigen-binding fragment of the present disclosure may comprise, for example, a human IgG1 constant region or a fragment thereof. In another exemplary embodiment, the antibody or antigen-binding fragment of the present disclosure may comprise, for example, a human IgG2 constant region or a fragment thereof. Constant regions of other antibody subtypes are also contemplated.

[0231] The light chain and heavy chain of the human antibody IgG isotype each contain a variable region, which has three hypervariable regions called complementarity determining regions (CDRs). Light chain CDRs are identified herein as CDRL1 or L1, CDRL2 or L2 and CDRL3 or L3. Heavy chain CDRs are identified herein as CDRH1 or H1, CDRH2 or H2 and CDRH3 or H3. The complementarity determining regions are flanked by framework regions (FRs) in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The light and heavy chain variable regions are responsible for binding to the target antigen, and therefore can show significant sequence diversity between antibodies. The constant region shows less sequence diversity and is responsible for binding to many natural proteins to trigger important biochemical events. The variable region of an antibody contains the antigen binding determinant of the molecule, and therefore determines the specificity of the antibody to its target antigen. Most of the sequence variability occurs in the CDRs, which combine to form an antigen binding site and contribute to the binding and recognition of the antigenic determinant. The framework region may play a role in the proper positioning and alignment of the three dimensions of the CDR to achieve optimal antigen binding. The specificity and affinity of an antibody to its antigen depends on the structure of the hypervariable region, as well as the size, shape, and chemical properties of the surface on which they are presented to the antigen. There are a variety of schemes that can be used to identify regions of high variability, and the two most common schemes are the schemes of Kabat and Chothia and Lesk. Kabat et al. (1991) defined "complementarity determining regions" (CDRs) based on the sequence variability of the antigen-binding regions of the VH and VL domains. Chothia and Lesk (1987) defined "hypervariable loops" (H or L) based on the position of the structural loop regions in the VH and VL domains. These separate schemes define adjacent or overlapping CDR and hypervariable loop regions, and those skilled in the art of antibodies often use the terms "CDR" and "hypervariable loops" interchangeably, and they can be used as such in this article. CDR / loops are identified herein according to the Kabat scheme.

[0232] Recombinant DNA technology now allows the design of various antibody formats, such as single-chain antibodies (e.g., single domains), diabodies, minibodies, nanobodies, etc., which are encompassed by the present disclosure.

[0233] "Antigen binding fragments" referred to herein may include any suitable antigen binding fragments known in the art. Antigen binding fragments may be naturally occurring fragments, or may be obtained by manipulating naturally occurring antibodies or by using recombinant methods. For example, antibody fragments may include, but are not limited to: Fv, single-chain Fv (scFv; a molecule consisting of VL and VH connected by a peptide linker), Fab, F(ab')2, single domain antibodies (sdAb; a fragment consisting of a single VL or VH), and multivalent presentations of any of these. Antibody fragments (such as those just described) may require linker sequences, disulfide bonds, or other types of covalent bonds to connect different parts of the fragment; those skilled in the art will be familiar with the requirements of different types of fragments and various schemes and various schemes for constructing them.

[0234] Antigen-binding fragments of the present disclosure encompass molecules having an antigen-binding site comprising amino acid residues (eg, one or more CDRs) that confer specific binding to an antigen.

[0235] Thus, exemplary embodiments of antigen-binding fragments of the present disclosure include, but are not limited to: (i) Fab fragments, i.e., L 、V H , C L and C H1 (ii) a F(ab')2 fragment, i.e. a bivalent fragment comprising two Fab fragments connected by a disulfide bond in the hinge region; (iii) a fragment consisting of V H and C H1 (iv) a single-arm V L and V H (v) dAb fragments (Ward et al., (1989) Nature 341:544-546), which are composed of V H domain composition; and (vi) isolated complementarity determining regions (CDRs), e.g., V H CDR3.

[0236] Specific embodiments of antigen binding fragments may include, for example, scFv, Fab, Fab', or (Fab')2.

[0237] The term "humanized antibody" encompasses fully humanized antibodies (i.e., the framework is 100% humanized) and partially humanized antibodies (e.g., at least one variable region contains one or more amino acids from a human antibody, and the other amino acids are amino acids from a non-human parent antibody). Typically, a "humanized antibody" contains a CDR of a non-human parent antibody (e.g., mouse, rat, rabbit, non-human primate, etc.) and a framework identical to the framework of a natural human antibody or a human antibody consensus sequence. In this case, those "humanized antibodies" are characterized as fully humanized. A "humanized antibody" may also contain one or more amino acid substitutions that have no corresponding relationship with those of a human antibody or a human antibody consensus sequence. Such substitutions include, for example, back mutations (e.g., reintroduction of non-human amino acids) that can retain antibody characteristics (e.g., affinity, specificity, etc.). Such substitutions are typically in the framework region. A "humanized antibody" also typically comprises a constant region (Fc), which is typically a constant region of a human antibody. Typically, the constant region of a "humanized antibody" is identical to that of a human antibody. Humanized antibodies can be obtained by CDR grafting (Tsurushita et al., 2005; Jones et al., 1986; Tempest et al., 1991; Riechmann et al., 1988; Queen et al., 1989). Such antibodies are considered fully humanized.

[0238] The term "chimeric antibody" refers to an antibody having a constant region from an origin different from that of the parent antibody. The term "chimeric antibody" encompasses antibodies having a human constant region. Typically, a "chimeric antibody" is composed of a variable region derived from a mouse antibody and a human constant region.

[0239] The term "hybrid antibody" refers to an antibody that comprises one of its heavy or light chain variable regions (its heavy chain or light chain) from a certain type of antibody (e.g., humanized antibody), while the other of the heavy or light chain variable regions (heavy chain or light chain) is from another type (e.g., murine, chimeric).

[0240] The antibodies and / or antigen-binding fragments of the present disclosure can be derived from, for example, mice, rats, or any other mammals, or from other sources, such as by recombinant DNA technology. The antibodies or antigen-binding fragments of the present disclosure can include, for example, synthetic antibodies, non-naturally occurring antibodies, antibodies obtained after immunization of non-human mammals, etc.

[0241] The antibodies or antigen-binding fragments thereof of the present disclosure may be isolated and / or substantially purified.

[0242] Variant Antigen Binding Agents

[0243] The present disclosure also encompasses variants of the antigen binding agents described herein.

[0244] More specifically, the disclosure encompasses variants of antibodies or antigen binding fragments, CARs and BiTEs described herein. Variants (e.g., antibodies or antigen binding fragments, CARs, BiTEs, etc.) include those with variations in their amino acid sequences, such as in one or more CDRs, in one or more framework regions and / or in constant regions. Variants (e.g., antibodies or antigen binding fragments, CARs, BiTEs, etc.) included in the disclosure are those with, for example, similar or improved binding affinity compared to the original antibodies or antigen binding fragments.

[0245] Variants encompassed by the present disclosure are those that may contain insertions, deletions or amino acid substitutions (conservative or non-conservative). These variants may remove at least one amino acid residue in their amino acid sequence and insert a different residue in its place.

[0246] More specifically, variants encompassed by the present disclosure include those having a light chain variable region and / or a heavy chain variable region that has at least 80% sequence identity with the light chain variable region and / or the heavy chain variable region of an antibody or antigen-binding variant disclosed herein. The CDRs of the variant antibody may be identical to the CDRs of the antibody or antigen-binding fragment disclosed herein.

[0247] The present disclosure also encompasses variants having CDR amino acid residues identical to those of the antibodies or antigen-binding fragments disclosed herein and having framework regions at least 80% identical in sequence to the framework regions of the antibodies or antigen-binding fragments disclosed herein.

[0248] Conservative substitutions can be achieved by replacing an amino acid residue from one of the following groups (Groups 1 to 6) (amino acid residues in CDR, variable chain, framework region or constant region, etc.) with another amino acid from the same group.

[0249] Other exemplary embodiments of conservative substitutions are shown in the table below.

[0250] (Group 1) Hydrophobic: norleucine, methionine (Met), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile)

[0251] (Group 2) Neutral hydrophilic: Cysteine ​​(Cys), Serine (Ser), Threonine (Thr)

[0252] (Group 3) Acidic: Aspartic acid (Asp), Glutamic acid (Glu)

[0253] (Group 4) Basic: Asparagine (Asn), Glutamine (Gln), Histidine (His), Lysine (Lys), Arginine (Arg)

[0254] (Group 5) Residues that affect chain orientation: glycine (Gly), proline (Pro); and

[0255] (Group 6) Aromatic: Tryptophan (Trp), Tyrosine (Tyr), Phenylalanine (Phe)

[0256] Non-conservative substitutions will entail exchanging a member of one of these groups for another.

[0257] Original residue Exemplary substitutions Conservative substitution Ala(A) Val,Leu,Ile Val Arg(R) Lys,Gln,Asn Lys Asn(N) Gln,His,Lys,Arg,Asp Gln Asp(D) Glu,Asn Glu Cys(C) Ser,Ala Ser Gln(Q) Asn;Glu Asn Glu(E) Asp,Gln Asp Gly(G) Ala Ala His(H) Asn,Gln,Lys,Arg, Arg Ile(I) Leu,Val,Met,Ala,Phe,Norleucine Leu Leu(L) Norleucine,Ile,Val,Met,Ala,Phe Ile Lys(K) Arg,Gln,Asn Arg Met(M) Leu, Phe, Ile Leu Phe(F) Leu,Val,Ile,Ala,Tyr Tyr Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr,Phe Tyr Tyr(Y) Trp,Phe,Thr,Ser Phe Val(V) Ile,Leu,Met,Phe,Ala,Norleucine Leu

[0258] The percent identity is an indication of the amino acids that are identical and that may occupy the same or similar positions compared to the original peptide. The percent similarity will be an indication of the identical amino acids at the same or similar positions compared to the original peptide and the amino acids that have been replaced by conservative amino acid substitutions.

[0259] Generally, the degree of similarity and identity between variable chains has been determined herein using the Blast2 sequence program (Tatiana A. Tatusova, Thomas L. Madden (1999), "Blast 2 sequences-a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174: 247-250), using default settings, i.e., the blastp program, the BLOSUM62 matrix (open gap 11 and extension gap penalty 1; gapxdropoff 50, expectation 10.0, word length 3) and activated filters.

[0260] "Substantially identical" sequences may contain one or more conservative amino acid mutations, or amino acid deletions that allow maintenance of biological functional activity. It is known in the art that one or more conservative amino acid mutations to a reference sequence may produce variant peptides that have no substantial changes in physiological, chemical, physicochemical or functional properties compared to the reference sequence; in such cases, the reference sequence and the variant sequence will be considered as "substantially identical" polypeptides.

[0261] Thus, variants of the present disclosure include those that may have at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the original sequence or a portion of the original sequence.

[0262] Nucleic acids, vectors and cells

[0263] Antibodies are typically produced in cells that allow for the expression of light and heavy chains expressed from one or more vectors that contain nucleic acid sequences encoding the light and / or heavy chains.

[0264] The present disclosure thus encompasses nucleic acids capable of encoding any of the CDRs, light chain variable regions, heavy chain variable regions, light chains, heavy chains described herein.

[0265] The term "nucleic acid" as used herein refers to RNA, DNA, cDNA and the like.

[0266] Due to the inherent degeneracy of the genetic code, other nucleic acid sequences encoding the same amino acid sequence can be generated and used to express antibodies or antigen-binding fragments thereof of the present disclosure. Nucleotide sequences can be engineered using methods generally known in the art to alter the nucleotide sequence for a variety of purposes, including, but not limited to, modification of cloning, processing and / or expression of gene products. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to engineer nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis can be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preferences, create splice variants, and the like.

[0267] In another aspect, the disclosure relates to a vector comprising a nucleic acid described herein.

[0268] According to the present disclosure, the vector may be an expression vector.

[0269] Expression vectors typically contain elements that control the transcription and translation of the inserted coding sequence in a particular host. These elements may include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3' untranslated regions. Methods well known to those skilled in the art can be used to construct such expression vectors. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.

[0270] The light chain variable region and heavy chain variable region of the antibody or antigen-binding fragment thereof can be encoded by the same nucleic acid molecule (eg, the same vector) or by separate molecules (eg, separate vectors).

[0271] Thus, the present disclosure provides a collection of vectors, wherein one of the vectors is capable of expressing a light chain or a light chain variable region, and another vector is capable of expressing a heavy chain or a heavy chain variable region.

[0272] Additional aspects of the present disclosure relate to a kit comprising a first bottle containing a nucleic acid or vector encoding a light chain or light chain variable region of an antibody or antigen-binding fragment thereof of the present disclosure, and a second bottle containing a nucleic acid or vector encoding a heavy chain or heavy chain variable region of the antibody or antigen-binding fragment thereof.

[0273] In another aspect, the disclosure relates to isolated cells, which may contain a nucleic acid, vector, antibody or antigen-binding fragment described herein.

[0274] The cell of described separation can be included in the nucleic acid of encoding light chain variable region and the nucleic acid of encoding heavy chain variable region on separate carrier or on the same carrier.The cell of described separation can also be included in the nucleic acid of encoding light chain and the nucleic acid of encoding heavy chain on separate carrier or on the same carrier.

[0275] According to the present disclosure, the cell may be capable of expressing, assembling and / or secreting an antibody or antigen-binding fragment thereof.

[0276] Furthermore, according to the present disclosure, the cell may comprise and / or may express the antibody described herein.

[0277] According further to the present disclosure, the cell may comprise a nucleic acid encoding a light chain variable region and a nucleic acid encoding a heavy chain variable region.

[0278] Production of antibodies or antigen-binding fragments in cells

[0279] The antibodies disclosed herein can be prepared by a variety of methods familiar to those skilled in the art, including hybridoma methods or recombinant DNA methods.

[0280] Conventional hybridoma technology requires immunizing rodents with antigens, isolating splenocytes and fusing them with myeloma cells lacking HGPRT expression, and selecting hybrid cells by culture medium containing hypoxanthine, aminopterin and thymidine (HAT). Hybridomas are screened to identify hybridomas that produce antibodies specific to a given antigen. Hybridomas are amplified and cloned. The nucleotide sequences of the light chain and heavy chain variable regions are obtained by standard sequencing methods, and expression vectors containing the light chain and heavy chain nucleotide sequences of the antibody are generated.

[0281] For recombinant expression of the antibody, the host cell is transformed with a vector or collection of vectors comprising the nucleic acid sequences for the light and heavy chains of the antibody or antigen-binding fragment thereof (on the same vector or on separate vectors).

[0282] In order to produce recombinant proteins in mammalian systems for a long time, cell lines that stably express proteins can be obtained. For example, using expression vectors that may contain viral replication origins and / or endogenous expression elements and selectable or visible marker genes on the same or separate vectors, nucleotide sequences that can encode any of the immunoglobulin light chains and heavy chains described herein can be transformed into cell lines. The present disclosure is not limited by the vectors or host cells used. In certain embodiments of the present disclosure, nucleotide sequences that can encode any of the immunoglobulin light chains and heavy chains described herein can each be connected to a separate expression vector and each chain is expressed separately. In another embodiment, the light chain and heavy chain that can encode any of the immunoglobulin light chains and heavy chains described herein can be connected to a single expression vector and expressed simultaneously.

[0283] Immunological methods for detecting and measuring polypeptide expression are known in the art. Examples of such techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence activated cell sorting (FACS) or flow cytometry. Those skilled in the art can easily apply these methods to the present disclosure.

[0284] Different host cells (e.g., Chinese Hamster Ovary (CHO), HeLa, MDCK, HEK293, and WI-38) with characteristic mechanisms for specific cellular and post-translational activities are commercially available from the American Type Culture Collection (ATCC) and can be selected to ensure the correct modification and processing of the expressed polypeptide.

[0285] Typically, in CHO cells, NS0 mouse myeloma cells, Antibodies or antigen-binding fragments thereof are produced in human cells.

[0286] The present disclosure relates to methods of preparing antibodies or antigen-binding fragments thereof, comprising expressing the light and heavy chains of the antibodies or antigen-binding fragments of the present disclosure in cultured cells.

[0287] The method may further comprise purifying or isolating the antibody or antigen-binding fragment of the present disclosure.The method may further comprise conjugating the antibody or antigen-binding fragment of the present disclosure to a cargo molecule, such as a therapeutic moiety or a detectable moiety.

[0288] Antibody conjugates

[0289] The antibodies or antigen-binding fragments thereof of the present disclosure can be attached to cargo molecules. Exemplary embodiments of cargo molecules include, but are not limited to, therapeutic moieties, detectable moieties, polypeptides (e.g., peptides, enzymes, growth factors), polynucleotides, liposomes, nanoparticles, nanowires, nanotubes, quantum dots, etc.

[0290] More specifically, the antibodies or antigen-binding fragments thereof of the present disclosure can be conjugated to a therapeutic moiety. The therapeutic moiety is typically attached to the antibody via a linker, which can be cleavable or non-cleavable.

[0291] Included in the list of therapeutic moieties are cytotoxic agents, cytostatic agents, anticancer agents (chemotherapeutic agents), and radiotherapeutic agents (eg, radioisotopes).

[0292] Exemplary embodiments of cytotoxic agents include, but are not limited to, alpha-amanitine, nostoc cyclotide, duocarmazine, duocarmycin, chalicheamicin, deruxtecan, pyrrolobenzodiazepines, (PBD), dolastatin, Pseudomonas endotoxin, ricin, auristatins (e.g., monomethyl auristatin E, monomethyl auristatin F), maytansinoids (e.g., mertansine), pyrrolobenzodiazepines (PBD) and the like.

[0293] Exemplary embodiments of radiotherapeutic agents include, but are not limited to, yttrium-90, scandium-47, rhenium-186, iodine-131, iodine-125, and many other agents recognized by those skilled in the art (e.g., lutetium (e.g., Lu 177 ), bismuth (e.g., Bi 213 ), copper (e.g., Cu 67 ), astatine-211 (211At), actinium 225 (Ac-225), etc.).

[0294] Exemplary embodiments of chemotherapeutic agents include, but are not limited to, 5-fluorouracil, doxorubicin, irinotecan, taxanes, carboplatin, cisplatin, and the like.

[0295] The antibodies or antigen-binding fragments of the disclosure may also be conjugated to a detectable moiety (ie, for detection or diagnostic purposes).

[0296] "Detectable moieties" include agents detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, and / or other physical means. Detectable moieties can be coupled to the antibodies and antigen-binding fragments thereof of the disclosure directly and / or indirectly (e.g., by linkage, such as, but not limited to, DOTA or NHS linkage) using methods well known in the art. A variety of detectable moieties can be used, selected based on the desired sensitivity, ease of conjugation, stability requirements, and available instrumentation. Suitable detectable moieties include, but are not limited to, fluorescent labels, radioactive labels (such as, but not limited to, 125 I.In 111 , Tc 99 ,I 131 , and including positron-emitting isotopes for PET scanners, etc.), nuclear magnetic resonance active labels, luminescent labels, chemiluminescent labels, chromophore labels, enzyme labels (such as, but not limited to horseradish peroxidase, alkaline phosphatase, etc.), quantum dots and / or nanoparticles. The detectable moiety can cause and / or generate a detectable signal, thereby allowing the signal from the detectable moiety to be detected.

[0297] Chimeric Antigen Receptors and Other Immunotherapeutic Agents

[0298] The sequences of the antibodies and antigen-binding fragments thereof of the present disclosure can be used to generate chimeric antigen receptors (CARs), bispecific T-cell engagers (BiTEs), or other immunotherapeutic agents, such as, but not limited to, bispecific killer cell engagers (BiKEs), trispecific killer cell engagers (TriKEs), or any immunotherapeutic compounds.

[0299] The CAR of the present disclosure may comprise, for example, a) an antigen binding domain of an antibody that specifically binds to epidermal growth factor receptor variant III (EGFRvIII), b) an optional spacer, c) a transmembrane domain, d) optionally at least one co-stimulatory domain, and e) at least one intracellular signaling domain.

[0300] The chimeric antigen receptor may also comprise a hinge region or spacer connecting the antigen binding domain and the transmembrane domain. The spacer may allow for better presentation of the antigen binding domain on the cell surface.

[0301] According to the present disclosure, the spacer may be optional. Alternatively, the spacer may comprise, for example, 1 to 200 amino acid residues, typically 10 to 100 amino acid residues, more typically 25 to 50 amino acid residues. The spacer may be derived from a human protein.

[0302] According to the present disclosure, the spacer or hinge region can be, for example, but not limited to, a CD8 hinge (eg, mouse, human CD8) or an IgG hinge (human immunoglobulin hinge) or a combination thereof.

[0303] Exemplary embodiments of transmembrane domains include, for example, but are not limited to, the alpha, beta or CD3zeta chains of the T cell receptor complex, CD28, CD27, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.

[0304] In certain embodiments, the transmembrane domain may include at least, for example, KIRDS2, OX40, CD2, CD27, LFA-1 (CD 11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 160, CD 19, IL2Rβ, IL2Rγ, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB 1. One or more transmembrane regions of CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Touch), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKG2D, and NKG2C.

[0305] A specific embodiment of a transmembrane domain is the transmembrane domain of CD28.

[0306] The co-stimulatory domain can be derived from, for example, but not limited to, CD28, CD27, 4-1BB, OX40, CD7, B7-1 (CD80), B7-2 (CD86), CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1d, ITGAE, CD103, ITGAL, CD11 a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46 and NKG2D or a combination thereof.

[0307] The intracellular signaling domain can be derived from, for example, but not limited to, CD3ζ, CD3γ, CD3δ, CD3ε, common FcRγ (FCERIG), FcRβ (FcεRib), CD79a, CD79b, FcγRlla, DAP10 or DAP12.

[0308] To target the secretory pathway, the chimeric antigen receptor may also comprise a signal peptide, such as the signal peptide of CD28 or any other signal peptide suitable for immune cells. The signal peptide is cleaved (cleavable).

[0309] BiTE, BiKE and TriKE molecules can include an antigen binding domain (e.g., scFv) that specifically binds to EGFRvIII and another domain (scFv) that binds to specific immune cells, including, but not limited to, molecules (e.g., CD3) specific to T cells and NK cell surface molecules (e.g., CD16). These typically include multiple scFvs connected in series by flexible joints.

[0310] Pharmaceutical composition

[0311] The present disclosure also relates to pharmaceutical compositions comprising the antibodies or antigen-binding fragments (conjugated or unconjugated) disclosed herein.

[0312] In addition to the active ingredient, the pharmaceutical composition may contain a pharmaceutically acceptable carrier, including, but not limited to, water, PBS, saline solution, gelatin, oil, alcohol, and other excipients and adjuvants that facilitate processing of the active compound into a pharmaceutically usable preparation. In other cases, such preparations may be sterilized.

[0313] As used herein, "pharmaceutical composition" refers to a therapeutically effective amount of a pharmaceutical agent and a pharmaceutically acceptable diluent, preservative, solubilizer, emulsifier, adjuvant and / or carrier. As used herein, "therapeutically effective amount" means an amount that provides a therapeutic effect for a given condition and administration regimen. Such compositions are liquid or lyophilized or otherwise dried preparations and include diluents of varying buffer content (e.g., Tris-HCl, acetate, phosphate), pH, and ionic strength, additives such as albumin or gelatin to prevent adsorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile salts), solubilizers (e.g., glycerol, polyethylene glycerol), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal, benzyl alcohol, parabens), fillers or tonicity adjusters (e.g., lactose, mannitol), covalent linkage of polymers such as polyethylene glycol to proteins, complexation with metal ions, or incorporation of the material into or on the surface of particulate preparations of polymeric compounds (e.g., polylactic acid, polyglycolic acid, hydrogels, etc.), or on the surface of liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. Such compositions will affect physical state, solubility, stability, in vivo release rate and in vivo clearance rate. Controlled or sustained release compositions include formulations in lipophilic reservoirs (e.g., fatty acids, waxes, oils). The present disclosure also includes microparticle compositions coated with polymers (e.g., poloxamers or poloxamines). Other embodiments of the compositions of the present disclosure include protective coatings, protease inhibitors or penetration enhancers in granular form for various routes of administration, including parenteral, pulmonary, nasal, oral, vaginal, rectal routes. In one embodiment, the pharmaceutical composition is administered parenterally, paracancerally, transmucosally, transdermally, intramuscularly, intravenously, intradermally, subcutaneously, intraperitoneally, intraventricularly, intracranially and intratumorally.

[0314] In addition, "pharmaceutically acceptable carriers" or "pharmaceutical carriers" used herein are known in the art and include, but are not limited to, 0.01-0.1M or 0.05M phosphate buffer or 0.8% saline. In addition, such pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil) and injectable organic esters (e.g., ethyl oleate). Aqueous carriers include water, alcohol / water solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient supplements, electrolyte supplements such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, for example, antimicrobial agents, antioxidants, collating agents, inert gases, and the like.

[0315] For any compound, the therapeutically effective dose can be estimated initially in cell culture assays or in animal models (such as mice, rats, rabbits, dogs, or pigs). Animal models can also be used to determine concentration ranges and routes of administration. Such information can then be used to determine useful doses and routes of administration in humans. These techniques are well known to those skilled in the art, and a therapeutically effective dose indicates the amount of active ingredient that improves a symptom or condition. Therapeutic efficacy and toxicity can be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating and comparing the ED 50 (the dose that is therapeutically effective for 50% of the population) and LD 50 (50% lethal dose to a population) statistics. Any of the above therapeutic compositions may be applied to any subject in need of such therapy, including, but not limited to, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and humans.

[0316]

[00136] The pharmaceutical compositions used in the present disclosure may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal.

[0317] Additional aspects of the disclosure relate to kits that can include one or more vials containing one or more antibodies or antigen-binding fragments or antibody drug conjugates described herein.

[0318] How to use

[0319] Aspects of the present disclosure include administering an antibody or antigen-binding fragment thereof, CAR, BiTE, BiKE, or TriKE molecule to a subject in need thereof.

[0320] Other aspects of the disclosure include administering to a subject in need thereof immune cells engineered to express a CAR, BiTE, BiKE or TriKE molecule.

[0321] The CAR, BiTE, BiKE or TriKE constructs of the present disclosure can be used to retarget engineered immune cells to EGFRvIII-positive tumors.

[0322] The engineered immune cells can be administered to a subject in need thereof.

[0323] According to one aspect of the present disclosure, immune cells are isolated from a subject, engineered to express a CAR, BiTE, BiKE or TriKE construct and re-administered to the subject.

[0324] The antibodies or antigen-binding fragments thereof of the present disclosure can be used to treat cancer in unconjugated form or in a form conjugated to a therapeutic moiety.

[0325] More specifically, the antibodies or antigen-binding fragments thereof of the present disclosure can be used to inhibit the growth of tumor cells expressing EGFRvIII.Antibody drug conjugates and radioimmunoconjugates are particularly contemplated for such purposes.

[0326] The present disclosure more particularly relates to methods of treating a subject having or suspected of having cancer by administering an antibody or antigen-binding fragment thereof or antibody drug conjugate disclosed herein.

[0327] The antibody or antigen-binding fragment thereof or antibody drug conjugate can be administered alone or in combination with other anti-cancer drugs as a pharmaceutical composition.

[0328] The term "subject" as used herein encompasses humans and animals such as non-human primates, cows, rabbits, mice, rats, sheep, goats, horses, birds, etc. The term "subject" specifically encompasses humans.

[0329] Subjects in need who will benefit from treatment include the mankind with the tumor cells expressing EGFRvIII.More specifically, the antibody or its Fab or antibody drug conjugates can be administered to a subject suspected of having glioblastoma multiforme (GBM).Subjects in need also encompass those suffering from or suspected of having cancer, such as those from breast cancer, head and neck cancer or oral cancer.

[0330] For the purposes of this disclosure, the term "treatment" refers to both therapeutic treatment and prophylactic or preventive measures, wherein the goal is to slow down (mitigate) the targeted pathological condition or disorder. Subjects in need of treatment include subjects who already have the disorder as well as subjects who are susceptible to the disorder or subjects in whom the disorder is to be prevented. Specifically, subjects in need include subjects with elevated levels of one or more cancer markers.

[0331] Alternatively, to practice the methods of the present disclosure and as known in the art, the antibodies or antigen-binding fragments of the present disclosure (conjugated or unconjugated) can be used in conjunction with a second molecule (e.g., a second antibody, etc.) that is capable of specifically binding to the antibodies or antigen-binding fragments of the present disclosure and can carry a desired detectable moiety, diagnostic moiety, or therapeutic moiety.

[0332] The antibodies or antigen-binding fragments thereof of the present disclosure can be used in assays or methods involving the detection of EGFRvIII in unconjugated form or in a form conjugated to a detectable moiety.

[0333] Particularly contemplated are methods of treating a subject suffering from a cancer associated with EGFRvIII expression.Such methods may comprise administering an antigen binding agent disclosed herein or a cell expressing such an antigen binding agent.

[0334] In an exemplary embodiment, the method may include administering an antibody-drug conjugate.

[0335] In another exemplary embodiment, the method may include administering a cell expressing a chimeric antigen receptor, a bispecific T-cell engager, a bispecific killer cell engager, or a trispecific killer cell engager.

[0336] Another aspect of the disclosure relates to the method for detecting EGFRvIII, and the method described may include contacting and measuring the combination with antibody described herein or Fab by the cell expressing EGFRvIII or comprising or the suspected sample (biopsy, body fluid such as serum, blood plasma, urine etc.) comprising EGFRvIII. Sample may be derived from and may suffer from cancer (for example, glioblastoma multiforme or cancer) or may be suspected of suffering from the mammal (for example, people) of this type of cancer. Sample may be a tissue sample or the cell culture supernatant obtained from mammal.

[0337] According to the present disclosure, the sample may be a serum sample, a plasma sample, a blood sample, or ascites fluid obtained from a mammal.

[0338] Other scopes, applicability, and advantages of the present disclosure will be apparent from the non-limiting detailed description given below.It should be understood, however, that the detailed description, while indicating exemplary embodiments of the present disclosure, is given by way of example only with reference to the accompanying drawings.

[0339] Example

[0340] Example 1: Generation of EGFRvIII-specific monoclonal antibodies

[0341] Monoclonal antibodies (mAbs) against EGFRvIII were generated by immunizing mice with the extracellular domain of the recombinant protein.

[0342] Immunization

[0343] Mice were bled (pre-immune serum) and injected intraperitoneally and subcutaneously on day 0 in TITERMAX TM 100 μg of recombinant EGFRvIII protein emulsified in adjuvant (Cedarlane Labs, Burlington, ON), and protein in PBS without adjuvant was injected on day 22. Blood was collected in microvette CB 300Z (Sarstedt, Montreal, QC) on day 29, and serum was stored at -20°C until further use.

[0344] ELISA (Serum titer determination)

[0345] By the ELISA for recombinant EGFRvIII protein assessment animal's pre-immune and post-immune serum titer.Unless otherwise noted, all incubations were performed at room temperature.In brief, half-area 96-well plates (Costar#3690) were coated with the immunogen of 5 μg / ml in PBS of 25 μl / well, and incubated overnight at 4 °C. Microplates were washed three times in PBS, and blocked 30 minutes with PBS containing 1% bovine serum albumin (BSA, Sigma catalog number A7030). Remove blocking buffer and add the serial dilutions of 25 μl serum samples.After incubation for 2 hours, the microplates were washed with PBS-TWEEN TM20 0.05% washed 4 times, and added 25 μl alkaline phosphatase conjugated F (ab ') 2 goat anti-mouse IgG (H + L, # 115-056-062, Jackson Immunoresearch, Cedarlane, Burlington, ON) 1 / 5,000 dilution in blocking buffer. After incubation for 1 hour, the microplate was washed 4 times, and 25 μl of 1 mg / ml p-nitrophenyl phosphate (pNPP) substrate (Sigma-Aldrich Canada Co., Oakville, ON) in carbonate buffer pH 9.6 was added and further incubated for 30 minutes. The absorbance was read at 405nm using a SpectraMax 340PC plate reader (Molecular Devices, Sunnyvale, CA). All pre-immune blood draws were negative for recombinant protein, and all post-immune blood draws were very strong (titer greater than 1 / 51200).

[0346] Hybridoma production

[0347] Mice receive the last strengthening of 100 μ g recombinant EGFRvIII protein, and gather their spleen in the crops after 3 to 4 days.All operations are carried out under aseptic condition.Splenocytes are collected in the Dulbecco's culture medium (IMDM, Gibco catalog number (Cat. No.) 31980-030) of Iscoveshi improvement, and use electric fusion scheme to merge with NS0 myeloma cell line.

[0348] Splenocytes and myeloma cells were washed in IMDM, respectively. Cells were washed in Isoosmolar buffer (Eppendorf catalog number 4308070536), and then washed in Cytofusion Medium C (BTX catalog number 47-0001). Myeloma and lymphocytes were mixed together in a 1:1 ratio and fused using the ECM2001 Cell Fusion System (BTX, Harvard Bioscience Inc.) according to the manufacturer's instructions.

[0349] After fusion, cells were plated at 2-4 × 10 5The input myeloma cells were suspended at a concentration of 1000 cells / mL in HAT selection medium (IMDM containing 20% ​​heat-inactivated FBS, penicillin-streptomycin (Sigma catalog number P7539), 1 ng / ml mouse IL-6 (Biolegend catalog number 575706), HAT medium supplement (Sigma catalog number H0262), and L-glutamine (Hy-Clone catalog number SH30034.01)) and incubated at 37°C, 5% CO2. The next day, the hybridoma cells were washed and cultured at 2-5X10 cells / mL. 5 The concentration of input myeloma cells was suspended in semi-solid medium D (StemCell Technologies catalog number 03804) supplemented with 5% heat-inactivated FBS, 1 ng / ml mouse IL-6 and 10 μg / ml FITC-F(ab')2 goat anti-mouse IgG (Fcγ specific, Jackson #115-096-071). The cell mixture was plated in Omnitray culture dishes (Nunc catalog number 242811) and further incubated at 37°C, 5% CO2 for 6-7 days. Then, the cells were cloned using a mammalian cell cloner (ClonepixFL TM , Molecular Devices) were transferred to sterile 96-well plates (Costar #3595) containing 200 μl of IMDM supplemented with 20% heat-inactivated FBS, penicillin-streptomycin, 1 ng / ml mouse IL-6, HT medium supplement (Sigma catalog number H0137) and L-glutamine and incubated at 37°C, 5% CO2 for 2-3 days.

[0350] Use recombinant EGFRvIII or EGFR wild-type protein to screen five thousand (5000) hybridoma supernatants from seven (7) fusion experiments to detect specific binding substances by ELISA.For this purpose, half-area 96-well plates (Costar#3690) are coated with the immunogen of 5 μg / ml in PBS of 25 μl / well, and incubated overnight at 4 ℃.Microplates are washed three times in PBS, and blocked 30 minutes with PBS containing 1% bovine serum albumin (BSA, Sigma catalog number A7030). Remove blocking buffer and add 25 μl hybridoma supernatants.After incubation for 2 hours, the microplates are washed with PBS-TWEEN TM200.05% was washed 4 times and 25 μl of alkaline phosphatase-conjugated F(ab')2 goat anti-mouse IgG (Fc specific, #115-056-071, Jackson Immunoresearch, Cedarlane, Burlington, ON) was added at a 1 / 5,000 dilution in blocking buffer. After incubation for 1 hour, the microplate was washed 4 times and 25 μl of 1 mg / ml p-nitrophenyl phosphate (pNPP) substrate (Sigma-Aldrich Canada Co., Oakville, ON) in carbonate buffer pH 9.6 was added and further incubated at 37°C for 1 hour. The absorbance was read at 405 nm using a SpectraMax 340PC plate reader (Molecular Devices, Sunnyvale, CA).

[0351] ELISA-positive antibodies were selected and further characterized by flow cytometry on U87MG cells overexpressing wt EGFR or EGFRvIII to confirm their specificity. For this purpose, 15-ml supernatant from each positive clone was generated.

[0352] Example 2: Determination of cell surface binding by flow cytometry

[0353] The binding properties of the anti-EGFRvIII monoclonal antibodies selected in Example 1 were evaluated by flow cytometry on the human glioblastoma cell line U87MG overexpressing wild-type EGFR (U87MG-EGFR wt or U87 WT) and U87MG overexpressing EGFRvIII mutation (Δ2-7 deletion mutation of EGFR; U87MG-EGFRvIII or U87vIII).

[0354] In brief, cells overexpressing full-length wt EGFR or EGFRvIII were obtained from the laboratory of W. Cavanee (Ludwig Institute for Cancer Research, University of California at San Diego). Cells were grown in DMEM high glucose medium containing 10% FBS and 400 μg / ml G418. Prior to analysis, cells were plated to a confluence of no more than 80% on the day of analysis, washed in PBS, and harvested by adding cell dissociation buffer (Sigma). After centrifugation, cells were plated at 2x10 6The cells were resuspended in complete medium at a cell density of 10 cells / mL. 50 μL / well of cells were distributed in a polypropylene v-bottom 96-well plate, and an equal volume of hybridoma supernatant was added and incubated for 2 hours. The cells were washed twice by centrifugation and further incubated for 1 hour with FITC-labeled F(ab')2 goat anti-mouse antibody (Fc specific, #115-096-071, Jackson Immunoresearch, Cedarlane, Burlington, ON). The cells were washed and resuspended in a culture medium containing propidium iodide to exclude dead cells from the analysis. The sample was filtered through a 60 μm nylon mesh filter plate (Millipore, Ireland) to remove cell aggregates. According to the manufacturer's instructions, BD FACS Diva TM Flow cytometric analysis of 2,000 live single-cell events gated on forward scatter, side scatter parameters, and propidium iodide dye exclusion was performed using a BD-LSRFortessa flow cytometer (Becton-Dickinson Biosciences, CA, USA) with standard filter sets.

[0355] Cells were stained with negative control anti-GFP 3E6 mAb supernatants (open histograms) or tested hybridoma supernatants (grey histograms). Specific binding was reflected by the increase in the mean fluorescence intensity of antibodies bound to U87 cells expressing EGFRvIII but not wt EGFR.

[0356] Of the 36 positive cell-based binding antibodies derived from seven independent fusion experiments, we selected nine hybridoma supernatants for further investigation and found that their binding was specific to U87MG cells overexpressing EGFRvIII, including 5G6 (Figure 1A), 1A8 (Figure 1B), 4B3 (Figure 1C), 4E11 (Figure 1D), 5D8 (Figure 1E), 9C9 (Figure 1F), 11B1 (Figure 1G), 11C8 (Figure 1H), and 11H3 (Figure 1I). We used 225mAb (ATCC HB-8508), a positive control mAb that binds to both EGFR WT and vIII, as shown in Figure 1J. The 13.1.2 antibody was generated and used as a positive control recombinant mAb that specifically binds to the vIII isoform, as shown in Figure 1K shown.

[0357] Example 3: Evaluation of Binding on Purified Denatured Antigen

[0358] In order to evaluate whether monoclonal antibody combines conformational epitope, ELISA analysis was carried out to natural and denatured recombinant human wild-type EGFR and EGFRvIII protein. 13.1.2 antibodies (Hamblett KJ, et al., 2015, U.S. Patent number 7,736,644) and 225 antibodies (i.e., identifying the mouse mAb of wt EGFR and EGFRvIII) to EGFRvIII mutation specificity were used as controls (Mendelson J et al., 2015, U.S. Patent number 4,943,533, Sato JD et al., 1983).

[0359] 1-2 mg / ml of antigens were incubated in PBS containing 40 mM final concentration of DTT at 95°C for 5 minutes. They were then incubated on ice for 5 minutes and diluted at their final coating concentration for ELISA purposes.

[0360] mAb is purified using HiTrap ProteinG HP 1mL column GE Healthcare catalog number 17-0404-01, and desalted using Zeba- centrifugal desalting column 5mL (Pierce) pre-equilibrated in PBS, and sterilized by filtration through 0.22 μM membrane (Millipore). Nano-drop 2000 (ThermoScientific) is used to determine the final concentration of the antibody solution, using IgG as the sample type. ELISA is performed as described above (serum titer determination) using 25 μl of mAb supernatant (experiment 1) or 1 μg / ml of purified mAb (experiment 2).

[0361] Table 1 shows the ELISA results (n=2) of different mAb clones assessed for recombinant EGFRvIII or wt EGFR under native or denaturing conditions. As expected, 225 antibodies only bind wt EGFR and EGFRvIII under native conditions. 13.1.2 Antibody binds to the EGFRvIII in native and denatured conformation, but does not bind to the EGFR wild type of native or denatured. The monoclonal antibody produced by our immunization scheme binds to the EGFRvIII in native and denatured conformation. It should be noted that several of our anti-EGFRvIII monoclonal antibodies (4B3, 4E11, 5D8 and 9C9) are positive for wt EGFR native antigen, although as demonstrated in Example 2, these do not bind to the U87MG overexpressing wt EGFR.

[0362] Table 1. Binding to native or denatured recombinant proteins as determined by ELISA

[0363]

[0364] ND: Not determined

[0365] Example 4: Internalization assessment of anti-EGFRvIII mAb

[0366] Use wherein anti-mouse Fc second antibody is coupled to the alternative mensuration of pHrodo dyestuff (Thermo Fisher Scientific), about their internalization into the anti-EGFRvIII monoclonal antibody of the ability evaluation purification in the cell expressing EGFRvIII.Due to the fluorescence that these pH-sensitive dyestuffs enhance in the low pH environment of endosome and lysosome, they can be used for specifically detecting the endocytosis in the living cell.

[0367] For this reason, glioblastoma U87-MG cells overexpressing human EGFR WT or vIII mutations are used. Typically, cells are passaged once or twice a week and used for all experiments within 4-6 weeks. U87MG cells are seeded in 96-well plates (Corning3721) in 100 μl culture medium at a density of 12,500 cells / well. The next day, 20nM of mouse first antibody (anti-EGFRvIII monoclonal antibody) and 30nM of chemically conjugated pHrodo Red (Thermofisher Scientific) anti-mouse second antibody are pre-incubated for 30 minutes, and the pHrodo Red is a pH-sensitive dye that hardly fluoresces at neutral pH and emits bright fluorescence in an acidic environment as it is internalized. Cell culture medium is replaced with 50 μl of fresh culture medium, and 50 μl of antibody complexes are added to cells, and its fluorescence is measured after incubation for 24 hours at 37°C, 5% CO2. Incubations without primary antibody (secondary antibody alone) or with an irrelevant primary antibody (control mouse IgG or anti-GFP mAb) were used to assess non-target internalization. 560nm / Em 590nm (5 nm bandwidth) the microplate was read and the data were blank subtracted.

[0368] Table 2 shows the results of the surrogate antibody internalization of anti-EGFRvIII monoclonal antibodies for U87MG cells overexpressing EGFRvIII or wild-type EGFR. The results are expressed as the percentage of relative fluorescence units (RFU) compared with 225 antibody positive controls (wild-type EGFR internalization mAbs that can bind wtEGFR and EGFRvIII antigens) calculated according to Formula I:

[0369] % internalization = RFU test mAb / RFU 225 mAb X 100 (Formula I)

[0370] The selected mAb panel showed significant internalization when incubated with U87MG cells overexpressing EGFRvIII, but no internalization in cells overexpressing wt EGFR.

[0371]

[0372] a This mAb was evaluated at 1 nM final concentration.

[0373] Anti-EGFRvIII monoclonal antibodies that showed internalization potency in cells overexpressing EGFRvIII, but not in EGFR-WT, in the second conjugate pHrodo-based internalization screen were selected for further analysis.

[0374] Example 5: Functional Characterization of Antibody Drug Conjugate (ADC) Potential

[0375] Use wherein by non-cleavable joint by anti-mouse Fc second antibody coupled to DM1 maytansine drug alternative assay, about its ability to cause growth inhibition in cells expressing EGFRvIII evaluated purified anti-EGFRvIII monoclonal antibody.Once internalized, the joint catabolism in lysosome will release active DM1 drug, which destroys the stability of microtubules and causes growth inhibition.Use the U87MG glioblastoma cell line overexpressing wt EGFR or EGFRvIII.Usually, cells are passaged once or twice per week, and used for all experiments in 4-6 weeks.

[0376] U87MG cells (EGFR WT or EGFRvIII) were seeded in 96-well plates (Corning 3917) at 2,000 cells / 100 μL / well the day before. 1 nM of mouse primary antibody (anti-EGFRvIII monoclonal antibody) was preincubated for 30 minutes with 1.5 nM of anti-mouse secondary antibody chemically conjugated to maytansine (DM1), a microtubule inhibitor that requires internalization to cause cell death. The antibody complexes were then added to the designated cells and their effects on cell viability were measured after 5 days of incubation at 37°C. Incubations without primary antibody (secondary antibody alone) or with irrelevant primary antibody (control mouse IgG or 3E6 anti-GFP mAb) were used to evaluate non-target directed cytotoxicity. Using CellTiterGlo TMCell viability was determined based on the quantification of ATP present in each well (which indicates the presence of metabolically active cells) using the ELISA kit (Promega, Madison). Signal output was measured on a luminescence plate reader (Envision, PerkinElmer) set at 0.1 second integration time. Integration time was adjusted to minimize signal saturation at high ATP concentrations.

[0377] Data expressed as relative luminescence units (RLU) were normalized to mouse IgG control wells and expressed as percent survival compared to mouse IgG, which was calculated according to Formula II:

[0378] % survival = RLU mAb / RLU mouse IgG x 100 (Formula II)

[0379] mAbs that showed high potency in cells overexpressing EGFRvIII, but not in EGFR-WT, in the cytotoxicity screen based on the second conjugate DM1 were selected.

[0380] Table 3 shows the result of the ADC replacement screening assay of anti-EGFRvIII monoclonal antibody for the U87MG cell of overexpressing EGFRvIII or wt EGFR.Results are expressed as the survival percentage relative to non-specific mouse IgG control (being set as 100%).The antibody of test is confirmed to cause the cell of overexpressing EGFRvIII to significantly reduce (>15%) with respect to the survival rate of the cell of overexpressing EGFR wild type.

[0381] As expected, the 225 antibody positive control caused cytotoxicity in both cell lines.

[0382]

[0383]

[0384] The degree of cytotoxicity associated with these antibodies selected from the 36 mouse primary mAbs tested confirmed that they exhibited a high degree of internalization and appropriate intracellular trafficking pathways to achieve activation of DM1 or MMAE drugs, making them suitable for ADC development. Therefore, mAbs that exhibited cytotoxicity in the alternative ADC assay were selected for direct conjugation to DM1 or MMAE.

[0385] Selected hybridomas were recloned by limiting dilution to ensure their monoclonality.

[0386] Example 6: DM1 conjugation and ADC testing of mouse monoclonal mAbs

[0387] The anti-EGFRvIII monoclonal antibody purified in Example 3 was conjugated to trans-4-[maleimidomethyl] cyclohexane-1-carboxylic acid succinimidyl ester (SMCC) linked to N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine (DM1) through a lysine residue, or conjugated to a cathepsin-cleavable linker (valine-citrulline) monomethyl auristatin E (MMAE) through a reduced interchain cysteine ​​residue. Product purity and drug: antibody ratio were determined by size exclusion chromatography (SEC) based on UPLC.

[0388] For conjugation, purified anti-EGFRvIII monoclonal antibodies (5G6 1A8, 4B3, 4E11, 5D8, 9C9, 11B1, 11C8, 11H3; Example 3) were buffer exchanged into conjugation buffer (100mM sodium phosphate, 20mM NaCl, 2mM EDTA pH 7.2) using a pre-equilibrated centrifugal desalting column. The concentration of each monoclonal antibody was adjusted to 2mg / mL using conjugation buffer, and 200μg of each monoclonal antibody in total was used for conjugation. A stock solution of SMCC-DM1 was prepared in dimethylacetamide (DMA). SMCC-DM1 from DMA stock solution was added to each monoclonal antibody to reach a SMCC-DM1:mAb molar ratio of 10.0. The solution was thoroughly mixed and incubated at 37°C for 3 hours. The reaction was terminated by passing the reaction mixture through two centrifugal desalting columns equilibrated in conjugation buffer (with 0.02% w / v polysorbate-20).

[0389] ADC is selected for suitable drug-antibody ratio (DAR) scope (usually 3-5 drugs / antibody) and monomer purity (using analytical SEC, >95% monomer content).By integrating the monomer peaks from UPLC-SEC chromatogram at 280nm and 252nm and comparing these with the ratio of the extinction coefficient of unconjugated antibody and free drug at the same wavelength, DAR is determined.Monomer percentage is determined from the total integrated area of ​​monomer, high molecular weight substance and low molecular weight substance observed in the chromatogram.The in vitro growth inhibition efficacy results of the ADCs of these DM-1 conjugations are presented in Table 4.Specifically, about their impact on the viability of the U87MG cells overexpressing EGFR WT or EGFRvIII, the anti-EGFRvIII ADC prepared above was tested.After exposure for 5 days, CellTiterGlo reagent was used to assess cell growth / viability, and a dose-response curve was generated to measure their efficacy (IC) relative to response-variable slope (four parameters) model using log (inhibitor) from GraphPad Prism v6.0 software. 50 ) and potency (% maximal inhibition).

[0390] Compared with the negative control of the unrelated anti-GFP mouse IgG-DM1 conjugate of non-targeting, the anti-EGFRvIII antibodies of all DM1-conjugated tested showed good efficacy (maximum growth inhibition of 76-97%) and strong potency (IC50<0.37-4.1nM) (Table 4) to the U87MG cells expressing EGFRvIII. In addition, this effect is specific to the cells expressing EGFRvIII, because the activity observed in the U87MG cells expressing EGFRwt (IC50 range = 15-30nM) is not significantly different from the activity observed for the unrelated ADC control (IC50~25nM).

[0391] Table 4 provides the ADC test results of mAb anti-EGFRvIII, %Maximum Inhibition = Percentage of Maximum Inhibition.

[0392]

[0393] Example 7: Evaluation of apparent affinity by flow cytometry

[0394] Purified anti-EGFRvIII monoclonal antibodies were evaluated for their binding activity by flow cytometry in dose-dependent binding curves using U87MG glioblastoma cell lines overexpressing either vIII or wt EGFR.

[0395] Prior to analysis, cells were plated so that they did not exceed 80% confluence on the day of analysis. Unless otherwise stated, all media were maintained at 4°C and all incubations were performed on wet ice. Cells were washed in PBS, harvested by adding cell dissociation buffer (Sigma), centrifuged and plated at 2×10 6 The cells were resuspended in complete medium at a cell density of 10 cells / mL. 50 μL / well of cells were distributed in a polypropylene v-bottom 96-well plate, and a series of 1 / 3 dilutions of purified mAb starting from 100 nM were added and incubated for 2 hours. The cells were washed twice by centrifugation and further incubated for 1 hour with FITC-labeled F(ab')2 goat anti-mouse antibody (Fc specific, #115-096-071, Jackson Immunoresearch, Cedarlane, Burlington, ON). The cells were washed and resuspended in a medium containing propidium iodide to exclude dead cells from the analysis. The samples were filtered through a 60 μm nylon mesh filter plate (Millipore, Ireland) to remove cell aggregates. According to the manufacturer's instructions, BD FACSDiva TMFlow cytometric analysis of 2,000 live single-cell events gated on forward scatter, side scatter parameters, and propidium iodide dye exclusion was performed using a BD-LSRFortessa flow cytometer (Becton-Dickinson Biosciences, CA, USA) with standard filter sets.

[0396] Specific detection of antibody binding was calculated as the mean fluorescence intensity of binding to each primary antibody after subtracting the background level of mean fluorescence intensity of binding to the mouse IgG control. Data were analyzed using a single-site specific binding with a Hill slope nonlinear regression curve fit model using GraphPad Prism v 6.0 software to determine the Bmax (maximum specific binding) and Kd for each mAb tested. app (Concentration required to achieve half maximal binding at equilibrium). The model used is based on Formula III:

[0397] Y=Bmax*X h / (Kd h +X h )(Formula III)

[0398] Bmax is the maximum specific binding, expressed in the same units as Y.

[0399] Kd is the concentration of ligand required to achieve half-maximal binding at equilibrium, expressed in the same units as X.

[0400] The variable "h" is the hill slope.

[0401] Fig. 2 shows the result of the flow cytometry experiment of the binding performance of EGFRvIII (Fig. 2 A and 2C) or wt EGFR (Fig. 2 B and 2D) of determining anti-EGFRvIII monoclonal antibody and cell surface expression.225 is the mouse mAb in conjunction with wild-type and vIII mutant EGFR, is used as the positive control of two kinds of cell lines.All anti-EGFRvIII mAbs all demonstrate the strong and comparable combination (Fig. 2 A and 2C) with the cell of overexpressing EGFRvIII variant, and do not have detectable combination (ndb) (Fig. 2 B and 2D) on the cell of overexpressing wild-type EGFR.Except 9C9 (data not shown), all monoclonal antibodies all combine the cell (table 5) of overexpressing EGFR vIII variant with low nM affinity in flow cytometry analysis.

[0402] Table 5: Apparent K of anti-EGFR vIII mAbs determined by flow cytometry analysis D .

[0403]

[0404] ndb: no detectable binding

[0405] Example 8. Epitope binning determined by flow cytometry competition

[0406] To evaluate whether the anti-EGFRvIII monoclonal antibodies bind to the same epitope region, flow cytometry competition experiments were performed on U87MG-vIII cells.

[0407] To this end, purified monoclonal antibodies 4E11 and 13.1.2 were conjugated to AlexaFluor 488 fluorescent dye (Thermofisher, Burlington, ON, Canada) using NHS ester derivatives according to the manufacturer's instructions.

[0408] Fixed concentrations of labeled mAbs (1-10 nM) were incubated with increasing concentrations (or 10-fold concentrations) of our unlabeled monoclonal antibodies to assess whether their binding capacity was impaired in the presence of unlabeled mAbs.

[0409] As can be seen from the results shown in Table 6, the selected anti-EGFRvIII monoclonal antibodies were divided into 2 major epitope regions, classified according to the binding of the 2 prototype antibodies; 1) the bin1 antibody was found to compete with the labeled 13.1.2 including antibodies 5G6, 1A8, 11B1, 11C8 and 11H3, and 2) the bin2 antibody was found to compete with the labeled 4E11 antibody including antibodies 4B3 and 5D8. The affinity of antibody 9C9 was lower and it did not compete with any of the labeled mAbs in this experiment.

[0410] Table 6. Competition assay by flow cytometry

[0411]

[0412] "NC" = no competition; "SC" = self-competition; "C" = competition; "ND" = not determined.

[0413] Example 9. Epitope mapping by yeast surface display

[0414] Use yeast surface display method (Feldhaus MJ et al., 2003Nat Biotechnol.2003 February; 21 (2): 163-70) draw the epitope of our anti-EGFRvIII monoclonal antibody.This technology allows the selected protein or peptide of clone to be expressed and displayed on the cell surface by covalent connection with cell wall.Can explore the antibody binding of the protein / peptide displayed.

[0415] A total of 36 different hEGFRvIII fragments of different sizes ranging from 10 to 414 amino acid residues were cloned into the pPNL6 vector (derived from The Pacific Northwest National Laboratory, USA) to express and display as fusion proteins as Aga2-HA-hEGFRvIII-MYC. The displayed hEGFRvIII fragments were used to identify the minimum fragment required for the binding of each anti-hEGFRvIII mAb.

[0416] By flow cytometry analysis, assessment anti-EGFRvIII mAb and the combination of the fusion protein expressed on the yeast cell surface.In brief, yeast cells are marked with anti-hEGFRvIII mAb and the anti-Myc antibody of chicken, the latter is used to monitor the expression level of fusion protein.After washing step, use respectively to mouse and chicken antibody specific second antibody, by dual-color indirect fluorescent labeling detection combination of the first antibody.

[0417] The anti-hEGFRvIII mAbs bound to the full-length hEGFRvIII protein and small peptides of the same protein, as well as native and heat-denatured yeast-displayed antigen fragments with similar signal intensities, suggesting that the epitopes of these mAbs are contained within a continuous peptide fragment (linear).

[0418] Figure 4A and 4B Results showing the binding properties of anti-hEGFRvIII monoclonal antibodies to various fragments of hEGFRvIII and wtEGFR proteins displayed on yeast cells.

[0419] Table 7 shows the minimum fragment (peptide) required for various anti-hEGFRvIII mAb binding. Four different epitope groups were identified: 1) amino acid 1-18 identified by 11B1, 11C8 (H5) and 11H3 and 13.1.2 control mAbs, 2) amino acid 3-18 identified by 5G6 and 1A8, 3) amino acid 15-37 identified by 4B3, 5D8 and 9C9, and 4) amino acid 19-37 identified by 4E11. Although there is no binding on the cells expressing U87MG-EGFR WT, 4E11 does bind to yeast cells (engineered to express EGFR WT fragments 266-482 and 1-623). Figure 4A and 4B ).

[0420]

[0421] Example 10. Fine epitope display using yeast surface

[0422] In order to further characterize the mAb epi-position in the EGFRvIII 15-37 zone, alanine scanning in this zone was carried out, and the modified fragment was expressed on the yeast surface.Except corresponding to the original Ala19 and the amino acid residue 5 and 8 of Ala22 of EGFRvIII, all amino acids of SEQ ID NO.:6 were mutated into alanine.The resulting peptide was expressed on the yeast surface, and every kind of anti-EGFRvIII mAb was tested to corresponding yeast mutant by flow cytometry.Therefore, the contribution of each amino acid in the monoclonal antibody combination was determined in this mensuration.Table 8 has shown the result that 4B3, 5D8, 9C9 and 4E11 monoclonal antibodies obtain in flow cytometry.The previous result that shows in the result obtained and the table 7 is consistent, and the combination of the mAb in bin 15-37 is subjected to the strong inhibition of an amino acid mutation between position 15 and 19.

[0423] Binding of mAbs 4B3 and 5D8 was abolished by mutation of any of the amino acid residues Arg18, Cys20, Gly21, or Cys35, and was attenuated by mutation of Gly31 to alanine. Binding of mAb 5D8 was attenuated by mutation of Glu26 to alanine. Binding of 9C9 was impaired by mutation of any of Cys16, Glu26, Gly31, Val32, Arg33, Lys34, Cys35, or Lys36, and was attenuated by mutation of Cys20 or Asp30 to alanine. Binding of 4E11 was strongly inhibited by mutation of any of Cys20, Glu26, Asp30, Gly31, Arg33, or Cys35, and was attenuated by mutation of Asp23 or Val32 to alanine. Based on these results, amino acid residues Cys20 and Cys35 appear to be important for the binding of antibodies targeting amino acid residues 15-37 of EGFRvIII.

[0424] Table 8. Flow cytometric evaluation of mAb binding to yeast expressing mutant amino acids within fragment 15-37 of EGFRvIII. Data represent percent binding of mAbs on yeast displaying mutant sequences compared to binding on yeast displaying wild-type sequence, normalized for Myc-tag expression.

[0425]

[0426]

[0427] *Original Ala 19 and Ala 22 without mutation

[0428] Legend Values ​​between 0% and 15%: No binding Values ​​between 16% and 59%: Partial binding Values ​​at or above 60%: Fully bound

[0429] Example 11: Antibody Sequencing

[0430] The sequences of the VH and VL domains of the thirteen anti-EGFRvIII monoclonal antibodies of Examples 2-5 were analyzed.

[0431] In brief, total RNA was extracted from hybridoma clones (Qiagen, RNEasy) and reversely transcribed into cDNA (SuperScriptTM, ThermoFisher Scientific, Waltham, MA, USA). DNA encoding VH and VL domains was PCR amplified (Platinum Taq or equivalent) using a mixture of a degenerate forward primer annealed in FR1 and a single reverse primer annealed in CH1 (Novagen / EMD Millipore catalog number 69831-3). The resulting amplicon was sequenced using the Sanger method on an ABI3730xl instrument or determined using a 2×250 bp read length on an Illumina MiSeq instrument.

[0432] The sequences of VH and VL domains and CDR regions are shown in the sequence table. CDR is usually indicated in bold in light chain or heavy chain variable region amino acid sequence. The sequence analysis of the common binding sequence in the CDR 1-3 region of VH and VL chain is carried out using Kabat numbering scheme (Kabat et al. 1992, Johnson et al. 2004). The results indicate that the CDR regions in the VH and VL regions of 11B1, 11C8 and 11H3 monoclonal antibodies are identical. There is 1 amino acid difference in VH between 11B1 and 11C8 (FR2 region), 1 amino acid difference in VH between 11C8 and 11H3 (FR3 region), and 2 amino acid differences between 11B1 and 11H3 (FR2 and FR3). These antibodies cluster together and recognize the common linear epitope (table 7) in fragment 1-18.

[0433] An alignment between the heavy chain variable regions of 11B1 (SEQ ID NO: 77), 11C8 (SEQ ID NO: 92), and 11H3 (SEQ ID NO: 102) is presented in Figure 5A The consensus sequence obtained from this alignment is provided in SEQ ID NO: 117. The 11B1 (SEQ ID NO: 67), 11C8 (SEQ ID NO: 82) and 11H3 (SEQ ID NO: 97) light chains are identical.

[0434] 4B3 and 5D8 have identical VL CDR, and in FR1, there are 2 amino acid differences, and in FR4, there is 1 amino acid difference.They have similar VH CDR, and in CDRH1, there is an amino acid difference, in CDRH2, there is an amino acid difference, and in each in FR1 and FR3, there is an amino acid difference.As shown in Table 8, they all combine the same epi-position on EGFRvIII.

[0435] An alignment between the 4B3 (SEQ ID NO: 27) and 5D8 (SEQ ID NO: 47) light chain variable regions is provided in Figure 5B. The consensus sequence obtained from the alignment is provided in SEQ ID NO: 115. An alignment between the 4B3 (SEQ ID NO: 32) and 5D8 (SEQ ID NO: 52) heavy chain variable regions is provided in Figure 5C. The consensus sequence obtained from the alignment is provided in SEQ ID NO: 116.

[0436] 9C9 has a VL similar to those of 4B3 and 5D8 (one amino acid difference in CDRH1, and another four amino acid differences in FR3 and FR4) and a unique VH, and binds a unique epitope (Table 8).

[0437] An alignment between the light chain variable regions of 4B3 (SEQ ID NO:27), 5D8 (SEQ ID NO:47), and 9C9 (SEQ ID NO:57) is presented in Figure 5D The consensus sequence obtained from this alignment is provided in SEQ ID NO:118.

[0438] Sequence analysis also revealed that clone 11B1 expressed two light chains; one was predominant (variable region represented by SEQ ID NO:67), and one was a less abundant transcript (SEQ ID NO:72). Similarly, clone 11C8 expressed two light chains; one was predominant (SEQ ID NO:82), and one was a less abundant transcript (SEQ ID NO:87). Clones 11G8 and 11H5 also expressed both transcripts (data not shown).

[0439] Sequence analysis revealed that clones 5G6, 1A8 and 4E11 possess unique VH and VL CDRs.

[0440] Example 12: Recombinant Antibody Production and Purification

[0441] To facilitate large-scale antibody production and consistency across production, the anti-EGFRvIII monoclonal antibodies identified in Example 11 were recombinantly produced in CHO cells.

[0442] The VH and VL regions were cloned into the pTT5 vector as fusions with human IgG1 constant regions (human IgG1 heavy chain and human kappa light chain, respectively) to generate chimeric antibodies.

[0443] The amino acid sequences of the chimeric antibodies are provided in the sequence listing: hFC-5G6, hFC-4E11 and hFC-13.1.2. All light chain sequences contain the signal sequence MVLQTQVFISLLLWISGAYG (SEQ ID NO: 113) at the N-terminus, while the heavy chain sequences contain the signal sequence MDWTWRILFLVAAATGTHA (SEQ ID NO: 114) at the N-terminus.

[0444] By 2mL expression exploration (scout), verify chimeric antibody expression.In short, with the construct (1: 1 ratio) transient transfection CHO cells containing VL and VH; Harvest conditioned medium (CM) at the 7th day, and evaluate chimeric antibody expression level by SDS-PAGE (data not shown).Chimeric antibody expresses well, and by starting small-scale production (50mL) with the same construct ratio transient transfection CHO cells.Harvest conditioned medium (CM) at the 7th day, purification (ProtA) chimeric antibody, quantitative and evaluated by SDS-PAGE.Data show, the CHO cells of transient transfection express all four kinds of chimeric antibodies well.

[0445] In order to confirm that the chimeric antibody of recombinant expression has the behavior similar to the monoclonal antibody expressed by hybridoma, the U87MG of overexpressing EGFRvIII is carried out flow cytometry binding experiment to ensure that EGFRvIII is not damaged in combination. In addition, the glioblastoma cell line of overexpressing EGFRvIII obtained from Cellther Polska (Cat. No. CL 01008-CLTH) is also used to test the combination and ADC activity. These cells are grown in RPMI and 10% fetal bovine serum using standard culture conditions.

[0446] Evaluation of apparent affinity was performed by flow cytometry analysis as described in Example 7, except that a secondary AF488-labeled F(ab')2 donkey anti-human antibody (Fc specific, #709-546-098 Jackson Immunoresearch, Cedarlane, Burlington, ON) was used to detect primary antibody binding.

[0447] In flow cytometry analysis, all chimeric antibodies all combine the glioblastoma cells (table 9) of overexpression EGFRvIII sudden change with specificity and similar affinity.Importantly, all chimeric antibodies (comprising benchmark 13.1.2) can not combine wt EGFR, as seen by lacking cell surface combination on the U87 cell of overexpression wtEGFR.This specificity is different from seen with ABT-806, and ABT-806 has been confirmed to significantly combine U87MG EGFRwt cells (people such as Panousis 2005) and may explain the toxicity caused by wt EGFR expression on normal tissue.

[0448] Table 9. Cell binding on glioblastoma cells expressing EGFRvIII

[0449]

[0450] ndb = no detectable binding

[0451] Example 13: DM1 and MMAE conjugation and ADC testing of recombinantly expressed chimeric mAbs

[0452] The chimeric antibodies produced in Example 12 were conjugated with DM1 or MMAE and tested for glioblastoma cells overexpressing wt EGFR or EGFRvIII proteins as described in Example 6. The conjugation of the reduced interchain cysteine ​​of the antibody and EGFRvIII, or the conjugation of the monomethyl auristatin E (MMAE) drug of the control mAb and the valine-citrulline linker cleavable by the peptide was performed using the following method. Lyophilized MC-vc-PAB-MMAE was dissolved in dimethylacetamide to a final concentration of 10mM. The reserve was divided and stored at -20°C for standby use. Before conjugation, each protein sample must reduce the disulfide bonds accessible on its surface to generate reactive free thiols. The degree of reduction of the disulfide bonds is adjusted by increasing the concentration of the reducing agent to control the level of DAR. In order to achieve the desired specified DAR, a three-point exploration experiment was performed using three different concentrations of TCEP (24, 40 and 55 μM), and the final concentration of the antibody was 10 μM. Reduction was started by adding TCEP from 25X working stock solution to the antibody solution in the following buffer: 100mM sodium phosphate, 20mM NaCl, 2mM EDTA, pH 7.2. The mixture was incubated at 37°C for 3 hours. MC-vc-PAB-MMAE with a 10-fold molar excess (100 μM) was added to the reaction mixture of 20X working stock solution from DMA. The final concentration of the co-solvent in the reactant was 5% v / v. The reactant was incubated at 25°C for 1 hour. During this incubation, 3x 0.5mL7K MWCO Zeba centrifugal columns were pre-equilibrated in standard formulation buffer (20mM succinate, 0.02% w / v polysorbate-20, pH 6.0). After the reaction was complete, polysorbate-20 was added to a final concentration of 0.02% w / v before the reaction mixture was continuously passed through the pre-equilibrated Zeba column. To the eluate was added 1 / 5 volume of 36% trehalose solution (in formulation buffer). The DAR was calculated using absorbance measurements of the conjugate at 248 nm and 280 nm, and the monomer purity of the sample was measured by HPLC-Superdex size exclusion chromatography. The DAR for each of the three TCEP concentrations was plotted against the DAR, and the slope of the linear relationship was used to determine the optimal concentration to achieve the desired DAR. The above procedure was then repeated using these optimal conditions at the specified scale. The final concentration reported for the conjugate was adjusted to compensate for the effect of the attached drug at 280 nm.

[0453] Prior to conjugation, absorbance scans of protein (antibody) samples (2 μL) were measured on a Nanodrop 2000 instrument. The absorbance ratio at 248 / 280 nm was determined and used to calculate the extinction coefficient of the antibody to be conjugated at 252 nm (for MCC-DM1 conjugates) or 248 nm (for val-cit MMAE conjugates). This value will be used to determine the drug: antibody ratio (DAR) of the resulting conjugate.

[0454] The growth inhibitory activity of the drug (DM1- and MMAE-) conjugated antibodies on glioblastoma cell lines (U87 and / or DKMG) expressing EGFRvIII or wt EGFR was tested. Typical results are shown in Figure 6 and the average values ​​are shown in Tables 10 and 11.

[0455] In the U87 cell line expressing EGFRvIII analyzed, compared with the benchmark mAb (hFc13.1.2-DM1, IC50=9.3nM) used, the chimeric anti-EGFRvIII antibody of DM1-conjugation showed good efficacy (maximum growth inhibition of 77-80%) and strong potency (IC50 range 0.9 -3.9nM). In addition, the activity of two test antibodies (especially hFc-4E11-DM1) seems to be very effective for cells expressing EGFRvIII, i.e. 18 times more potent than the efficacy seen on cells expressing EGFRwt, and 25 times more potent than the efficacy seen with DM1-conjugated palivizumab (non-targeted irrelevant control ADC) (Table 10). Similar results were observed in DKMG cells expressing EGFRvIII.

[0456] Table 10 provides the results of DM1-ADC testing of chimeric anti-EGFRvIII antibodies.

[0457] DAR: Drug-Antibody Ratio

[0458] %Maximum Inhibition = percent maximal inhibition.

[0459] Table 10: Results of DM1-ADC testing of chimeric anti-EGFRvIII antibodies

[0460]

[0461] When tested, MMAE-conjugated chimeric anti-EGFRvIII antibodies showed good efficacy (75-95% maximal growth inhibition) and strong potency (IC50 <0.2-0.4 nM) in cell lines overexpressing EGFRvIII compared to benchmark 13.1.2 (IC50 of 1.9-2.5) or a non-targeted, irrelevant chimeric palivizumab conjugate negative control (Table 11).

[0462] Table 11 provides the results of MMAE-ADC testing of chimeric anti-EGFRvIII antibodies.

[0463]

[0464] It is noteworthy that hFc-4E11 and hFc-5G6 of DM1- and MMAE conjugation all show excellent functional activity, because they are 10 times more effective than the corresponding benchmark 13.1.2 antibody conjugates to cells overexpressing EGFRvIII. In all cases, it is confirmed that the activity of cells expressing EGFRvIII is significantly greater than the activity of non-targeted irrelevant ADC controls. In addition, this effect is specific for cells expressing EGFRvIII, because the activity observed in the cells expressing EGFRwt U87MG is not significantly different from the activity observed in the corresponding irrelevant ADC controls.

[0465] Example 14: In vivo tumor growth inhibition by ADC in a U87MG-vIII tumor xenograft mouse model

[0466] To further assess in vivo efficacy, anti-EGFRvIII antibody-drug conjugates (ADCs) were evaluated for their ability to inhibit tumor growth in a xenograft model expressing EGFRvIII.

[0467] The ADCs were tested in a subcutaneous glioblastoma xenograft model (Study No. SP.03: 5 mg / kg ADC and Study No. SP.06: 3 mg / kg ADC).

[0468] In brief, the U87MG cells expressing EGFRvIII were cultivated as described in Example 2. Cells were passaged twice weekly and used in 4-6 weeks after thawing. They were harvested by trypsinization (0.25% trypsin / EDTA, Gibco15090-046), then washed in cold phosphate-buffered saline (PBS), and assessed for viability by the ability to exclude trypan blue dye. All cell colonies grown as xenografts in mice had at least 98% survival rate.

[0469] Six-week-old, 25-g male CD1 Albino mice (Crl:CD1-Foxn1 nu The care and use of animals were in accordance with the guidelines of the Canadian Council on Animal Care (CCAC).

[0470] Under sterile conditions, cells (5×10 cells / injection site) were subcutaneously inoculated on the left flank of nude mice (n=12) anesthetized with isoflurane. 6 cells in a volume of 0.1 mL PBS or HBSS). Allow tumors to grow to 80-100 mm. 3 Animals were then randomized one day prior to administration to ensure that each group contained animals with different tumor sizes.

[0471] Tumor growth was monitored by caliper measurement every three days for 29 days after treatment or until they were euthanized for ethical reasons (humane endpoint): 1) more than 10% body weight loss within 48 hours without weight recovery; 2) tumor volume exceeded 2500 mm 3 ; 3) tumor ulceration; 4) obvious signs of distress, such as immobility and reduced grooming behavior.

[0472] Tumor volume was calculated according to Formula IV:

[0473] Estimated tumor volume [mm 3 ]=π / 6(length [mm])×(width [mm]x(height [mm]))(Formula IV)

[0474] The test articles were prepared in ADC buffer and were shown to contain <5% aggregates (by HPLC-SEC) and <1 EU / mg endotoxin. Mice were injected intravenously (iv) via the tail vein at 5 mg / kg (study SP.03) or 3 mg / kg (study SP.06) of the DAR3-based test article on days 0 and 4 (96 hours apart). A control group was treated with ADC buffer.

[0475] All animals were observed once daily for mortality and signs of disease. Individual body weights and tumor sizes of animals were measured and recorded on the day of treatment and every three days thereafter.

[0476] The tumor volume in each group is shown as mean ± SEM, and is plotted as the function of measuring time after U87MG-EGFRvIII cell inoculation. The group contrast of tumor volume data was carried out using a two-factor analysis of variance (ANOVA) with Tukey's multiple comparison test using GraphPad Prism 7.0 version. The difference between the treatment group and the control group was statistically significant at p < 0.05.

[0477] Figure 7 Results from study SP.03 are represented and show the effect of mouse anti-EGFRvIII ADC on tumor growth (assessed as tumor volume).

[0478] From day 4 to day 20, a statistically significant (p<0.05) effect on tumor growth was observed between the 5 mg / kg anti-EGFRvIII-DM1 treatment group and the ADC buffer treatment group. 3 Compared with the control group, the average tumor volume of the anti-EGFRvIII treated group of mice increased from about 90 to 195-287mm 3 No statistically significant differences in tumor growth inhibition were observed between the different anti-ADCs at 5 mg / kg (Table 12). This significant difference in tumor volume suggests that our mouse anti-EGFRvIII ADC can greatly inhibit tumor growth in the glioblastoma tumor model.

[0479] Body weight was measured as an indicator of potential off-target toxicity. No significant differences in body weight were observed between control animals (ADC buffer group) and ADC-treated animals. Both groups of mice gained approximately 5-10% body weight during the experiment.

[0480] Table 12: Percent reduction in tumor volume compared to the control group (ADC buffer). The percentage reduction in tumor volume (TV) compared to the control was calculated based on the following equation:

[0481] % = (ΔTV 对照- ΔTV 治疗 ) / ΔTV 对照 *100; ΔTV = TV d28 -TV d0

[0482]

[0483] Significant tumor growth inhibition was also observed in animals treated with 2 mg / kg mouse anti-EGFRvIII ADCs, which included 3E6-DM1, 11H3-DM1, 1A8-DM1 in addition to the 5G6-DM1, 4E11-DM1, and benchmark 13.1.2-DM1 groups, with tumor volume reduction ranging from 20-50% compared to the anti-GFP-DM1 irrelevant ADC control (data not shown).

[0484] Figure 8A and Figure 8B represent the results of study SP.06 and show the effect of chimeric anti-EGFRvIIIDM1-ADC or MMAE-ADC on tumor growth (assessed by tumor volume). Tumor growth curves in mice bearing U87MG-EGFRvIII tumors treated twice (day 0 and day 4) with ADC buffer (control) or 3mg / kg ADC, with tumor volume (mm) recorded every three days. 3) for 17 days. For ADC buffer (circle), hFc-5G6-DM1 (diamond), hFc-4E11-DM1 (open circle), benchmark hFc-13.1.2-DM1 (triangle) and negative control Palivizumab-DM1 (square) groups, each data point represents the mean ± SEM. Palivizumab is a humanized mAb against respiratory syncytial virus (RSV).

[0485] Compared with the ADC buffer treatment group, animals treated with palivizumab-DM1 (Fig. 8A) and palivizumab-MMAE (Fig. 8B) all showed a significant reduction in tumor growth (p<0.05), with tumor reductions of 31.0% and 22.8% on the 17th day (Table 13). However, from day 1 to day 17, compared with the buffer treatment group, animals treated with chimeric anti-EGFRvIII ADC showed a more significant (p<0.05) tumor growth reduction, with tumor volume reductions of 37.2% to 60.0% on the 17th day. No statistically significant differences in tumor growth inhibition were observed in different chimeric anti-EGFRvIII ADCs. There were no statistically significant differences (p>0.05) between the corresponding DM1 and MMAE conjugates. Tumors of U87MGvIII xenografts tended to ulcerate during the experiment. Both the control and ADC-treated groups showed ulceration rates exceeding 50%, with the only exception being the hFc-4E11-DM1-treated animals, which exhibited a significantly lower ulceration rate (-16.7%, data not shown).

[0486] Table 13: Percent reduction in tumor volume compared to the control group (ADC buffer) on day 17 after the start of treatment (mean ± SEM). The percentage reduction in tumor volume (TV) compared to the control was calculated based on the following equation:

[0487] % = (ΔTV 对照- ΔTV 治疗 ) / ΔTV 对照 *100; ΔTV = TV d17 -TV d0

[0488]

[0489] In summary, during the study, chimeric anti-EGFRvIII ADC treated animals showed a more significant (p < 0.05) tumor growth reduction compared to the buffer treatment group, and confirmed that the efficacy of the 13.1.2 benchmark (when evaluated as MMAE or DM1 conjugates, respectively) was comparable or more effective. It is worth noting that at day 17, compared to 4E11-DM1 (p < 0.05), the benchmark 13.1.2-DM1 induced significantly lower tumor growth inhibition in the U87MGvIII model.

[0490] Example 15: Chimeric Antigen Receptor T-Cell Function Test

[0491] Various experiments were performed to confirm the activity of the novel EGFRvIII-specific single chain variable fragments in the context of a chimeric antigen receptor (CAR). Briefly, a single-chain variable fragment consisting of heavy and light chains of the 5G6 (SEQ ID NO: 12 and SEQ ID NO: 7, respectively, such as in the scFv of SEQ ID NO: 166) or 4E11 (SEQ ID NO: 42 and SEQ ID NO: 37, respectively, such as provided in the scFv of SEQ ID NO: 167) sequence (connected using a flexible linker sequence and restriction sites (e.g., GTGGGGSGGGGSGGGGSDV (SEQ ID NO: 171), it should be noted that any suitable linker known in the art can be used in the scFv constructs provided herein) is combined with a standard CAR construct. The CAR construct used for testing comprises one of the human CD28-signal peptides, the scFv sequence just mentioned above, the mouse CD8-hinge domain, the human CD28-transmembrane domain, the CD28-intracellular signaling domain, and the CD3-ζ intracellular signaling domain (5G6-CAR SEQ ID NO: 168, 4E11-CAR SEQ ID NO: 169). NO:169).

[0492] The in vitro function of EGFRvIII CAR constructs was tested using novel flow cytometry based on high-throughput screening platforms developed by the applicant; in some cases it is referred to as CAR-J assay. In short, EGFRvIII-specific (just described above) or control (targeting CD19) CAR plasmids were electroporated into Jurkat human CD8+T- cell lines. Then the cells expressing CAR were exposed to various target cell lines (with or without EGFRvIII expression) at different doses and maintained under standard culture conditions. After being incubated with target cells for 24 hours, the cell activation of CAR-T cells was examined by flow cytometry by the surface expression of T- cell activation marker CD69. The CD69 expression levels of Jurkat cells expressing CAR that were not stimulated or incubated with irrelevant target cells were also quantified, and the level of automatic activation (enhanced signaling) associated with each CAR was checked ( Fig.10 ).

[0493] Human primary peripheral blood derived T cells are used to confirm the in vitro and in vivo activity of 4E11 or 5G6 CAR constructs targeting EGFRvIII in the context of primary human immune cells. In short, EGFRvIII-CAR or control (FMC63-CAR targeting human CD19) lentivirus is produced in HEK293 using standard production schemes, and concentrated using ultracentrifugation. Primary T cells are separated from donor blood samples using magnetic separation, and activated using anti-CD3 / CD28 beads. Primary T cells are then transduced with CAR lentivirus and expanded in culture for several days.

[0494] In vitro function of primary human EGFRvIII-specific CAR-transduced T cells was evaluated using an in vivo fluorescence microscopy protocol. In brief, CAR-T or mock-transduced T cells targeting EGFRvIII were generated as described just above, without introducing lentiviral constructs into cells treated under similar conditions. The cells were then co-cultured with target cells expressing EGFRvIII that were modified to also express a nuclear localized form of mKate2 fluorescent protein. Using Incucyte TM The co-cultures were continuously monitored over 6 days using an automated intravital fluorescence microscopy device (Sartorius, USA). The relative growth of target cells was then assessed using automated counting of mKate2+ cells ( Fig.11 ). The data showed that the growth of U87MG cells overexpressing EGFRvIII was effectively inhibited by 4E11 or 5G6 CAR-T compared with control CAR-T (FMC63) or mock-transduced T-cells.

[0495] Nod-SCID-IL2γR2 carrying U87vIII tumors expressing EGFRvIII was also used - / -( The in vivo function of primary CAR-T cells targeting EGFRvIII was tested in NSG mice (Jackson Laboratory, Barr Harbor, ME). Briefly, mice were injected subcutaneously with 1x10 6 Eight days after tumor cell injection, cryopreserved CAR-T cells were thawed, washed with PBS, and a total of 1x10 7 T cells (with 20-25% CAR transduction) were immediately delivered intratumorally to ensure uniform distribution of tumor size between groups. Tumor growth was assessed three times a week using calipers by trained animal technicians who were blinded to the specific treatment groups ( Fig. 12A ). The primary endpoint was more than 2000mm 3 Tumor size, and secondary endpoints determined by overall animal health and well-being ( Fig. 12B The tumor volume was calculated using the following formula: tumor volume = (0.4)(ab 2 ), where a = major diameter and b = minor diameter.

[0496] In this study, better tumor growth control and increased survival were observed in animals receiving target-specific 4E11 primary CAR-T compared with untreated control animals ( Fig. 12A and Fig. 12B ).

[0497] Example 16: Bispecific immune cell engager functional testing

[0498] Various experiments were carried out to prove the activity of the single-chain variable fragment of novel EGFRvIII-specificity under the background of bispecific T cell adapter.Construct was produced using synthetic DNA, wherein 4E11scFV sequence (SEQ ID NO:167) was connected to form bispecific adapter molecule (SEQ ID NO:170) with the scFv sequence (OKT3) of previously demonstrated CD3 engagement. The schematic diagram (Figure 13) of exemplary bispecific immune cell adapter was provided. The plasmid expressing this bispecific construct was transfected into human embryonic kidney cells (HEK293T), and supernatant was collected after 2 to 4 days of cultivation. The supernatant from HEK293T was transferred to the hole of the target cell (U87vIII) containing Jurkat cells and different doses of expressed antigen. By using human CD69-specific antibody staining and flow cytometry to check CD69 expression level, the activation ( Fig.14). The data showed that only the 4E11 bispecific construct targeting EGFRvIII could induce a highly target-specific T-cell activation response compared to the control bispecific molecule.

[0499] The in vitro function of the specific bispecific immune cell adapter of the primary human EGFRvIII-interacting with primary human T cells was evaluated using the in vivo fluorescence microscopy scheme.In brief, the human T cells (which are allowed to be restored to a resting state in the culture of several weeks) amplified polyclonally were co-cultured with the target cells expressing EGFRvIII, and the target cells were modified to the mKate2 fluorescent protein also expressing nuclear localization form.The supernatant of the bispecific immune cell adapter of the HEK293T supernatant of different doses or the bispecific immune cell adapter of the 4E11-CD3 targeting of the cell secretion control CD19-CD3 or EGFRvIII-specific targeting was transferred to the T-cell target cell co-culture.Then use Incucyte TM The co-cultures were continuously monitored for more than 6 days using an automated intravital fluorescence microscopy device (Sartorius, USA). The relative growth of target cells was then assessed using automated counting of mKate2+ cells ( Fig.15 The results confirmed that the 4E11-CD3 bispecific immune cell engager could actively induce T cell-mediated inhibition of EGFRvIII-positive tumor cell growth compared with the bispecific CD19-CD3 control molecule or mock supernatant.

[0500] The embodiments and examples described herein are exemplary and are not intended to limit the scope of the disclosure claimed. The inventor intends to cover the changes of the aforementioned embodiments with the claims, including alternatives, modifications and equivalents. The citations listed in this application are incorporated herein by reference.

[0501] References

[0502] All patents, patent applications, and publications mentioned throughout this application are incorporated herein by reference.

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[0511] -Jones PT,Dear PH,Foote J,Neuberger MS,Winter G(1986)Nature 321,522-525.

[0512] -Kabat EA, Wu TT. Identical V region amino acid sequences and segments of sequences in antibodies of different specificities. Relative contributions of VH and VL genes, minigenes, and complementarity-determining regions to binding of antibody-combining sites. J Immunol. 1991;147:1709-19.

[0513] -Mendelsohn J, et al., Clin Cancer Res. January 15, 2015;21(2):227-9.

[0514] -Reilly, EB., Molecular Cancer Therapeutics, Volume 14(5), pp. 1141-51, 2015.201

[0515] -Jones PT, Dear PH, Foote J, Neuberger MS, Winter G(1986) Nature 321, 522-525

[0516] -Riechmann L, Clark M, Waldmann H, Winter G(1988) Nature 332, 323-327.

[0517] -Sato JD, et al., Mol. Biol. Med. 1:511-529, 1983.

[0518] -Tempest PR, Bremmer P, Lambert M, Taylor G, Furze JM, Carr FJ, Harris WJ(1991) Biotechnology 9, 266-271.

[0519] -Tsurushita N, Hinton, RP, Kumar S(2005) Design of humanized antibodies: From anti-Tac to Zenapax. Methods 36, 69-83.

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[0527] Sequence Listing

[0528] In some sequences, the CDRs are underlined and indicated in bold.

[0529]

[0530]

[0531]

[0532]

[0533]

[0534]

[0535]

[0536]

[0537]

[0538]

[0539]

[0540]

[0541]

[0542] This application provides the following implementation schemes:

[0543] 1. An antigen binding agent comprising an antigen binding domain of an antibody that specifically binds to epidermal growth factor receptor variant III (EGFRvIII), wherein the antigen binding domain comprises:

[0544] a. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a CDRL1 having an amino acid sequence of SEQ ID NO: 8, a CDRL2 sequence of SEQ ID NO: 9, and a CDRL3 sequence of SEQ ID NO: 10; and the heavy chain variable region comprises a CDRH1 sequence of SEQ ID NO: 13, a CDRH2 sequence of SEQ ID NO: 14, and a CDRH3 sequence of SEQ ID NO: 15;

[0545] b. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises CDRL1 having the amino acid sequence shown in SEQ ID NO: 18, CDRL2 having the amino acid sequence shown in SEQ ID NO: 19, and CDRL3 having the amino acid sequence shown in SEQ ID NO: 20; and the heavy chain variable region comprises CDRH1 having the amino acid sequence shown in SEQ ID NO: 23, CDRH2 having the amino acid sequence shown in SEQ ID NO: 24, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 25;

[0546] c. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises CDRL1 having the amino acid sequence shown in SEQ ID NO: 28, CDRL2 having the amino acid sequence shown in SEQ ID NO: 29, and CDRL3 having the amino acid sequence shown in SEQ ID NO: 30; and the heavy chain variable region comprises CDRH1 having the amino acid sequence shown in SEQ ID NO: 33, CDRH2 having the amino acid sequence shown in SEQ ID NO: 34, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 35;

[0547] d. a light chain variable region and a heavy chain variable region, the light chain variable region comprising CDRL1 having the amino acid sequence shown in SEQ ID NO: 38, CDRL2 having the amino acid sequence shown in SEQ ID NO: 39, and CDRL3 having the amino acid sequence shown in SEQ ID NO: 40; the heavy chain variable region comprising CDRH1 having the amino acid sequence shown in SEQ ID NO: 43, CDRH2 having the amino acid sequence shown in SEQ ID NO: 44, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 45;

[0548] e. a light chain variable region and a heavy chain variable region, the light chain variable region comprising CDRL1 having the amino acid sequence shown in SEQ ID NO: 48, CDRL2 having the amino acid sequence shown in SEQ ID NO: 49, and CDRL3 having the amino acid sequence shown in SEQ ID NO: 50; the heavy chain variable region comprising CDRH1 having the amino acid sequence shown in SEQ ID NO: 53, CDRH2 having the amino acid sequence shown in SEQ ID NO: 54, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 55;

[0549] f. a light chain variable region and a heavy chain variable region, the light chain variable region comprising CDRL1 having the amino acid sequence shown in SEQ ID NO: 58, CDRL2 having the amino acid sequence shown in SEQ ID NO: 59, and CDRL3 having the amino acid sequence shown in SEQ ID NO: 60; the heavy chain variable region comprising CDRH1 having the amino acid sequence shown in SEQ ID NO: 63, CDRH2 having the amino acid sequence shown in SEQ ID NO: 64, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 65;

[0550] g. a light chain variable region and a heavy chain variable region, the light chain variable region comprising CDRL1 having the amino acid sequence shown in SEQ ID NO: 68, CDRL2 having the amino acid sequence shown in SEQ ID NO: 69, and CDRL3 having the amino acid sequence shown in SEQ ID NO: 70; the heavy chain variable region comprising CDRH1 having the amino acid sequence shown in SEQ ID NO: 78, CDRH2 having the amino acid sequence shown in SEQ ID NO: 79, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 80; and

[0551] h. a light chain variable region and a heavy chain variable region, the light chain variable region comprising CDRL1 having the amino acid sequence shown in SEQ ID NO:73, CDRL2 having the amino acid sequence shown in SEQ ID NO:74, and CDRL3 having the amino acid sequence shown in SEQ ID NO:75; the heavy chain variable region comprising CDRH1 having the amino acid sequence shown in SEQ ID NO:78, CDRH2 having the amino acid sequence shown in SEQ ID NO:79, and CDRH3 having the amino acid sequence shown in SEQ ID NO:80.

[0552] 2. The antigen binding agent of embodiment 1, wherein the antigen binding agent comprises:

[0553] a. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 7 or substantially the same as SEQ ID NO: 7; and the heavy chain variable region comprises a sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 12 or substantially the same as SEQ ID NO: 12;

[0554] b. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 17 or substantially the same as SEQ ID NO: 17; and the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 22 or substantially the same as SEQ ID NO: 22;

[0555] c. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 27 or substantially the same as SEQ ID NO: 27; and the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 32 or substantially the same as SEQ ID NO: 32;

[0556] d. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 37 or substantially the same as SEQ ID NO: 37; and the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 42 or substantially the same as SEQ ID NO: 42;

[0557] e. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 47 or substantially the same as SEQ ID NO: 47; and the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 52 or substantially the same as SEQ ID NO: 52;

[0558] f. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 57 or substantially the same as SEQ ID NO: 57; and the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 62 or substantially the same as SEQ ID NO: 62;

[0559] g. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO:67 or substantially the same as SEQ ID NO:67; the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO:77 or substantially the same as SEQ ID NO:77, an amino acid sequence set forth in SEQ ID NO:92 or substantially the same as SEQ ID NO:92, or an amino acid sequence set forth in SEQ ID NO:102 or substantially the same as SEQ ID NO:102; or

[0560] h. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO:72 or substantially the same as SEQ ID NO:72; and the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO:77 or substantially the same as SEQ ID NO:77, or an amino acid sequence shown in SEQ ID NO:92 or substantially the same as SEQ ID NO:92.

[0561] 3. The antigen binding agent of embodiment 1 or 2, wherein the antigen binding agent is an antibody or antigen binding fragment thereof, a chimeric antigen receptor, a bispecific T-cell engager, a bispecific killer cell engager, a trispecific killer cell engager or any immunotherapeutic compound.

[0562] 4. An antibody or an antigen-binding fragment thereof that specifically binds to EGFRvIII and comprises:

[0563] a. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a CDRL1 having an amino acid sequence of SEQ ID NO: 8, a CDRL2 sequence of SEQ ID NO: 9, and a CDRL3 sequence of SEQ ID NO: 10; and the heavy chain variable region comprises a CDRH1 sequence of SEQ ID NO: 13, a CDRH2 sequence of SEQ ID NO: 14, and a CDRH3 sequence of SEQ ID NO: 15;

[0564] b. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises CDRL1 having the amino acid sequence shown in SEQ ID NO: 18, CDRL2 having the amino acid sequence shown in SEQ ID NO: 19, and CDRL3 having the amino acid sequence shown in SEQ ID NO: 20; and the heavy chain variable region comprises CDRH1 having the amino acid sequence shown in SEQ ID NO: 23, CDRH2 having the amino acid sequence shown in SEQ ID NO: 24, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 25;

[0565] c. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises CDRL1 having the amino acid sequence shown in SEQ ID NO: 28, CDRL2 having the amino acid sequence shown in SEQ ID NO: 29, and CDRL3 having the amino acid sequence shown in SEQ ID NO: 30; and the heavy chain variable region comprises CDRH1 having the amino acid sequence shown in SEQ ID NO: 33, CDRH2 having the amino acid sequence shown in SEQ ID NO: 34, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 35;

[0566] d. a light chain variable region and a heavy chain variable region, the light chain variable region comprising CDRL1 having the amino acid sequence shown in SEQ ID NO: 38, CDRL2 having the amino acid sequence shown in SEQ ID NO: 39, and CDRL3 having the amino acid sequence shown in SEQ ID NO: 40; the heavy chain variable region comprising CDRH1 having the amino acid sequence shown in SEQ ID NO: 43, CDRH2 having the amino acid sequence shown in SEQ ID NO: 44, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 45;

[0567] e. a light chain variable region and a heavy chain variable region, the light chain variable region comprising CDRL1 having the amino acid sequence shown in SEQ ID NO: 48, CDRL2 having the amino acid sequence shown in SEQ ID NO: 49, and CDRL3 having the amino acid sequence shown in SEQ ID NO: 50; the heavy chain variable region comprising CDRH1 having the amino acid sequence shown in SEQ ID NO: 53, CDRH2 having the amino acid sequence shown in SEQ ID NO: 54, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 55;

[0568] f. a light chain variable region and a heavy chain variable region, the light chain variable region comprising CDRL1 having the amino acid sequence shown in SEQ ID NO: 58, CDRL2 having the amino acid sequence shown in SEQ ID NO: 59, and CDRL3 having the amino acid sequence shown in SEQ ID NO: 60; the heavy chain variable region comprising CDRH1 having the amino acid sequence shown in SEQ ID NO: 63, CDRH2 having the amino acid sequence shown in SEQ ID NO: 64, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 65;

[0569] g. a light chain variable region and a heavy chain variable region, the light chain variable region comprising CDRL1 having the amino acid sequence shown in SEQ ID NO: 68, CDRL2 having the amino acid sequence shown in SEQ ID NO: 69, and CDRL3 having the amino acid sequence shown in SEQ ID NO: 70; the heavy chain variable region comprising CDRH1 having the amino acid sequence shown in SEQ ID NO: 78, CDRH2 having the amino acid sequence shown in SEQ ID NO: 79, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 80; and

[0570] h. a light chain variable region and a heavy chain variable region, the light chain variable region comprising CDRL1 having the amino acid sequence shown in SEQ ID NO:73, CDRL2 having the amino acid sequence shown in SEQ ID NO:74, and CDRL3 having the amino acid sequence shown in SEQ ID NO:75; the heavy chain variable region comprising CDRH1 having the amino acid sequence shown in SEQ ID NO:78, CDRH2 having the amino acid sequence shown in SEQ ID NO:79, and CDRH3 having the amino acid sequence shown in SEQ ID NO:80.

[0571] 5. The antibody or antigen-binding fragment thereof of embodiment 4, wherein the antibody is a monoclonal antibody, a polyclonal antibody, a humanized antibody, a chimeric antibody, a human antibody, a single-chain antibody or a multispecific antibody.

[0572] 6. An antibody or an antigen-binding fragment thereof that specifically binds to EGFRvIII, comprising:

[0573] a. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 118 or substantially the same as SEQ ID NO: 118; and the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 116 or substantially the same as SEQ ID NO: 116;

[0574] b. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 115 or substantially identical to SEQ ID NO: 115; and the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 116 or substantially identical to SEQ ID NO: 116; or

[0575] c. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO:118 or substantially the same as SEQ ID NO:118; and the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO:62 or substantially the same as SEQ ID NO:62.

[0576] 7. An antibody or an antigen-binding fragment thereof that specifically binds to EGFRvIII and comprises:

[0577] a. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 7 or substantially the same as SEQ ID NO: 7; and the heavy chain variable region comprises a sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 12 or substantially the same as SEQ ID NO: 12;

[0578] b. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 17 or substantially the same as SEQ ID NO: 17; and the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 22 or substantially the same as SEQ ID NO: 22;

[0579] c. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 27 or substantially the same as SEQ ID NO: 27; and the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 32 or substantially the same as SEQ ID NO: 32;

[0580] d. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 37 or substantially the same as SEQ ID NO: 37; and the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 42 or substantially the same as SEQ ID NO: 42;

[0581] e. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 47 or substantially the same as SEQ ID NO: 47; and the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 52 or substantially the same as SEQ ID NO: 52;

[0582] f. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 57 or substantially the same as SEQ ID NO: 57; and the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 62 or substantially the same as SEQ ID NO: 62;

[0583] g. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO:67 or substantially the same as SEQ ID NO:67; the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO:77 or substantially the same as SEQ ID NO:77, an amino acid sequence set forth in SEQ ID NO:92 or substantially the same as SEQ ID NO:92, or an amino acid sequence set forth in SEQ ID NO:102 or substantially the same as SEQ ID NO:102; or

[0584] h. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO:72 or substantially the same as SEQ ID NO:72; and the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO:77 or substantially the same as SEQ ID NO:77, or an amino acid sequence shown in SEQ ID NO:92 or substantially the same as SEQ ID NO:92.

[0585] 8. The antibody or antigen-binding fragment thereof of any one of embodiments 4-7, wherein the antibody or antigen-binding fragment thereof comprises a human IgG1 constant region.

[0586] 9. The antibody or antigen-binding fragment thereof of any one of embodiments 4-8, wherein the antibody or antigen-binding fragment thereof comprises a human IgG2 constant region.

[0587] 10. An antibody or an antigen-binding fragment thereof that specifically binds to EGFRvIII and comprises a sequence selected from the group consisting of:

[0588] a. a light chain comprising an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 108 or substantially the same as SEQ ID NO: 108; and a heavy chain comprising an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 107 or substantially the same as SEQ ID NO: 107, and

[0589] b. a light chain comprising an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 110 or substantially the same as SEQ ID NO: 110; and a heavy chain comprising an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 109 or substantially the same as SEQ ID NO: 109.

[0590] 11. The antibody or antigen-binding fragment thereof of any one of embodiments 4-10, wherein the antigen-binding fragment comprises scFv, Fab, Fab' or (Fab')2.

[0591] 12. The antibody or antigen-binding fragment thereof of any one of embodiments 4-11, wherein the antibody or antigen-binding fragment thereof is linked to a cargo molecule.

[0592] 13. The antibody or antigen-binding fragment thereof of embodiment 12, wherein the cargo molecule comprises a therapeutic moiety.

[0593] 14. The antibody or antigen-binding fragment of embodiment 13, wherein the therapeutic moiety comprises a cytotoxic agent, a cytostatic agent, an anticancer agent, or a radiotherapeutic agent.

[0594] 15. The antibody or antigen-binding fragment thereof of embodiment 13, wherein the antibody or antigen-binding fragment thereof is conjugated to a detectable portion.

[0595] 16. A pharmaceutical composition comprising the antigen-binding agent, antibody or antigen-binding fragment of any one of embodiments 1-15 and a pharmaceutically acceptable carrier, diluent or excipient.

[0596] 17. A nucleic acid molecule encoding the light chain variable region and / or heavy chain variable region of the antigen binding agent or antibody or antigen binding fragment thereof according to any one of embodiments 1-11.

[0597] 18. The nucleic acid molecule of embodiment 17, wherein the nucleic acid is selected from the group consisting of:

[0598] a. A nucleic acid molecule comprising a sequence having at least 80% identity with the nucleotide sequence shown in SEQ ID NO: 11 and / or a nucleic acid molecule comprising a sequence having at least 80% identity with the nucleotide sequence shown in SEQ ID NO: 16;

[0599] b. A nucleic acid molecule comprising a sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 21 and / or a nucleic acid molecule comprising a sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 26;

[0600] c. A nucleic acid molecule comprising a sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 31 and / or a nucleic acid molecule comprising a sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 36;

[0601] d. a nucleic acid molecule comprising a sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 41 and / or a nucleic acid molecule comprising a sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 46;

[0602] e. A nucleic acid molecule comprising a sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 51 and / or a nucleic acid molecule comprising a sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 56;

[0603] f. A nucleic acid molecule comprising a sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 61 and / or a nucleic acid molecule comprising a sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 66;

[0604] g. a nucleic acid molecule comprising a sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 71 and / or a nucleic acid comprising a sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 81;

[0605] h. a nucleic acid comprising a sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 76 and / or a nucleic acid molecule comprising a sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 81;

[0606] i. A nucleic acid molecule comprising a sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 86 and / or a nucleic acid molecule comprising a sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 96;

[0607] j. a nucleic acid molecule comprising a sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 91 and / or a nucleic acid molecule comprising a sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 96; or

[0608] k. A nucleic acid molecule comprising a sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 101 and / or a nucleic acid molecule comprising a sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 106.

[0609] 19. A kit comprising at least one of the antigen-binding agents or antibodies or antigen-binding fragments thereof according to any one of embodiments 1-15.

[0610] 20. A vector or a collection of vectors comprising a nucleic acid sequence encoding the light chain variable region and the heavy chain variable region of the antigen-binding agent or antibody or antigen-binding fragment thereof according to any one of embodiments 1-11.

[0611] 21. An isolated cell comprising the vector or vector collection of embodiment 20.

[0612] 22. The isolated cell of embodiment 21, wherein the cell is capable of expressing, assembling and / or secreting antibodies or antigen-binding fragments thereof.

[0613] 23. A kit comprising a first bottle and a second bottle, wherein the first bottle comprises nucleotides or vectors encoding the light chain of the antigen-binding agent or antibody or antigen-binding fragment thereof of any one of embodiments 1-11, and the second bottle comprises nucleotides or vectors encoding the heavy chain of the antibody or antigen-binding fragment thereof of any one of embodiments 1-11.

[0614] 24. A method of treating a cancer comprising cells expressing EGFRvIII, the method comprising administering to a subject in need thereof the antibody or antigen-binding fragment thereof of any one of embodiments 4-15.

[0615] 25. The method of embodiment 24, wherein the antibody is used in combination with a chemotherapeutic agent.

[0616] 26. The method of any one of embodiments 24 or 25, wherein the subject in need thereof has or is suspected of having glioblastoma multiforme.

[0617] 27. The method of any one of embodiments 24 or 25, wherein the subject in need thereof has or is suspected of having cancer.

[0618] 28. The method of embodiment 27, wherein the cancer comprises breast cancer or HNSCC.

[0619] 29. A method for detecting EGFRvIII, the method comprising contacting a sample comprising or suspected of comprising EGFRvIII with the antibody or antigen-binding fragment of any one of embodiments 1-15.

[0620] 30. A method for preparing the antibody or antigen-binding fragment thereof according to any one of embodiments 4-11, the method comprising culturing a cell comprising a nucleic acid encoding the antibody or antigen-binding fragment, thereby producing the antibody or antigen-binding fragment thereof.

[0621] 31. The method of embodiment 30, further comprising conjugating the antibody or antigen-binding fragment thereof to a cargo molecule.

[0622] 32. The method of embodiment 31, wherein the cargo molecule comprises a therapeutic moiety.

[0623] 33. The method of embodiment 31, wherein the cargo molecule comprises a detectable portion.

[0624] 34. A method for treating a subject suffering from a cancer relevant to EGFRvIII expression, the method comprising administering a cell expressing the antigen binding agent of any one of embodiments 1-3, wherein the antigen binding agent is a chimeric antigen receptor, a bispecific T-cell engager, a bispecific killer cell engager, or a trispecific killer cell engager.

[0625] 35. The method of embodiment 34, wherein the subject in need thereof suffers from or is suspected of suffering from glioma.

[0626] 36. The method of embodiment 35, wherein the glioma is glioblastoma multiforme.

[0627] 37. The method of embodiment 34, wherein the subject in need thereof has or is suspected of having cancer.

[0628] 38. The method of embodiment 37, wherein the cancer comprises breast cancer, oral cancer, or HNSCC.

[0629] 39. The method of any one of embodiments 34-38, wherein the cell is a T-cell.

[0630] 40. The method of any one of embodiments 34-38, wherein the cells are NK cells.

[0631] 41. The method of any one of embodiments 34-40, wherein the cells are autologous immune cells of the subject.

[0632] 42. An isolated population of cells engineered to express the antigen binding agent of any one of embodiments 1-3.

[0633] 43. The isolated cell population described in embodiment 42, wherein the isolated cell population is of human origin.

[0634] 44. The isolated cell population of embodiment 42 or 43, wherein the isolated cell population comprises T cells, natural killer (NK) cells, cytotoxic T cells, regulatory T cells, and combinations thereof.

[0635] 45. The isolated cell population of embodiment 44, wherein the isolated cell population comprises T cells.

[0636] 46. ​​The isolated cell population of embodiment 45, wherein the T-cells comprise CD4+ T-cells, CD8+ T-cells, or a combination thereof.

[0637] 47. The isolated cell population described in embodiment 44, wherein the isolated cell population comprises NK cells.

[0638] 48. The isolated cell population of any one of embodiments 42-47, wherein the isolated cell population is engineered to express another chimeric antigen receptor having affinity for another antigen of the same target or a different target.

[0639] 49. The isolated cell population of any one of embodiments 42-47, wherein the isolated cell population comprises immune cells of the host.

[0640] 50. A pharmaceutical composition comprising the isolated cell population of any one of embodiments 42-49 and a pharmaceutically acceptable buffer or excipient.

Claims

1. An antigen binding agent comprising an antigen binding domain of an antibody that specifically binds to epidermal growth factor receptor variant III (EGFRvIII), wherein the antigen binding domain comprises: a. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a CDRL1 having an amino acid sequence of SEQ ID NO: 8, a CDRL2 sequence of SEQ ID NO: 9, and a CDRL3 sequence of SEQ ID NO: 10; and the heavy chain variable region comprises a CDRH1 sequence of SEQ ID NO: 13, a CDRH2 sequence of SEQ ID NO: 14, and a CDRH3 sequence of SEQ ID NO: 15; b. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises CDRL1 having the amino acid sequence shown in SEQ ID NO: 18, CDRL2 having the amino acid sequence shown in SEQ ID NO: 19, and CDRL3 having the amino acid sequence shown in SEQ ID NO: 20; and the heavy chain variable region comprises CDRH1 having the amino acid sequence shown in SEQ ID NO: 23, CDRH2 having the amino acid sequence shown in SEQ ID NO: 24, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 25; c. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises CDRL1 having the amino acid sequence shown in SEQ ID NO: 28, CDRL2 having the amino acid sequence shown in SEQ ID NO: 29, and CDRL3 having the amino acid sequence shown in SEQ ID NO: 30; and the heavy chain variable region comprises CDRH1 having the amino acid sequence shown in SEQ ID NO: 33, CDRH2 having the amino acid sequence shown in SEQ ID NO: 34, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 35; d. a light chain variable region and a heavy chain variable region, the light chain variable region comprising CDRL1 having the amino acid sequence shown in SEQ ID NO: 38, CDRL2 having the amino acid sequence shown in SEQ ID NO: 39, and CDRL3 having the amino acid sequence shown in SEQ ID NO: 40; the heavy chain variable region comprising CDRH1 having the amino acid sequence shown in SEQ ID NO: 43, CDRH2 having the amino acid sequence shown in SEQ ID NO: 44, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 45; e. a light chain variable region and a heavy chain variable region, the light chain variable region comprising CDRL1 having the amino acid sequence shown in SEQ ID NO: 48, CDRL2 having the amino acid sequence shown in SEQ ID NO: 49, and CDRL3 having the amino acid sequence shown in SEQ ID NO: 50; the heavy chain variable region comprising CDRH1 having the amino acid sequence shown in SEQ ID NO: 53, CDRH2 having the amino acid sequence shown in SEQ ID NO: 54, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 55; f. a light chain variable region and a heavy chain variable region, the light chain variable region comprising CDRL1 having the amino acid sequence shown in SEQ ID NO: 58, CDRL2 having the amino acid sequence shown in SEQ ID NO: 59, and CDRL3 having the amino acid sequence shown in SEQ ID NO: 60; the heavy chain variable region comprising CDRH1 having the amino acid sequence shown in SEQ ID NO: 63, CDRH2 having the amino acid sequence shown in SEQ ID NO: 64, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 65; g. a light chain variable region and a heavy chain variable region, the light chain variable region comprising CDRL1 having the amino acid sequence shown in SEQ ID NO: 68, CDRL2 having the amino acid sequence shown in SEQ ID NO: 69, and CDRL3 having the amino acid sequence shown in SEQ ID NO: 70; the heavy chain variable region comprising CDRH1 having the amino acid sequence shown in SEQ ID NO: 78, CDRH2 having the amino acid sequence shown in SEQ ID NO: 79, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 80; and h. a light chain variable region and a heavy chain variable region, the light chain variable region comprising CDRL1 having the amino acid sequence shown in SEQ ID NO:73, CDRL2 having the amino acid sequence shown in SEQ ID NO:74, and CDRL3 having the amino acid sequence shown in SEQ ID NO:75; the heavy chain variable region comprising CDRH1 having the amino acid sequence shown in SEQ ID NO:78, CDRH2 having the amino acid sequence shown in SEQ ID NO:79, and CDRH3 having the amino acid sequence shown in SEQ ID NO:

80.

2. The antigen binding agent of claim 1, wherein the antigen binding agent comprises: a. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 7 or substantially the same as SEQ ID NO: 7; and the heavy chain variable region comprises a sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 12 or substantially the same as SEQ ID NO: 12; b. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 17 or substantially the same as SEQ ID NO: 17; and the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 22 or substantially the same as SEQ ID NO: 22; c. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 27 or substantially the same as SEQ ID NO: 27; and the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 32 or substantially the same as SEQ ID NO: 32; d. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 37 or substantially the same as SEQ ID NO: 37; and the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 42 or substantially the same as SEQ ID NO: 42; e. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 47 or substantially the same as SEQ ID NO: 47; and the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 52 or substantially the same as SEQ ID NO: 52; f. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 57 or substantially the same as SEQ ID NO: 57; and the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 62 or substantially the same as SEQ ID NO: 62; g. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO:67 or substantially the same as SEQ ID NO:67; the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO:77 or substantially the same as SEQ ID NO:77, an amino acid sequence set forth in SEQ ID NO:92 or substantially the same as SEQ ID NO:92, or an amino acid sequence set forth in SEQ ID NO:102 or substantially the same as SEQ ID NO:102; or h. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO:72 or substantially the same as SEQ ID NO:72; and the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO:77 or substantially the same as SEQ ID NO:77, or an amino acid sequence shown in SEQ ID NO:92 or substantially the same as SEQ ID NO:

92.

3. The antigen binding agent of claim 1 or 2, wherein the antigen binding agent is an antibody or antigen binding fragment thereof, a chimeric antigen receptor, a bispecific T-cell engager, a bispecific killer cell engager, a trispecific killer cell engager, or any immunotherapeutic compound.

4. An antibody or an antigen-binding fragment thereof that specifically binds to EGFRvIII and comprises: a. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a CDRL1 having an amino acid sequence of SEQ ID NO: 8, a CDRL2 sequence of SEQ ID NO: 9, and a CDRL3 sequence of SEQ ID NO: 10; and the heavy chain variable region comprises a CDRH1 sequence of SEQ ID NO: 13, a CDRH2 sequence of SEQ ID NO: 14, and a CDRH3 sequence of SEQ ID NO: 15; b. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises CDRL1 having the amino acid sequence shown in SEQ ID NO: 18, CDRL2 having the amino acid sequence shown in SEQ ID NO: 19, and CDRL3 having the amino acid sequence shown in SEQ ID NO: 20; and the heavy chain variable region comprises CDRH1 having the amino acid sequence shown in SEQ ID NO: 23, CDRH2 having the amino acid sequence shown in SEQ ID NO: 24, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 25; c. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises CDRL1 having the amino acid sequence shown in SEQ ID NO: 28, CDRL2 having the amino acid sequence shown in SEQ ID NO: 29, and CDRL3 having the amino acid sequence shown in SEQ ID NO: 30; and the heavy chain variable region comprises CDRH1 having the amino acid sequence shown in SEQ ID NO: 33, CDRH2 having the amino acid sequence shown in SEQ ID NO: 34, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 35; d. a light chain variable region and a heavy chain variable region, the light chain variable region comprising CDRL1 having the amino acid sequence shown in SEQ ID NO: 38, CDRL2 having the amino acid sequence shown in SEQ ID NO: 39, and CDRL3 having the amino acid sequence shown in SEQ ID NO: 40; the heavy chain variable region comprising CDRH1 having the amino acid sequence shown in SEQ ID NO: 43, CDRH2 having the amino acid sequence shown in SEQ ID NO: 44, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 45; e. a light chain variable region and a heavy chain variable region, the light chain variable region comprising CDRL1 having the amino acid sequence shown in SEQ ID NO: 48, CDRL2 having the amino acid sequence shown in SEQ ID NO: 49, and CDRL3 having the amino acid sequence shown in SEQ ID NO: 50; the heavy chain variable region comprising CDRH1 having the amino acid sequence shown in SEQ ID NO: 53, CDRH2 having the amino acid sequence shown in SEQ ID NO: 54, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 55; f. a light chain variable region and a heavy chain variable region, the light chain variable region comprising CDRL1 having the amino acid sequence shown in SEQ ID NO: 58, CDRL2 having the amino acid sequence shown in SEQ ID NO: 59, and CDRL3 having the amino acid sequence shown in SEQ ID NO: 60; the heavy chain variable region comprising CDRH1 having the amino acid sequence shown in SEQ ID NO: 63, CDRH2 having the amino acid sequence shown in SEQ ID NO: 64, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 65; g. a light chain variable region and a heavy chain variable region, the light chain variable region comprising CDRL1 having the amino acid sequence shown in SEQ ID NO: 68, CDRL2 having the amino acid sequence shown in SEQ ID NO: 69, and CDRL3 having the amino acid sequence shown in SEQ ID NO: 70; the heavy chain variable region comprising CDRH1 having the amino acid sequence shown in SEQ ID NO: 78, CDRH2 having the amino acid sequence shown in SEQ ID NO: 79, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 80; and h. a light chain variable region and a heavy chain variable region, the light chain variable region comprising CDRL1 having the amino acid sequence shown in SEQ ID NO:73, CDRL2 having the amino acid sequence shown in SEQ ID NO:74, and CDRL3 having the amino acid sequence shown in SEQ ID NO:75; the heavy chain variable region comprising CDRH1 having the amino acid sequence shown in SEQ ID NO:78, CDRH2 having the amino acid sequence shown in SEQ ID NO:79, and CDRH3 having the amino acid sequence shown in SEQ ID NO:

80.

5. The antibody or antigen-binding fragment thereof of claim 4, wherein the antibody is a monoclonal antibody, a polyclonal antibody, a humanized antibody, a chimeric antibody, a human antibody, a single-chain antibody or a multispecific antibody.

6. An antibody or an antigen-binding fragment thereof that specifically binds to EGFRvIII, comprising: a. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 118 or substantially the same as SEQ ID NO: 118; and the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 116 or substantially the same as SEQ ID NO: 116; b. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 115 or substantially identical to SEQ ID NO: 115; and the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 116 or substantially identical to SEQ ID NO: 116; or c. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO:118 or substantially the same as SEQ ID NO:118; and the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO:62 or substantially the same as SEQ ID NO:

62.

7. An antibody or an antigen-binding fragment thereof that specifically binds to EGFRvIII and comprises: a. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 7 or substantially the same as SEQ ID NO: 7; and the heavy chain variable region comprises a sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 12 or substantially the same as SEQ ID NO: 12; b. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 17 or substantially the same as SEQ ID NO: 17; and the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 22 or substantially the same as SEQ ID NO: 22; c. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 27 or substantially the same as SEQ ID NO: 27; and the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 32 or substantially the same as SEQ ID NO: 32; d. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 37 or substantially the same as SEQ ID NO: 37; and the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 42 or substantially the same as SEQ ID NO: 42; e. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 47 or substantially the same as SEQ ID NO: 47; and the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 52 or substantially the same as SEQ ID NO: 52; f. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 57 or substantially the same as SEQ ID NO: 57; and the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 62 or substantially the same as SEQ ID NO: 62; g. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO:67 or substantially the same as SEQ ID NO:67; the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO:77 or substantially the same as SEQ ID NO:77, an amino acid sequence set forth in SEQ ID NO:92 or substantially the same as SEQ ID NO:92, or an amino acid sequence set forth in SEQ ID NO:102 or substantially the same as SEQ ID NO:102; or h. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO:72 or substantially the same as SEQ ID NO:72; and the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO:77 or substantially the same as SEQ ID NO:77, or an amino acid sequence shown in SEQ ID NO:92 or substantially the same as SEQ ID NO:

92.

8. The antibody or antigen-binding fragment thereof of any one of claims 4 to 7, wherein the antibody or antigen-binding fragment thereof comprises a human IgG1 constant region.

9. The antibody or antigen-binding fragment thereof of any one of claims 4-8, wherein the antibody or antigen-binding fragment thereof comprises a human IgG2 constant region.

10. An antibody or an antigen-binding fragment thereof that specifically binds to EGFRvIII and comprises a sequence selected from the group consisting of: a. a light chain comprising an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 108 or substantially the same as SEQ ID NO: 108; and a heavy chain comprising an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 107 or substantially the same as SEQ ID NO: 107, and b. a light chain comprising an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO:110 or substantially the same as SEQ ID NO:110; and a heavy chain comprising an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO:109 or substantially the same as SEQ ID NO:109.

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