Anti-EGFRvIII antibody-drug conjugates and uses thereof

Abstract

The present disclosure provides antibody-drug conjugates (ADCs) comprising an antibody that binds to the class III variant of EGFR (EGFRvIII) conjugated to tesirin, and methods of using them. According to certain embodiments, the antibodies or antigen-binding fragments thereof useful herein bind to human EGFRvIII with high affinity. The antibodies or antigen-binding fragments thereof useful herein may be fully human antibodies. The ADCs provided herein are useful for the treatment of various cancers.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 213,478, filed June 22, 2021, and U.S. Provisional Patent Application No. 63 / 242,929, filed September 10, 2021, each of which is incorporated by reference in its entirety herein.

[0002] Field of Disclosure The present disclosure relates to antibody-drug conjugates (ADCs), including human antibodies and antigen-binding fragments of human antibodies, that specifically bind to deletion mutants of the human epidermal growth factor receptor (EGFR), specifically the class III deletion mutant EGFRvIII, in which the antibody or antigen-binding fragment is conjugated to tesirin, and methods of treatment using those ADCs.

[0003] Sequence Listing A public copy of the Sequence Listing has been submitted electronically via EFS-Web contemporaneously herewith as an ASCII-formatted Sequence Listing with the filename "10966WO01_Sequence_Listing_ST25.TXT", a creation date of June 21, 2022, and a size of approximately 49,152 bytes. The Sequence Listing contained in this ASCII-formatted document is a part of this specification and is incorporated herein by reference in its entirety. [Background technology]

[0004] background Overexpression and / or gene amplification of epidermal growth factor (EGF) receptor or EGFR has been reported in several human tumors, including those in breast, ovarian, bladder, brain, and various squamous cell carcinomas (Wong, AJ et al., 1987, Proc. Natl. Acad. Sci. USA, 84:6899-6903 (Non-Patent Document 1); Harris et al., 1992, Natl. Cancer Inst. Monogr. 11:181-187 (Non-Patent Document 2)). However, targeting EGFR as an anti-neoplastic therapeutic approach has been problematic, since many normal tissues also express their receptors and can be targeted along with neoplastic targets. On the other hand, it has been reported that many glioblastomas with EGFR gene amplification often contain gene rearrangements (Ekstrand, AJ et al., 1992, Proc. Natl. Acad. Sci. USA, 89: 4309-4313 (Non-Patent Document 3), Wong AJ et al., 1992, Proc. Natl. Acad. Sci. USA, 89: 2965-2969 (Non-Patent Document 4)). In one study, 17 of 44 glioblastomas were found to have one or more changes in the EGFR coding sequence, and all of these cases contained amplified EGFR, but none of the 22 cases without gene amplification showed tumor-specific sequence abnormalities (Frederick, L. et al., 2000, Cancer Res 60: 1383-1387 (Non-Patent Document 5)). The same study also showed that multiple types of EGFR mutations can be detected in individual tumors.

[0005] Class III variants of EGFR (EGFRvIII) are the most frequent EGFR variants in glioblastoma (Bigner et al., 1990, Cancer Res 50:8017-8022 (Non-Patent Document 6); Humphrey et al., 1990, Proc Natl Acad Sci USA 87:4207-4211 (Non-Patent Document 7); Yamazaki et al., 1990, Jap J Cancer Res 81:773-779 (Non-Patent Document 8); Ekstrand et al., 1992, Proc Natl Acad Sci USA 89:4309-4313 (Non-Patent Document 3); Wikstrand et al., 1995, Cancer Res 55:3140-3148 (Non-Patent Document 9); and Frederick et al., 2000, Cancer Res 60:1383-1387 (Non-Patent Document 5). EGFRvIII is characterized by a deletion of exons 2-7 of the EGFR gene, resulting in an in-frame deletion of 801 base pairs in the coding region, i.e., a deletion of 6-273 amino acid residues (based on the number of residues in mature EGFR), as well as the generation of a new glycine at the fusion junction (Humphrey et al., 1988, Cancer Res 48:2231-2238 (Non-Patent Document 10); Yamazaki et al., 1990, supra). EGFRvIII has been shown to have ligand-independent, weak but constitutively active kinase activity, as well as enhanced tumorigenicity (Nishikawa et al., 1994, Proc Natl Acad Sci USA 91:7727-7731 (Non-Patent Document 11), and Batra et al., 1995, Cell Growth and Differentiation 6:1251-1259 (Non-Patent Document 12)).In addition to gliomas, EGFRvIII has been detected in ductal and intraductal carcinoma of the breast (Wikstrand et al., 1995, Cancer Res 55:3140-3148 (Non-Patent Document 9)), non-small cell lung cancer (Garcia de Palazzo et al., 1993, Cancer Res 53:3217-3220 (Non-Patent Document 13)), ovarian cancer (Moscatello et al., 1995, Cancer Res 55:5536-5539 (Non-Patent Document 14)), prostate cancer (Olapade-Olaopa et al., 2000, British J Cancer 82:186-194 (Non-Patent Document 15)), and squamous cell carcinoma of the head and neck (Tinhofer et al., 2011, Clin Cancer Res 17(15):5197-5204 (Non-Patent Document 16)). In contrast, these and other studies have reported that normal tissues do not express EGFRvIII (Garcia de Palazzo et al., 1993, supra; Wikstrand et al., 1995, supra; and Wikstrand et al., 1998, J Neuro Virol 4:148-158). The highly tumor-specific nature of EGFRvIII makes it a particularly useful target for treating cancers and tumors that express this molecule.

[0006] The amino acid sequence of human EGFR is shown in SEQ ID NO: 27, and the amino acid sequence of EGFRvIII is shown in SEQ ID NO: 28. Antibodies against EGFRvIII are described, for example, in US 5,212,290 (Patent Document 1), US 7,736,644 (Patent Document 2), US 7,589,180 (Patent Document 3), and US 7,767,792 (Patent Document 4).

[0007] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. [Prior art documents] [Patent documents]

[0008]

Patent Document 1

Patent document 2

Patent document 3

Patent document 4

Non-licensed literature

[0009] [Non-licensed document 1] Wong,AJet al.,1987,Proc.Natl.Acad.Sci.USA,84:6899-6903

Non-licensed Document 2

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

[0010] [Summary of the Disclosure] The present disclosure provides antibody-drug conjugates (ADCs) comprising antibodies and antigen-binding fragments thereof that bind to EGFRvIII, the antibodies and antigen-binding fragments thereof being conjugated to Tesiline, which contains a pyrrolobenzodiazepine (PBD) payload / warhead, SG3199 (Tiberghien et al., 2016, ACS Medicinal Chemistry Letters 7(11):983-987). The ADCs are particularly useful for targeting tumor cells expressing EGFRvIII.

[0011] Antibodies useful in the ADCs provided herein can be full-length (e.g., IgG1 or IgG4 antibodies) or may include only the antigen-binding portion (e.g., a Fab, F(ab')2, or scFv fragment) and may be modified to affect functionality, for example, to eliminate residual effector function (Reddy et al., 2000, J. Immunol. 164:1925-1933).

[0012] Exemplary anti-EGFRvIII antibodies useful herein are listed in Table 1. Table 1 lists the amino acid sequence identifiers of the heavy chain variable region (HCVR), light chain variable region (LCVR), heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3), and light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) of exemplary anti-EGFRvIII antibodies. Table 2 lists the complete heavy and light chain amino acid sequences of exemplary anti-EGFRvIII antibodies. Table 3 lists the nucleic acid sequence identifiers of the HCVR, LCVR, HCDR1, HCDR2 HCDR3, LCDR1, LCDR2, and LCDR3 of exemplary anti-EGFRvIII antibodies.

[0013] The present disclosure provides ADCs comprising an antibody or antigen-binding fragment thereof that specifically binds to EGFRvIII, comprising three complementarity determining regions (HCDR1, HCDR2, and HCDR3, respectively) within the HCVR comprising the amino acid sequence of SEQ ID NO:2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0014] The present disclosure also provides an ADC comprising an antibody or antigen-binding fragment thereof that specifically binds to EGFRvIII, comprising three complementarity determining regions (LCDR1, LCDR2, and LCDR3, respectively) within an LCVR comprising the amino acid sequence of SEQ ID NO: 10, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0015] The present disclosure provides ADCs comprising an antibody or antigen-binding fragment thereof that specifically binds to EGFRvIII, including a HCVR comprising the amino acid sequence of SEQ ID NO:2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0016] The present disclosure also provides an ADC comprising an antibody or antigen-binding fragment thereof that specifically binds to EGFRvIII, comprising an LCVR comprising the amino acid sequence of SEQ ID NO: 10, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0017] The present disclosure also provides an ADC comprising an antibody or antigen-binding fragment thereof that specifically binds to EGFRvIII, the antibody or antigen-binding fragment thereof comprising a HCVR comprising the amino acid sequence of SEQ ID NO:2 and a LCVR comprising the amino acid sequence of SEQ ID NO:10.

[0018] The present disclosure also provides ADCs comprising an antibody or antigen-binding fragment thereof that specifically binds to EGFRvIII, comprising a heavy chain CDR1 (HCDR1) comprising the amino acid sequence of SEQ ID NO:4, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0019] The present disclosure also provides an ADC comprising an antibody or antigen-binding fragment thereof that specifically binds to EGFRvIII, comprising a heavy chain CDR2 (HCDR2) comprising the amino acid sequence of SEQ ID NO:6, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0020] The present disclosure also provides an ADC comprising an antibody or antigen-binding fragment thereof that specifically binds to EGFRvIII, comprising a heavy chain CDR3 (HCDR3) comprising the amino acid sequence of SEQ ID NO:8, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0021] The present disclosure also provides an ADC comprising an antibody or antigen-binding fragment thereof that specifically binds to EGFRvIII, comprising a light chain CDR1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 12, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0022] The present disclosure also provides an ADC comprising an antibody or antigen-binding fragment thereof that specifically binds to EGFRvIII, comprising a light chain CDR2 (LCDR2) comprising the amino acid sequence of SEQ ID NO: 14, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0023] The present disclosure also provides an ADC comprising an antibody or antigen-binding fragment thereof that specifically binds to EGFRvIII, comprising a light chain CDR3 (LCDR3) comprising the amino acid sequence of SEQ ID NO: 16, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0024] The disclosure also provides ADCs comprising an antibody or antigen-binding fragment thereof that specifically binds to EGFRvIII, comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within the HCVR of SEQ ID NO:2 and the LCVR of SEQ ID NO: 10. In certain embodiments, the set of amino acid sequences of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 are SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, and SEQ ID NO:16, respectively.

[0025] Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the particular HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary rules that can be used to identify the boundaries of CDRs include, for example, the Kabat definition, the Chothia definition, and the AbM definition. In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within antibodies.

[0026] The present disclosure includes ADCs that include anti-EGFRvIII antibodies with modified glycosylation patterns. In some embodiments, modifications to remove undesired glycosylation sites or antibodies lacking fucose moieties present on the oligosaccharide chains may be useful, for example, to increase antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, modifications of galactosylation can be performed to modify complement-dependent cytotoxicity (CDC). In some embodiments, the antibody or antigen-binding fragment thereof is aglycosylated. Aglycosylated antibodies are point mutated at a suitable residue to prevent glycosylation. In some aspects, the antibody or antigen-binding fragment thereof comprises a heavy chain that is aglycosylated, for example, at N297 (according to EU index numbering) to improve conjugation efficiency. In certain embodiments, N297 is mutated to a glutamine (Q) residue, i.e., the antibody comprises an N297Q mutation.

[0027] In another aspect, the invention provides a conjugate comprising an anti-EGFRvIII-tesirin ADC, where the antibody or antigen-binding fragment thereof is conjugated to EGFRvIII.

[0028] In another aspect, the invention provides a pharmaceutical composition comprising an ADC comprising Tesirin and a recombinant human antibody or fragment thereof that specifically binds to EGFRvIII, and a pharma- ceutically acceptable carrier. In a related aspect, the invention features a composition that is a combination of an anti-EGFRvIII antibody-Tesirin ADC and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with the anti-EGFRvIII antibody-Tesirin ADC. Exemplary combination therapies and co-formulations that include the anti-EGFRvIII antibody-Tesirin ADC of the present disclosure are disclosed elsewhere herein.

[0029] In yet another aspect, the invention provides a therapeutic method for killing tumor cells or inhibiting or reducing tumor cell growth using an anti-EGFRvIII antibody-Tesirin conjugate or an antigen-binding portion of an antibody conjugated to Tesirin. The therapeutic method according to this aspect of the disclosure comprises administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising an antibody-Tesirin conjugate or an antigen-binding fragment of an antibody conjugated to Tesirin. The disorder to be treated is any disease or condition that is ameliorated, ameliorated, inhibited, or prevented by targeting an ADC to EGFRvIII.

[0030] Other embodiments will be apparent from a consideration of the following detailed description.Other embodiments will be apparent from a consideration of the following detailed description. [Brief description of the drawings]

[0031] [Figure 1] FIG. 1 shows a comparison of tumor volume and body weight 61 days after implantation of 0.5×10 6 MMT-EGFRvIII cells subcutaneously injected into the flank of female SCID mice with anti-EGFRvIII-tesirin conjugate or anti-EGFRvIII-maytansinoid DM1 conjugate. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] Detailed Description Before describing the present disclosure, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, and therefore, the methods and conditions may vary. It should also be understood that the terminology used herein is for the purpose of describing only specific embodiments, and is not intended to be limiting, since the scope of the present disclosure is limited only by the appended claims.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.As used herein, the term "about" when used in relation to a specific referenced numerical value means that the value may vary from the referenced value by 1% or less.For example, as used herein, the expression "about 100" includes 99 and 101, and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0034] It should be noted that, for purposes of describing and defining this disclosure, the use of relative terms such as "substantially," "generally," "approximately," and the like are utilized herein to express the degree of inherent uncertainty that may result from any quantitative comparison, value, measurement, or other representation. These terms are also utilized herein to express the extent to which a quantitative representation may vary from the stated reference without resulting in a change in the basic functionality of the subject matter at issue.

[0035] In some examples, the term "substantially" with respect to a given parameter, characteristic, or condition may mean and include to the extent that one of ordinary skill in the art would understand that the given parameter, characteristic, or condition is met with a small degree of variance, such as within acceptable manufacturing tolerances. As an example, depending on the particular parameter, characteristic, or condition that is substantially met, the parameter, characteristic, or condition may be at least 90%, at least 95%, at least 99% met, or may be completely met.

[0036] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, exemplary methods and materials are now described. All patents, patent applications, and non-patent publications mentioned herein are hereby incorporated by reference in their entirety.

[0037] definition As used herein, the term "EGFRvIII," unless otherwise indicated, refers to a human EGFR class III variant having the amino acid sequence set forth in SEQ ID NO:28, or a biologically active fragment thereof, that exhibits any characteristics specific to EGFRvIII as opposed to those in common with normally expressed EGFR. EGFRvIII lacks amino acid residues 6-273 of mature EGFR (i.e., SEQ ID NO:27 without the signal peptide (i.e., residues 1-24)) and contains a new glycine residue at position 6 between amino acid residues 5 and 274.

[0038] All references herein to proteins, polypeptides, and protein fragments are intended to refer to the human version of the respective protein, polypeptide, or protein fragment, unless expressly specified as being from a non-human species. Thus, the term "EGFRvIII" refers to human EGFRvIII, unless specified as being from a non-human species, e.g., "mouse EGFRvIII," "monkey EGFRvIII," etc.

[0039] As used herein, the phrase "cell surface expressed EGFRvIII" refers to one or more EGFRvIII proteins or extracellular domains thereof that are expressed on the surface of a cell in vitro or in vivo such that at least a portion of the EGFRvIII protein is exposed to the extracellular side of the cell membrane and accessible to an antigen-binding portion of an antibody. "Cell surface expressed EGFRvIII" can include or consist of an EGFRvIII protein expressed on the surface of a cell that normally expresses EGFRvIII protein. Alternatively, "cell surface expressed EGFRvIII" can include or consist of an EGFRvIII protein expressed on the surface of a cell that does not normally express human EGFRvIII on its surface but has been artificially engineered to express EGFRvIII on its surface.

[0040] The term "antibody" includes immunoglobulin molecules that contain four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and multimers thereof (e.g., IgM). Each heavy chain contains a heavy chain variable region (referred to herein as HCVR or V H The heavy chain constant region comprises a C H 1. C H 2, and C H Each light chain comprises three domains: a light chain variable region (herein LCVR or V L The light chain constant region comprises one domain (C L 1) is included. H Area and V L The regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), separated by regions that are relatively conserved, called framework regions (FRs). H and V L is composed of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the present disclosure, the FRs of the anti-EGFRvIII antibody (or antigen-binding portion thereof) may be identical to the human germline sequence or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on the parallel analysis of two or more CDRs.

[0041] As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc., include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies may be derived from intact antibody molecules using any suitable standard method, such as, for example, proteolytic or recombinant genetic engineering techniques, involving the manipulation and expression of DNA encoding the antibody variable regions and, optionally, the constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, to place one or more variable and / or constant domains in a suitable configuration, or to introduce codons, create cysteine ​​residues, modify, add, or delete amino acids, etc.

[0042] Non-limiting examples of antigen-binding fragments include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single chain Fv (scFv) molecules, (vi) dAb fragments, and (vii) minimal recognition units consisting of amino acid residues mimicking a hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Domain-specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other engineered molecules such as shark variable IgNAR domains are also encompassed by the term "antigen-binding fragment" as used herein.

[0043] Antigen-binding fragments of antibodies typically contain at least one variable domain, which may be of any size or amino acid composition and generally contains at least one CDR adjacent to, or in frame with, one or more framework sequences. L V associated with domain H In an antigen-binding fragment having a domain, H Domain and V L The domains can be arranged relative to each other in any suitable configuration. For example, the variable region is a dimer and the V H -V H , V H -V L Or V L -V L Alternatively, the antigen-binding fragment of the antibody may contain a dimer of monomeric V H Or V L It may contain domains.

[0044] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of the present disclosure include: (i) a V H -C H 1, (ii) V H -C H 2. (iii) V H -C H 3. (iv) V H -C H 1-C H 2. (v) V H -C H 1-C H 2-C H 3. (vi) V H -C H 2-C H 3. (vii) V H -C L、 (viii) V L -C H 1, (ix) V L -C H 2. (x)V L -C H 3. (xi) VL -C H 1-C H 2. (xii) V L -C H 1-C H 2-C H 3. (xiii) V L -C H 2-C H 3, and (xiv) V L -C L In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or may be linked by a full or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that provide a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the present disclosure may be linked to each other and / or to one or more monomeric V H Or V L It may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above in non-covalent association with the domains (eg, via disulfide bonds).

[0045] The antibodies useful herein may function via complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). "Complement-dependent cytotoxicity" (CDC) refers to the lysis of antigen-expressing cells by the antibodies of the present disclosure in the presence of complement. "Antibody-dependent cell-mediated cytotoxicity" (ADCC) refers to a cell-mediated reaction in which non-specific cytotoxic cells expressing Fc receptors (FcRs) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibodies on target cells, thereby resulting in the lysis of the target cells. CDC and ADCC can be measured using assays that are well known and available in the art. (See, e.g., U.S. Pat. Nos. 5,500,362 and 5,821,337, and Clynes et al. (1998) Proc. Natl. Acad. Sci. (USA) 95:652-656). The constant region of an antibody is important in the ability of the antibody to fix complement and mediate cell-dependent cytotoxicity. Thus, the isotype of the antibody can be selected based on whether it is desirable for the antibody to mediate cytotoxicity.

[0046] In certain embodiments of the present disclosure, the anti-EGFRvIII antibody used herein is a human antibody. The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences, for example in the CDRs, particularly in CDR3 (e.g., mutations introduced by in vitro random or site-specific mutagenesis or in vivo somatic mutation). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, are grafted onto human framework sequences.

[0047] The antibodies useful herein may in some embodiments be recombinant human antibodies. The term "recombinant human antibodies" as used herein is intended to include all human antibodies prepared, expressed, created, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transduced into a host cell (described in more detail below), antibodies isolated from recombinants, combinatorial human antibody libraries (described in more detail below), antibodies isolated from animals (e.g., mice) that are transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, created, or isolated by any other means, including splicing human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or in vivo somatic mutagenesis, when animals transgenic for human Ig sequences are used), so that the V H Area and V L The amino acid sequence of the region is H Sequence and V L While the sequences are derived from and related to the sequences, they may not naturally occur in the human antibody germline repertoire in vivo.

[0048] Human antibodies can exist in two forms related to hinge heterogeneity. In one form, the immunoglobulin molecule contains a stable four-chain construct of approximately 150-160 kDa, where the dimers are held together by interchain heavy chain disulfide bonds. In the second form, the dimers are not linked via interchain disulfide bonds, and the approximately 75-80 kDa molecule is composed of covalently linked light and heavy chains (half antibodies). These forms have been very difficult to separate, even after affinity purification.

[0049] The frequency of occurrence of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the antibody. A single amino acid substitution in the hinge region of a human IgG4 hinge can significantly reduce the occurrence of the second form to the level typically observed using a human IgG1 hinge (Angal et al. (1993) Molecular Immunology 30:105). The present invention provides a method for the identification of a hinge, C H 2nd Area or C H Antibodies with one or more mutations in three regions are included, which mutations may be desirable, for example, to improve the yield of a desired antibody type in production.

[0050] An antibody useful herein may be an isolated antibody. As used herein, an "isolated antibody" refers to an antibody that has been identified and separated and / or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which it is naturally present or produced, is an "isolated antibody" for the purposes of this disclosure. An isolated antibody also includes an antibody in situ within a recombinant cell. An isolated antibody is an antibody that has been subjected to at least one purification or isolation step. According to certain embodiments, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0051] Anti-EGFRvIII antibodies useful herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences from which the antibodies were derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The present disclosure includes ADCs comprising antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue in the germline sequence from which the antibody was derived, or to the corresponding residue in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as "germline mutations"). Starting with the heavy and light chain variable region sequences disclosed herein, one of skill in the art can readily produce numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, the V H and / or V LAll of the framework and / or CDR residues within the domain are mutated back to the residue found in the original germline sequence from which the antibody is derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only mutated residues found within the first 8 amino acids of FR1, or the last 8 amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residue in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody is originally derived). Furthermore, the antibodies useful herein may contain any combination of two or more germline mutations in the framework and / or CDR regions, e.g., certain individual residues are mutated to the corresponding residue in a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residue in a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonist or agonist biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed by the present disclosure.

[0052] The present disclosure also includes anti-EGFRvIII antibodies useful herein that include variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present disclosure includes anti-EGFRvIII antibodies having HCVR, LCVR, and / or CDR amino acid sequences that have, for example, 10 or less, 8 or less, 6 or less, or 4 or less conservative amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR amino acid sequences set forth in Table 1 herein.

[0053] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as the paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different regions on an antigen and have different biological effects. Epitopes may be either conformational or linear. Conformational epitopes are generated by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are epitopes generated by adjacent amino acid residues in a polypeptide chain. In certain circumstances, epitopes may include sugar, phosphoryl, or sulfonyl moieties on an antigen.

[0054] When referring to polypeptides, the term "substantial identity" or "substantially identical" refers to two peptide sequences that share at least 95% sequence identity, and even more preferably at least 98% or 99% sequence identity, when optimally aligned, such as by the programs GAP or BESTFIT, using default gap weights. In some embodiments, residue positions that are not identical differ only by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially alter the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331, incorporated herein by reference. Examples of groups of amino acids having side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine, (2) aliphatic-hydroxyl side chains: serine and threonine, (3) amide-containing side chains: asparagine and glutamine, (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan, (5) basic side chains: lysine, arginine, and histidine, (6) acidic side chains: aspartic acid and glutamic acid, and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix, as disclosed in Gonnet et al. (1992) Science 256:1443-1445, which is incorporated herein by reference. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0055] Sequence similarity for polypeptides, also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For example, GCG software contains programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between a wild-type protein and its mutant protein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignment and percent sequence identity of the regions of best overlap between the query sequence and the search sequence (Pearson (2000) supra). Another preferred algorithm for comparing the sequences of the present disclosure to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, with default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410, and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, each of which is incorporated herein by reference.

[0056] The subject is a mammal, preferably a human.

[0057] Anti-EGFRvIII antibodies, including Fc variants According to certain embodiments of the present disclosure, the anti-EGFRvIII antibodies useful herein comprise an Fc domain that comprises one or more mutations that enhance or reduce antibody binding to the FcRn receptor, e.g., at acidic pH compared to neutral pH. For example, the present disclosure provides an Fc domain that comprises one or more mutations that enhance or reduce antibody binding to the FcRn receptor, e.g., at acidic pH compared to neutral pH. H 2 or C HThe present invention includes ADCs that include anti-EGFRvIII antibodies that contain mutations in the 3 region that increase the affinity of the Fc domain for FcRn in acidic environments (e.g., in endosomes where the pH ranges from about 5.5 to about 6.0). Such mutations can extend the serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modifications at positions 250 (e.g., E or Q), 250 and 428 (e.g., L or F), 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D, or T), or modifications at positions 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., A, W, H, F, or Y [N434A, N434W, N434H, N434F, or N434Y]), or modifications at positions 250 and / or 428, or modifications at positions 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S), 428L, 259I (e.g., V259I), and 308F (e.g., V308F), 433K (e.g., H433K) and 434 (e.g., 434Y), 252, 254, and 256 (e.g., 252Y, 254T, and 256E), 250Q and 428L modifications (e.g., T250Q and M428L), and 307 and / or 308 modifications (e.g., 308F and / or 308P). In yet another embodiment, the modifications include 265A (e.g., D265A) and / or 297A (e.g., N297A) modifications.

[0058] For example, the disclosure includes ADCs that include anti-EGFRvIII antibodies that include an Fc domain that includes one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (e.g., T250Q and M248L), 252Y, 254T and 256E (e.g., M252Y, S254T and T256E), 428L and 434S (e.g., M428L and N434S), 434S and 434S (e.g., M434S and N434S), and 434S and 434S (e.g., M434S and N434S). 4S), 257I and 311I (e.g., P257I and Q311I), 257I and 434H (e.g., P257I and N434H), 376V and 434H (e.g., D376V and N434H), 307A, 380A and 434A (e.g., T307A, E380A and N434A), and 433K and 434F (e.g., H433K and N434F). All possible combinations of the foregoing Fc domain mutations, and other mutations in antibody variable domains disclosed herein, are contemplated as being within the scope of the present disclosure.

[0059] The present disclosure also provides a chimeric heavy chain constant (C H ) region, and H The region is composed of C H For example, antibodies useful herein include segments derived from C regions derived from human IgG1, human IgG2, or human IgG4 molecules. H C from a human IgG1, human IgG2, or human IgG4 molecule in combination with some or all of the three domains. H Chimera C containing part or all of the 2 domains H According to certain embodiments, the antibodies useful herein may comprise a chimeric C region having a chimeric hinge region. HFor example, the chimeric hinge may comprise an "upper hinge" amino acid sequence (amino acid residues at positions 216-227 according to EU numbering) derived from a human IgG1, IgG2 or IgG4 hinge region combined with a "lower hinge" sequence (amino acid residues at positions 228-236 according to EU numbering) derived from a human IgG1, IgG2 or IgG4 hinge region. According to a particular embodiment, the chimeric hinge region comprises amino acid residues derived from a human IgG1 upper hinge or a human IgG4 upper hinge and amino acid residues derived from a human IgG2 lower hinge. The chimeric hinges described herein may comprise an "upper hinge" amino acid sequence (amino acid residues at positions 216-227 according to EU numbering) derived from a human IgG1, IgG2 or IgG4 hinge region combined with a "lower hinge" sequence (amino acid residues at positions 228-236 according to EU numbering) derived from a human IgG1, IgG2 or IgG4 hinge region. According to a particular embodiment, the chimeric hinge region comprises amino acid residues derived from a human IgG1 upper hinge or a human IgG4 upper hinge and amino acid residues derived from a human IgG2 lower hinge. H Antibodies comprising the regions, in certain embodiments, exhibit altered Fc effector functions without negatively affecting the therapeutic or pharmacokinetic properties of the antibody (see, e.g., U.S. Provisional Application No. 61 / 759,578, filed February 1, 2013, the disclosure of which is incorporated herein by reference in its entirety).

[0060] In one embodiment of the invention, the Fc is an IgG4 with the mutation S108P.

[0061] Antibody-drug conjugates (ADCs) Provided herein is an antibody-drug conjugate (ADC) comprising an anti-EGFRvIII antibody, or an antigen-binding fragment thereof, conjugated to tesirin.

[0062] Tesilin has the following structure: TIFF2024527235000001.tif36152

[0063] Tesilin is also called SG3249.

[0064] Provided herein is a compound having the structure: TIFF2024527235000002.tif35155, where Ab comprises an anti-EGFRvIII antibody or antigen-binding fragment thereof and -S- is a sulfide bond at a cysteine ​​residue of the antibody or antigen-binding fragment thereof. In certain embodiments, the Ab comprises three heavy chain CDRs within the HCVR amino acid sequence comprising SEQ ID NO:2 and three light chain CDRs within the LCVR amino acid sequence of SEQ ID NO:10. In certain embodiments, the Ab comprises an HCDR1 amino acid sequence of SEQ ID NO:4, an HCDR2 amino acid sequence of SEQ ID NO:6, an HCDR3 amino acid sequence of SEQ ID NO:8, an LCDR1 amino acid sequence of SEQ ID NO:12, an LCDR2 amino acid sequence of SEQ ID NO:14, and an LCDR3 amino acid sequence of SEQ ID NO:16. In certain embodiments, the Ab comprises an HCVR amino acid sequence having at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:2, and an LCVR amino acid sequence having at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:10. In certain embodiments, the Ab comprises the HCVR amino acid sequence of SEQ ID NO:2, and / or the LCVR amino acid sequence of SEQ ID NO:10.

[0065] Also provided herein is a compound having the structure: TIFF2024527235000003.tif35155, where Ab comprises an anti-EGFRvIII antibody or antigen-binding fragment thereof, and -S- is a sulfide bond at a cysteine ​​residue of the antibody or antigen-binding fragment thereof. In certain embodiments, Ab is a complete antibody. In certain embodiments, Ab comprises a heavy chain and a light chain, and the heavy chain comprises the amino acid sequence of SEQ ID NO: 18. In certain embodiments, Ab comprises a heavy chain and a light chain, and the heavy chain comprises the amino acid sequence of SEQ ID NO: 20. In certain embodiments, Ab comprises a heavy chain and a light chain, and the light chain comprises the amino acid sequence of SEQ ID NO: 22. In certain embodiments, Ab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 18, and a light chain comprising the amino acid sequence of SEQ ID NO: 22. In certain embodiments, Ab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 20, and a light chain comprising the amino acid sequence of SEQ ID NO: 22.

[0066] In some embodiments, the DAR (drug-antibody ratio) is about 1 to about 4. In some embodiments, the DAR is about 2 to about 4. In some embodiments, the DAR is about 2 to about 3. In some embodiments, the DAR is about 3 to about 4. In some embodiments, the DAR is about 2. In some embodiments, the DAR is about 3. In some embodiments, the DAR is about 4.

[0067] The synthesis of Tesiline can be carried out, for example, using the procedure described in Tiberghien et al. (ACS Medicinal Chemistry Letters 2016,7(11):983-987). Tesiline contains the pyrrolobenzodiazepine warhead / payload moiety SG3199, which has the following structure: TIFF2024527235000004.tif16128

[0068] Epitope mapping and related techniques The epitope bound by the antibodies useful herein may consist of a single contiguous sequence of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids of the EGFRvIII protein. Alternatively, the epitope may consist of multiple non-contiguous amino acids (or amino acid sequences) of EGFRvIII. In some embodiments, the epitope is located on or near the ligand binding domain of EGFRvIII. In other embodiments, the epitope is located outside the ligand binding domain of EGFRvIII, e.g., at a location on the surface of EGFRvIII that does not interfere with ligand binding to EGFRvIII when an antibody binds to such an epitope.

[0069] According to certain embodiments, antibodies and antigen-binding fragments thereof useful herein include anti-EGFRvIII antibodies that specifically bind to EGFRvIII (and do not bind to EGFR), where the antibodies recognize an EGFRvIII junction peptide (e.g., SEQ ID NO: 23). Such antibodies may be referred to herein as "junction peptide binders," "EGFRvIII peptide binding antibodies," and the like. According to other embodiments, anti-EGFRvIII antibodies useful herein specifically bind to EGFRvIII (and do not bind to EGFR), where the antibodies do not recognize an EGFRvIII junction peptide (e.g., do not recognize the junction peptide of SEQ ID NO: 23 and / or do not recognize the peptide of SEQ ID NO: 24). Such antibodies may be referred to herein as "conformational binders," "EGFRvIII conformational epitope binders," and the like.

[0070] Antibodies and antigen-binding fragments thereof useful herein include anti-EGFRvIII antibodies that bind to or interact with one or more residues in hEGFRvIII ECD(L25-A380).mmH (SEQ ID NO:29), e.g., SEQ ID NO:25 or one or more residues corresponding to amino acids 64-82 GPCRKVCNGIGIGEFKDSL (SEQ ID NO:26) of SEQ ID NO:29.

[0071] Various techniques known to those skilled in the art can be used to determine whether an antibody or an antigen-binding fragment thereof "interacts with one or more amino acids" in a polypeptide or protein. Exemplary techniques include conventional cross-blocking assays, such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY), alanine scanning mutation analysis, peptide blot analysis (Reineke, 2004, Methods Mol Biol 248:443-463), and peptide truncation analysis. Additionally, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be used (Tomer, 2000, Protein Science 9:487-496). Another method that can be used to identify amino acids in a polypeptide that interact with an antibody is hydrogen / deuterium exchange detected by mass spectrometry. In general terms, hydrogen / deuterium exchange involves deuterium labeling of the protein of interest and then binding of the antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water, allowing hydrogen-deuterium exchange to occur at all residues except those protected by the antibody (which remain deuterium labeled). After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry analysis to reveal deuterium-labeled residues that correspond to the specific amino acids with which the antibody interacts. See, e.g., Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0072] The present disclosure further includes ADCs comprising anti-EGFRvIII antibodies that bind to the same epitope as any of the specific exemplary antibodies described herein (e.g., antibodies comprising any of the amino acid sequences described in Table 1 herein). Similarly, the present disclosure also includes ADCs comprising anti-EGFRvIII antibodies that compete for binding to EGFRvIII with any of the specific exemplary antibodies described herein (e.g., antibodies comprising any of the amino acid sequences described in Table 1 herein).

[0073] By using routine methods known in the art and exemplified herein, it is easy to determine whether an antibody binds to the same epitope as a reference anti-EGFRvIII antibody or competes for binding with the reference anti-EGFRvIII antibody. For example, to determine whether a test antibody binds to the same epitope as a reference anti-EGFRvIII antibody of the present disclosure, the reference antibody is bound to an EGFRvIII protein. The ability of the test antibody to bind to an EGFRvIII molecule is then evaluated. If the test antibody can bind to EGFRvIII after saturation binding with the reference anti-EGFRvIII antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-EGFRvIII antibody. On the other hand, if the test antibody cannot bind to an EGFRvIII molecule after saturation binding with the reference anti-EGFRvIII antibody, the test antibody may bind to the same epitope as the epitope bound by the reference anti-EGFRvIII antibody of the present disclosure. Additional routine experiments (e.g., peptide mutations and binding analysis) can then be performed to confirm whether the observed lack of binding of the test antibody is indeed due to binding to the same epitope as the reference antibody, or whether steric blocking (or another phenomenon) is responsible for the observed lack of binding. This type of experiment can be performed using ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art. According to certain embodiments of the present disclosure, two antibodies bind to the same (or overlapping) epitope if, for example, a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess of one antibody inhibits the binding of the other antibody by at least 50%, but preferably 75%, 90%, or even 99%, as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990:50:1495-1502). Alternatively, two antibodies are considered to bind the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody.Two antibodies are considered to have "overlapping epitopes" if only the subset of amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody.

[0074] To determine whether an antibody competes for binding (or cross-competes for binding) with a reference anti-EGFRvIII antibody, the above-mentioned binding method is carried out in two directions: in the first direction, the reference antibody is bound to the EGFRvIII molecule under saturating conditions, and then the binding of the test antibody to the EGFRvIII molecule is evaluated. In the second direction, the test antibody is bound to the EGFRvIII molecule under saturating conditions, and then the binding of the reference antibody to the EGFRvIII molecule is evaluated. In both directions, if only the first (saturating) antibody can bind to the EGFRvIII molecule, it is concluded that the test antibody and the reference antibody compete for binding to EGFRvIII. As will be understood by those skilled in the art, an antibody that competes for binding to a reference antibody may not necessarily bind to the same epitope as the reference antibody, but may sterically block the binding of the reference antibody by binding to an overlapping or adjacent epitope.

[0075] Biological characteristics of anti-EGFRvIII ADCs The present invention includes anti-EGFRvIII-Teciliin ADCs that specifically bind to EGFRvIII. In some embodiments, the ADCs comprise an anti-EGFRvIII antibody or antigen-binding fragment thereof and do not bind to (i) the conjugated peptide of SEQ ID NO: 23 or (ii) the peptide of SEQ ID NO: 24. In some embodiments, the ADCs have an equilibrium dissociation constant (K D ) for human EGFRvIII dimers of about 10 nM or less, as measured by a surface plasmon resonance assay at 37° C. In some embodiments, the ADC comprises an anti-EGFRvIII antibody or antigen-binding fragment thereof that exhibits an equilibrium dissociation constant (K DIn some embodiments, the ADC comprises an anti-EGFRvIII antibody, or antigen-binding fragment thereof, that does not bind to EGFR dimers at a level detectable by surface plasmon resonance assay.

[0076] In some embodiments, the anti-EGFRvIII-tesirin ADC exhibits one or more of the following characteristics: (a) reduces viability in EGFRvIII-expressing cells in vivo; (b) exhibits bystander cytotoxicity in vivo against non-EGFRvIII-expressing cells co-cultured with EGFRvIII-expressing cells; (c) exhibits prolonged survival in mice bearing intracranial glioblastoma multiforme expressing EGFRvIII; (d) exhibits anti-tumor efficacy in mice bearing EGFRvIII-expressing tumors in the absence of treatment-associated weight loss; (e) exhibits tumor regression in mice bearing patient-derived glioblastoma multiforme; (f) exhibits greater tumoricidal activity at lower doses compared to a control antibody conjugated to MMAF; and / or (g) exhibits greater anti-tumor efficacy in tumor-bearing mice than an anti-EGFRvIII-maytansinoid ADC.

[0077] Preparation of human antibodies The anti-EGFRvIII antibody or antigen-binding fragment thereof useful herein can be a fully human antibody. Methods for generating monoclonal antibodies, including fully human monoclonal antibodies, are known in the art. Any such known method can be used in the context of the present disclosure to generate human antibodies that specifically bind to human EGFRvIII.

[0078] For example, using VELOCIMMUNE™ technology, or any other similar known method for generating fully human monoclonal antibodies, high affinity chimeric antibodies against EGFRvIII are first isolated, having human variable regions and mouse constant regions. As in the experimental section below, the antibodies are characterized and selected for desired characteristics, including affinity, ligand blocking activity, selectivity, epitope, etc. If necessary, the mouse constant region is replaced with a desired human constant region, such as wild-type or modified IgG1 or IgG4, to generate a fully human anti-EGFRvIII antibody. While the constant region selected can vary depending on the particular application, the characteristics of high affinity antigen binding and target specificity reside in the variable region. In certain instances, fully human anti-EGFRvIII antibodies are isolated directly from antigen-positive B cells.

[0079] The present invention includes a method for producing an ADC comprising an antibody or antigen-binding fragment thereof of the present invention that specifically binds to EGFRvIII, the method comprising culturing a host cell comprising a polynucleotide encoding an immunoglobulin comprising the HCVR of the antibody or fragment and an immunoglobulin comprising the LCVR of the antibody or fragment in a culture medium under conditions favorable for expression of the polynucleotide. One or more of the immunoglobulins of the antibody or fragment so produced can then be conjugated to tesirin, for example, by reducing the immunoglobulin chains (e.g., in the presence of dithiothreitol) and incubating the tesirin with the reduced immunoglobulin chains. The host cell in which such an antibody or fragment can be expressed is a eukaryotic or prokaryotic host cell, for example, a mammalian cell. Such host cells are well known in the art and many are available from the American Type Culture Collection (ATCC). These host cells include, among others, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells, and many other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse, and hamster cells. Other cell lines that can be used are insect cell lines (e.g., Spodoptera frugiperda or Trichoplusia ni), amphibian cells, bacterial cells, plant cells, and fungal cells. Fungal cells include, for example, Pichia, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta), Pichia lindneri (Pichialindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia spp., Saccharomyces cerevisiae, Saccharomyces spp., Hansenula polymorpha, Kluyveromyces spp., Kluyveromyces lactis, Candida albicans Examples of fungal cells that may be used include yeast and filamentous fungal cells, including Aspergillus albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium species, Fusarium gramineum, Fusarium venenatum, Physcomitrella patens, and Neurospora crassa. ADCs produced by such methods form part of the invention.

[0080] biological equivalent Anti-EGFRvIII antibodies and antibody fragments useful herein include proteins having amino acid sequences that differ from the amino acid sequences of the described antibodies but retain the ability to bind to human EGFRvIII. Such variant antibodies and antibody fragments contain one or more amino acid additions, deletions, or substitutions compared to the parent sequence, but exhibit biological activity that is essentially equivalent to the biological activity of the described antibodies. Similarly, DNA sequences encoding the anti-EGFRvIII antibodies of such antibodies include sequences that contain one or more additions, deletions, or substitutions of nucleotides compared to the disclosed sequences, but encode anti-EGFRvIII antibodies or antibody fragments that are essentially biologically equivalent to the anti-EGFRvIII antibodies or antibody fragments of the present disclosure. Examples of such variant amino acid sequences and DNA sequences are discussed above.

[0081] Two antigen-binding proteins or antibodies are considered bioequivalent if they are pharmaceutical equivalents or pharmaceutical substitutes and do not show significant differences in their rate and extent of absorption when administered in the same molar dose, either in single or multiple doses, under similar experimental conditions. Some antibodies are considered to be equivalents, or pharmaceutical substitutes, if the extent of absorption is equivalent but the rate of absorption is not, but the antibodies can be considered bioequivalent because the difference in absorption rate is intentional, reflected in the labeling, is not essential, for example, to achieve effective body drug concentrations with chronic use, and is not considered medically significant for the particular drug product tested.

[0082] In one embodiment, two antigen binding proteins are bioequivalent if they have no clinically meaningful differences in their safety, purity, and potency.

[0083] In one embodiment, two antigen binding proteins are bioequivalent if a patient is able to make one or more switches between the reference product and the biological product without an expected increased risk of adverse effects, including clinically significant changes in immunogenicity, or a decrease in efficacy, compared to continuous therapy without such switches.

[0084] In one embodiment, two antigen binding proteins are bioequivalent if they both act by one or more common mechanisms for one or more conditions of use, to the known extent of such mechanisms.

[0085] Bioequivalence may be demonstrated by in vivo and in vitro methods, including, for example, (a) in vivo tests in humans or other mammals that measure the concentration of the antibody or its metabolites in blood, plasma, serum, or other biological fluid as a function of time, (b) in vitro tests that correlate with and reasonably predict human in vivo bioavailability data, (c) in vivo tests in humans or other mammals that measure the relevant acute pharmacological effects of the antibody (or its target) as a function of time, and (d) well-controlled clinical trials that demonstrate the safety, efficacy, or bioavailability or bioequivalence of the antibody.

[0086] Biologically equivalent variants of anti-EGFRvIII antibodies useful in the present invention may be constructed, for example, by making various substitutions of residues or sequences or by deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity can be deleted or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges during renaturation. In other situations, biologically equivalent antibodies may include anti-EGFRvIII antibody variants that contain amino acid changes that alter the glycosylation characteristics of the antibody, for example, mutations that eliminate or remove glycosylation.

[0087] Species selectivity and species cross-reactivity According to certain embodiments, the present disclosure provides anti-EGFRvIII antibodies useful herein that bind to human EGFRvIII but not to EGFRvIII from other species. The present disclosure also provides anti-EGFRvIII antibodies that bind to human EGFRvIII and EGFRvIII from one or more non-human species. For example, an anti-EGFRvIII antibody useful herein may bind to human EGFRvIII and, in some cases, may or may not also bind to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus monkey, marmoset, rhesus monkey, or chimpanzee EGFRvIII. According to certain exemplary embodiments, an anti-EGFRvIII antibody is provided that specifically binds to human EGFRvIII and cynomolgus monkey (e.g., Macaca fascicularis) EGFRvIII. Other anti-EGFRvIII antibodies of the present disclosure bind to human EGFRvIII but do not bind, or only weakly bind, to cynomolgus monkey EGFRvIII.

[0088] Therapeutic Formulations and Administration The present disclosure provides pharmaceutical compositions comprising anti-EGFRvIII antibody-Tesilin conjugates, i.e., anti-EGFRvIII antibody-Tesilin ADCs. The pharmaceutical compositions of the present disclosure are formulated with suitable carriers, excipients, and other agents that provide improved transport, delivery, tolerability, etc. Numerous suitable formulations can be found in a formulary known to every pharmacist: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipids (cationic or anionic) containing vesicles (e.g., LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998) J Pharm Sci Technol 52:238-311.

[0089] The dose of the ADC administered to a patient may vary depending on the age and size of the patient, the target disease, condition, route of administration, etc. The preferred dose is typically calculated according to body weight or body surface area. In adult patients, it may be beneficial to administer the antibody of the present disclosure intravenously at about 0.001 to about 20 mg / kg body weight, more preferably about 0.002 to about 7, about 0.003 to about 5, or about 0.005 to about 3 mg / kg body weight, usually in a single dose. Exemplary dosages include 1 ug / kg, 3.5 ug / kg, 7 ug / kg, and 10 ug / kg. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. Effective dosages and schedules for administering anti-EGFRvIII antibody conjugates can be empirically determined, for example, the patient's progress can be monitored by periodic evaluation and the dosage adjusted accordingly. Furthermore, interspecies scaling of dosages can be performed using methods well known in the art (eg, Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0090] A variety of delivery systems are known and can be used to administer the pharmaceutical compositions of the present disclosure, such as, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis, etc. (see, for example, Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (such as, for example, oral mucosa, rectal mucosa, and intestinal mucosa), and can be administered together with other bioactive agents. Administration can be systemic or local. Thus, a method for administering an anti-EGFRvIII antibody-tesirin ADC into the body of a subject is provided herein, the method comprising injecting the ADC into the body of a subject. In some embodiments, the ADC is injected subcutaneously into the subject's body. In some embodiments, the ADC is injected intravenously into the subject's body. In some embodiments, the ADC is injected intramuscularly into the subject's body.

[0091] The pharmaceutical composition of the present disclosure may be provided in a container. The pharmaceutical composition of the present disclosure may be provided in an injection device. The pharmaceutical composition may be delivered subcutaneously or intravenously using a standard needle and syringe. For subcutaneous delivery, a pen delivery device is easily adapted to deliver the pharmaceutical composition of the present disclosure. Such a pen delivery device may be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition inside the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device is sold pre-filled with the pharmaceutical composition held in a reservoir inside the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0092] A number of reusable pen and autoinjector delivery devices find use in the subcutaneous delivery of the pharmaceutical compositions of the present disclosure, including, but not limited to, the AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25™ pen, HUMALOG™ pen, HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II, and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN Examples of disposable pen delivery devices having application in subcutaneous delivery of pharmaceutical compositions of the present disclosure include, but are not limited to, the SOLOSTAR Pen (Sanofi-Aventis), FLEXPEN (Novo Nordisk), and KWIKPEN (Eli Lilly), SURECLICK Autoinjector (Amgen, Thousand Oaks, CA), PENLET (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA Pen (Abbott Labs, Abbott Park IL), to name a few.

[0093] In certain circumstances, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer, Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201, supra). In another embodiment, a polymeric material can be used. See Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, the controlled release system can be placed in the vicinity of the target of the composition. Thus, only a fraction of the systemic dose is required (see, for example, Goodson, 1984, Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0094] The injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injections, infusions, and the like. These injectable preparations may be prepared by known methods. For example, the injectable preparations may be prepared, for example, by dissolving, suspending, or emulsifying the above-described antibody or its salt in a sterile aqueous or oily medium conventionally used for injections. The aqueous medium for injection may be, for example, physiological saline, an isotonic solution containing glucose, and other auxiliary agents, which may be used in combination with a suitable solubilizing agent such as alcohol (e.g., ethanol), polyalcohol (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. The oily medium may be, for example, sesame oil, soybean oil, and the like, which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, and the like. The injection solution thus prepared is preferably filled into a suitable ampoule.

[0095] Advantageously, the above-mentioned pharmaceutical compositions for oral or parenteral use are prepared in dosage forms with unit doses suitable for adapting the dose of the active ingredient. Such dosage forms in unit doses include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the above-mentioned antibody contained is generally about 5 to about 500 mg per dosage form in unit dose, and particularly in the form of injection, the above-mentioned antibody is preferably contained in about 5 to about 100 mg, and for other dosage forms, it is preferably contained in about 10 to about 250 mg.

[0096] Accordingly, the invention includes methods for administering an ADC of the invention to a subject (e.g., a subject suffering from cancer), the method comprising the step of introducing the ADC into the subject's body, e.g., by injection or any of the methods discussed herein.

[0097] The invention also includes any container (e.g., a glass or plastic vial, or a bag such as an intravenous infusion bag) or such device that contains an ADC of the invention, for example, a syringe comprising a barrel, plunger, and needle.

[0098] Therapeutic Uses of Anti-EGFRvIII Antibody Conjugates The disclosure includes methods comprising administering to a subject in need thereof a therapeutic composition comprising an antibody-drug conjugate comprising an anti-EGFRvIII antibody conjugated to Tecilin (e.g., an anti-EGFRvIII antibody or ADC comprising any of the HCVR / LCVR or CDR sequences set forth herein in Table 1). The therapeutic composition can comprise an anti-EGFRvIII antibody conjugated to Tecilin, or any of its antigen-binding fragments, and a pharma- ceutically acceptable carrier or diluent.

[0099] The ADCs of the present disclosure are useful, inter alia, for the treatment, prevention, and / or amelioration of any disease or disorder associated with or mediated by expression or activity or overexpression of EGFRvIII or treatable by blocking the interaction between EGFRvIII and EGFR ligands or otherwise inhibiting EGFRvIII activity and / or signaling and / or promoting receptor internalization and / or decreasing the number of cell surface receptors. For example, the ADCs of the present disclosure are useful for treating tumors that express EGFRvIII and / or respond to ligand-mediated signaling. The ADCs of the present disclosure may also be used to treat primary and / or metastatic tumors arising in the brain and meninges, oropharynx, lung and bronchial tree, gastrointestinal tract, male and female reproductive organs, muscle, bone, skin and appendages, connective tissue, spleen, immune system, blood-forming cells and bone marrow, liver and urinary tract, and special sensory organs such as the eye. In certain embodiments, the ADCs of the disclosure are used to treat one or more of the following cancers: glioblastoma, renal cell carcinoma, pancreatic cancer, head and neck cancer, prostate cancer, malignant glioma, osteosarcoma, colorectal cancer, gastric cancer (e.g., gastric cancer with MET amplification), malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, breast cancer (ductal or intraductal), or melanoma.

[0100] In the context of the therapeutic methods described herein, the anti-EGFRvIII antibody-tesirin conjugate may be administered as a monotherapy (i.e., as the only therapeutic agent) or in combination with one or more additional therapeutic agents (examples of which are described elsewhere herein).

[0101] According to certain embodiments, the present disclosure provides a method for treating cancer, reducing tumor growth, and / or causing tumor regression in a patient. The method according to this aspect of the disclosure comprises administering to the patient a first antibody-drug conjugate (ADC), alone or in combination with a second anti-EGFRvIII antibody or ADC. The first ADC typically comprises an antibody or antigen-binding fragment of an antibody and tesirin, where the antibody or antigen-binding fragment of the first ADC specifically binds to EGFRvIII but does not bind to the conjugated EGFRvIII peptide of SEQ ID NO:23 or the peptide of SEQ ID NO:24 (i.e., the first ADC comprises a conformational EGFRvIII-binding antibody). In embodiments in which a second antibody or ADC is administered, the second antibody or ADC typically comprises an antibody or antigen-binding fragment of an antibody and a cytotoxin, where the second antibody or antigen-binding fragment specifically binds EGFRvIII and also binds the conjugated EGFRvIII peptide of SEQ ID NO: 23 and / or the peptide of SEQ ID NO: 24 (i.e., the second antibody or ADC comprises an EGFRvIII conjugated peptide-binding antibody). When two separate anti-EGFRvIII ADCs are used in the context of this aspect of the disclosure, both ADCs may, in certain embodiments, comprise the same cytotoxic agent, i.e., both may comprise tesirine, or the same class of cytotoxic agent. In other embodiments in which two separate anti-EGFRvIII ADCs are used, each ADC may comprise a different cytotoxic agent and / or a different class of cytotoxic agent. According to certain embodiments, the antibody or antigen-binding fragment (i.e., a conformational EGFRvIII binding antibody) of the first ADC comprises heavy and light chain complementarity determining regions comprising SEQ ID NOs: 4, 6, 8, 12, 14, and 16, or a heavy chain variable region comprising SEQ ID NO: 2 and a light chain variable region comprising SEQ ID NO: 10.

[0102] Combination Therapies and Formulations The present disclosure includes compositions and therapeutic formulations comprising any of the anti-EGFRvIII antibody-tesirin conjugates described herein in combination with one or more additional therapeutically active ingredients, as well as methods of treatment comprising administering such combinations to a subject in need thereof.

[0103] The anti-EGFRvIII antibody-tesirin conjugates useful herein may be co-formulated with and / or administered in combination with one or more additional therapeutically active ingredients selected from the group consisting of: a PRLR antagonist (e.g., an anti-PRLR antibody or a small molecule inhibitor of PRLR), an EGFR antagonist (e.g., an anti-EGFR antibody [e.g., cetuximab or panitumumab], or a small molecule inhibitor of EGFR [e.g., gefitinib or erlotinib]), an antagonist of another EGFR family member such as Her2 / ErbB2, ErbB3, or ErbB4 (e.g., an anti-ErbB2 [e.g., trastuzumab or T-DM1 {KADCYLA®}], an anti-ErbB3 or anti-ErbB4 antibody, or small molecule inhibitors of ErbB2, ErbB3, or ErbB4 activity), cMET antagonists (e.g., anti-cMET antibodies), IGF1R antagonists (e.g., anti-IGF1R antibodies), B-raf inhibitors (e.g., vemurafenib, sorafenib, GDC-0879, PLX-4720), PDGFR-α inhibitors (e.g., anti-PDGFR-α antibodies), PDGFR-β inhibitors (e.g., anti-PDGFR-β antibodies or small molecule kinase inhibitors, e.g., imatinib mesylate or sunitinib malate), PDGF ligand inhibitors (e.g., anti-PDGF-A, -B, -C, or -D antibodies, aptamers, siRNA, etc.), VEGF antagonists (e.g., VEGF-Trap such as aflibercept, e.g., US 7,087,See, e.g., 411 (also referred to herein as "VEGF inhibitor fusion proteins"), anti-VEGF antibodies (e.g., bevacizumab), small molecule kinase inhibitors of VEGF receptors (e.g., sunitinib, sorafenib, or pazopanib)), DLL4 antagonists (e.g., anti-DLL4 antibodies disclosed in US 2009 / 0142354, such as REGN421), Ang2 antagonists (e.g., anti-Ang2 antibodies disclosed in US 2011 / 0027286, such as H1H685P), FOLH1 antagonists (e.g., anti-FOLH1 antibodies), STEAP1 or STEAP2 antagonists (e.g., anti-STEAP1 antibodies or anti-STEAP2 antibodies), TMPRSS2 antagonists (e.g., For example, anti-TMPRSS2 antibodies), MSLN antagonists (e.g., anti-MSLN antibodies), CA9 antagonists (e.g., anti-CA9 antibodies), uroplakin antagonists (e.g., anti-uroplakin [e.g., anti-UPK3A] antibodies), MUC16 antagonists (e.g., anti-MUC16 antibodies), Tn antigen antagonists (e.g., anti-Tn antibodies), CLEC12A antagonists (e.g., anti-CLEC12A antibodies), TNFRSF17 antagonists (e.g., anti-TNFRSF17 antibodies), LGR5 antagonists (e.g., anti-LGR5 antibodies), monovalent CD20 antagonists (e.g., monovalent anti-CD20 antibodies such as rituximab), PD-1 antibodies, PD-L1 antibodies, CD3 antibodies, CTLA-4 antibodies, and the like. Other agents that may be beneficially administered in combination with the anti-EGFRvIII antibody-tecilin conjugates of the present disclosure include, for example, tamoxifen, aromatase inhibitors, and cytokine inhibitors, including small molecule cytokine inhibitors and antibodies that bind to cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17, IL-18, or their respective receptors.

[0104] The disclosure includes compositions and therapeutic formulations comprising any of the anti-EGFRvIII antibodies described herein in combination with one or more chemotherapeutic agents, including alkylating agents such as thiotepa and cyclophosphamide (Cytoxan™); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine. ethylenimines and methylamelamine, including methylamelamine;nitrogen mustards, such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichine, phenesterine, prednimustine, trofosfamide, uracil mustard;nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antioxidants, such as methylamelamine, methylamelamine, methylamelamine; Biological substances such as aclacinomycin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, mycobacterium, Enol acids, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate;Purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenishers such as folinic acid; aceglatone; aldophosphamide glycosides glycoside);aminolevulinic acid;amsacrine;bestrabucil;bisantrene;edatraxate;defofamine;demecolcine;diaziquone;elfornithine;elliptinium acetate;ethoglucide;gallium nitrate;hydroxyurea;lentinan;lonidamine;mitoguazone;mitoxantrone;mopidamol;nitracrine;pentostatin;phenamet;pirarubicin;podo Filic acid; 2-ethylhydrazide; procarbazine; PSK™; razoxane; schizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, e.g., paclitaxel (Taxol™, Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel (Taxotere™, Aventis Antony France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone;Teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicin; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above. Also included in this definition are antihormonal agents that act to regulate or inhibit the action of hormones on tumors, such as antiestrogens, including tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxyphene, keoxifene, LY117018, onapristone, and toremifene (Fareston); antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0105] The anti-EGFRvIII antibody conjugates of the present disclosure may also be administered and / or co-formulated in combination with antivirals, antibiotics, analgesics, corticosteroids, steroids, oxygen, antioxidants, COX inhibitors, cardioprotectants, metal chelators, IFN-gamma, and / or NSAIDs.

[0106] The additional therapeutically active ingredient, such as any of the agents listed above or derivatives thereof, may be administered immediately prior to, simultaneously with, or immediately following administration of the anti-EGFRvIII antibody-Tesirin conjugate of the present disclosure (for purposes of this disclosure, such administration regimens will be considered as administration of the anti-EGFRvIII antibody-Tesirin conjugate "in combination with" the additional therapeutically active ingredient). The present disclosure includes pharmaceutical compositions in which the anti-EGFRvIII antibody-Tesirin conjugate of the present disclosure is co-formulated with one or more of the additional therapeutically active ingredients described elsewhere herein.

[0107] Dosing regimen According to certain embodiments of the present disclosure, multiple doses of an anti-EGFRvIII antibody-Tesirin conjugate (or a pharmaceutical composition comprising an anti-EGFRvIII antibody-Tesirin conjugate and any combination of additional therapeutically active agents mentioned herein) may be administered to a subject over a defined time course. The method according to this aspect of the present disclosure includes sequentially administering multiple doses of an anti-EGFRvIII antibody-Tesirin conjugate of the present disclosure to a subject. As used herein, "sequentially administering" means that each dose of an anti-EGFRvIII antibody is administered to a subject at different times, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The present disclosure includes methods that include sequentially administering to a patient a single initial dose of an anti-EGFRvIII antibody-Tesirin conjugate, followed by one or more secondary doses of an anti-EGFRvIII antibody-Tesirin conjugate, and optionally followed by one or more tertiary doses of an anti-EGFRvIII antibody-Tesirin conjugate.

[0108] The terms "initial dose", "secondary dose", and "tertiary dose" refer to the temporal order of administration of the anti-EGFRvIII antibody-Tesirin conjugate of the present disclosure. Thus, the "initial dose" is the dose administered at the beginning of the treatment regimen (also referred to as the "baseline dose"), the "secondary dose" is the dose administered after the initial dose, and the "tertiary dose" is the dose administered after the secondary dose. The initial dose, secondary dose, and tertiary dose may all contain the same amount of anti-EGFRvIII antibody-Tesirin conjugate, but generally may differ from each other in terms of frequency of administration. However, in certain embodiments, the amount of anti-EGFRvIII antibody-Tesirin conjugate contained in the initial dose, secondary dose, and / or tertiary dose differs from each other during the course of treatment (e.g., adjusted upwards or downwards as appropriate). In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the start of the treatment regimen as a "loading dose," with subsequent doses administered on a less frequent basis (e.g., "maintenance doses").

[0109] In certain exemplary embodiments of the present disclosure, each secondary and / or tertiary dose is administered 1 to 26 weeks (e.g., 1 week, 1 1 / 2 weeks, 2 weeks, 2 1 / 2 weeks, 3 weeks, 3 1 / 2 weeks, 4 weeks, 4 1 / 2 weeks, 5 weeks, 5 1 / 2 weeks, 6 weeks, 6 1 / 2 weeks, 7 weeks, 7 1 / 2 weeks, 8 weeks, 8 1 / 2 weeks, 9 weeks, 9 1 / 2 weeks, 10 weeks, 10 1 / 2 weeks, 11 weeks, 11 1 / 2 weeks, 12 weeks, 12 1 / 2 weeks, 13 weeks) after the immediately preceding dose. After 12 weeks, 13 and a half weeks, 14 weeks, 14 and a half weeks, 15 weeks, 15 and a half weeks, 16 weeks, 16 and a half weeks, 17 weeks, 17 and a half weeks, 18 weeks, 18 and a half weeks, 19 weeks, 19 and a half weeks, 20 weeks, 20 and a half weeks, 21 weeks, 21 and a half weeks, 22 weeks, 22 and a half weeks, 23 weeks, 23 and a half weeks, 24 weeks, 24 and a half weeks, 25 weeks, 25 and a half weeks, 26 weeks, 26 and a half weeks, or more). As used herein, the phrase "immediately preceding dose" refers to a dose of anti-EGFRvIII antibody-tecilin conjugate administered to a patient in a multiple dose sequence prior to administration of the immediately succeeding dose in the sequence without an intervening dose.

[0110] The method according to this aspect of the disclosure may include administering any number of secondary and / or tertiary doses of anti-EGFRvIII antibody-tecilin conjugate to the patient. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient. Similarly, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient. The administration regimen may be administered indefinitely for the life of a particular subject or until such treatment is no longer therapeutically required or beneficial.

[0111] In embodiments including multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1-2 weeks or 1-2 months after the immediately preceding dose. Similarly, in embodiments including multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2-12 weeks after the immediately preceding dose. In certain embodiments of the present disclosure, the frequency at which the secondary and / or tertiary doses are administered to the patient may vary over the course of the treatment regimen. The frequency of administration may be adjusted during the course of treatment by the physician depending on the needs of the individual patient after clinical testing.

[0112] The present disclosure includes dosing regimens in which two to six loading doses are administered to a patient at a first frequency (e.g., once per week, once every two weeks, once per three weeks, once per month, once per two months, etc.), followed by two or more maintenance doses administered to the patient on a less frequent basis. For example, according to this aspect of the disclosure, if a loading dose is administered at a monthly frequency, maintenance doses may be administered to the patient once every six weeks, once every two months, once every three months, etc. EXAMPLES

[0113] The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the disclosed methods and compositions, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to the numbers used, but some experimental error and deviation should be accounted for. Unless otherwise indicated, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure.

[0114] Example 1. Generation of anti-EGFRvIII antibodies Anti-EGFRvIII antibodies were obtained by immunizing VELOCIMMUNE® mice (i.e., engineered mice containing DNA encoding the variable regions of human immunoglobulin heavy and kappa light chains) with an immunogen containing the extracellular domain of EGFRvIII.

[0115] The antibody immune response was monitored by EGFRvIII-specific immunoassay. When the desired immune response was obtained, splenocytes were harvested and fused with mouse myeloma cells to maintain their viability and form hybridoma cell lines. The hybridoma cell lines were screened and selected to identify cell lines that produce EGFRvIII-specific antibodies. Using this technique, an exemplary H1H1863N2 anti-EGFRvIII chimeric antibody (i.e., with human variable domains and mouse constant domains) was obtained. The variable domain sequence of this antibody was first disclosed in US9,475,875. This antibody is referred to herein as REGN1076. The aglycosylated version of the antibody (i.e., H1H1863N2-N297Q), in which the asparagine (N) at residue 297, as determined by EU index numbering of the REGN1076 antibody heavy chain, is mutated to glutamine (Q), is referred to herein as REGN3124. The variable region sequences and the complete heavy and light chain sequences are provided below.

[0116] Separately, a reduced fucosylated REGN1076 ["REGN1076(Fuc-)"] was prepared in the CHO host cell line described as "8088" in U.S. Patent Application Publication No. 2010 / 0304436A1, which is specifically incorporated by reference in its entirety. Mass spectrometry analysis of the resulting (Fuc-) antibody confirmed that the core fucose had been removed compared to the original antibody.

[0117] Table 1 lists the amino acid sequence identifiers for the heavy and light chain variable regions and CDRs of exemplary anti-EGFRvIII antibodies useful herein, while Table 2 provides the sequence identifiers for the full-length heavy and light chain amino acid sequences. The corresponding nucleic acid sequence identifiers are set forth in Table 3.

[0118] Table 1: Sequence identifiers for the variable region amino acid sequences of REGN1076 and REGN3124 TIFF2024527235000005.tif17133

[0119] Table 2: Sequence identifiers for the complete heavy and light chain amino acid sequences of REGN1076 and REGN3124 TIFF2024527235000006.tif22147

[0120] Table 3: Sequence identifiers for variable region nucleic acid sequences of REGN1076 and REGN3124 TIFF2024527235000007.tif17133

[0121] As will be appreciated by one of skill in the art, an antibody having a particular Fc isotype can be converted to an antibody having a different Fc isotype (e.g., an antibody having a murine IgG1 Fc can be converted to an antibody having a human IgG4 Fc, etc.), but in either case the variable domains (including the CDRs) indicated by the numerical identifiers shown in Table 1 will remain the same and the binding characteristics will be expected to be the same or substantially similar regardless of the nature of the Fc domain.

[0122] The antibodies were found to rapidly internalize into EGFRvIII-positive tumor cells. Certain additional biological properties of exemplary anti-EGFRvIII antibodies generated according to the methods of this example are described in detail in the Examples set forth below.

[0123] Control and comparative control constructs used in the following examples A control construct was included in the following experiments for comparison purposes. The comparative control antibody, referred to herein as COMP, is a humanized anti-EGFRvIII antibody (hIgG1) having heavy and light chain variable domains with amino acid sequences corresponding to SEQ ID NOs: 42 and 47, respectively, of the "hu806" antibody disclosed in US Patent Application Publication No. 2010 / 0056762. The antibody is also referred to as ABT-414. The "hu806" antibody is known to bind to residues 311-326 (SEQ ID NO: 24) of amplified or overexpressed EGFR (SEQ ID NO: 27) or residues 44-59 of EGFRvIII (SEQ ID NO: 28). COMP-MMAF refers to the ABT-414 antibody conjugated to monomethyl auristatin F (MMAF) via a non-cleavable linker.

[0124] Control 1932 and control 3892 are isotype control antibodies. Control 1932 has no Fc modification and control 3892 has the N297Q modification.

[0125] Example 2. Tesilin-Antibody Conjugation and Characterization Antibodies REGN1076 and REGN3124, and isotype control antibodies control 1932 (without Fc modification) and control 3892 (with N297Q modification) were each treated with 1 mM dithiothreitol at 10 mg / mL in 50 mM HEPES or PBS, 150 mM NaCl, pH 7.5 for 30 min at 37° C. After gel filtration (G-25, sodium acetate pH 4.5), maleimide linker payload Tecilin (a.k.a. SG3249, synthesized as disclosed in Tiberghein et al., 2016, ACS Medicinal Chemistry Letters 7(11):983-987) (1.2 equivalents / SH group) in DMSO (10 mg / mL) was added to the reduced antibody and the mixture was adjusted to pH 7.0 with 1 M HEPES (pH 7.4). The conjugate was purified by size exclusion chromatography and sterile filtered. Protein concentrations were determined by UV and payload-to-antibody ratios were determined by mass spectrometry. Size-exclusion HPLC confirmed that all conjugates used were greater than 95% monomeric, and LC-MS confirmed that there was less than 0.5% unconjugated linker payload. Payload-to-antibody ratios are shown in Table 4.

[0126] To determine the loading of tesilin on the antibody, the conjugate was deglycosylated, reduced and analyzed by LC-MS.

[0127] For this assay, 50 μg of conjugate was diluted with Milli-Q water to a final concentration of 1 mg / mL. 10 μL of PNGase F solution [PNGase F solution was prepared by adding 150 μL of PNGase F stock (New England Biolabs, Catalog No. P0704L) and 850 μL of Milli-Q water and mixing well] was added to the diluted conjugate solution, which was then incubated at 37 °C overnight. 2.4 μL of 0.5 M TCEP was added to the sample, which was then incubated at 50 °C for 30 min, so that the resulting material had a final TCEP concentration of 20 mM. 10 μL of each sample was injected onto an LC-MS (Waters Synat G2-Si) and eluted with a gradient mobile phase (20-40%) at 0.1 mL / min over 25 min (Mobile Phase A: 0.1% v / v FA in HO, Mobile Phase B: 0.1% v / v FA in acetonitrile). LC separation was performed on a Waters Acquity BEH C18 column (1.0×50 mm, 1.7 μM).

[0128] Mass spectrometry spectra were deconvoluted. Identified light and heavy chain peaks represent light chain (L) with linker-payload values ​​= 0 and 1, and heavy chain (H) with linker-payload values ​​= 0, 1, 2, and 3. From the various intensity values, the drug-to-antibody ratio (DAR) was calculated for the homodimeric antibody conjugates using the following Equation 1. The DAR for each conjugate is provided in Table 4. Equation 1: TIFF2024527235000008.tif17128

[0129] Table 4. Yield and payload to antibody ratio TIFF2024527235000009.tif29128

[0130] Example 3. Biacore binding kinetics of EGFRvIII monoclonal antibodies The equilibrium dissociation constant (K DValues) were determined using a real-time surface plasmon resonance biosensor assay on a Biacore 2000 or 3000 instrument. The Biacore sensor surface was derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody (GE Healthcare, #BR-1008-39) to capture anti-EGFRvIII antibody drug conjugates and parental unmodified antibody expressed with a human constant region. Biacore binding studies were performed in 0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, 0.05% v / v Surfactant P20 (HBS-EP running buffer). Different concentrations (3-fold dilutions) of human EGFRvIII extracellular domain (hEGFRvIII-MMH, SEQ ID NO: 29 (range 600 nM to 22.2 nM)) expressed with a C-terminal myc-myc-hexahistidine tag prepared in HBS-EP running buffer were injected over the anti-EGFRvIII antibody drug conjugate or antibody capture surface at a flow rate of 50 μL / min. Association of hEGFRvIII-MMH to the captured antibody drug conjugate and monoclonal antibody, respectively, was monitored for 4 min. hEGFRvIII-MMH dissociation was then monitored for 6–8 min in HBS-EP running buffer. The anti-human Fc surface was regenerated using an injection of 20 mM H3PO4. All binding rate experiments were performed at 25 °C. Kinetic association (k a ) and dissociation (k d ) rate constant was determined. All sensorgrams were double-referenced by subtracting the buffer injection sensorgram signal from the corresponding analyte sensorgram, thereby removing artifacts caused by dissociation of the antibody from the capture surface. The binding-dissociation equilibrium constant (K D ) and dissociation half-life (t 1 / 2 ) to the following: Calculated from the rate constant as shown in TIFF2024527235000010.tif5128.

[0131] The binding kinetic parameters of hEGFRvIII-MMH binding to anti-EGFRvIII antibody drug conjugates and antibodies at 25° C. are shown in Table 5. As shown, the parent antibodies and their corresponding antibody drug conjugates have similar binding K D The values ​​were shown.

[0132] Table 5. Biacore rates of human EGFRvIII-MMH binding to anti-EGFRvIII conjugates and parent unmodified antibody TIFF2024527235000011.tif48137

[0133] Example 4. Cell killing activity of anti-EGFRvIII antibody-Tesilin ADC To determine the relative cell killing potential of the anti-EGFRvIII antibody drug conjugates of the present invention, cell killing assays were performed on cell lines expressing human EGFRvIII. To develop the cell lines, Lipofectamine LTX with Plus Reagent was used to generate U251 cells (Sigma, #9063001) expressing human EGFRvIII (hEGFRvIII; amino acids 1-380 of Accession No. NP_005219.2, including deletion of amino acids 30-297 and creation of a conjugated glycine residue, i.e., SEQ ID NO: 25), herein referred to as U251MG / hEGFRvIII. The U251 line was maintained in complete growth medium (MEM Earle's salts + 10% FBS + 1% L-glutamine / penicillin / streptomycin + 1% non-essential amino acids + sodium pyruvate).

[0134] To measure the in vitro cytotoxicity of anti-EGFRvIII antibody drug conjugates, nuclei counts after 6 days of treatment with antibody drug conjugates were assessed. For U251MG and U251 / hEGFRvIII cells, cells were seeded at 3000 cells / well in 96-well plates (PerkinElmer, #6055308) in complete growth medium and grown overnight at 37°C in 5% CO2. For cell viability curves, serially diluted antibody drug conjugates and payloads were added to cells at final concentrations ranging from 100 nM to 1.5 pM (based on toxin concentration) and then incubated for 6 days at 37°C in 5% CO2. The last wells in each dilution series (untreated wells) served as blank controls containing either media alone (ADC) or media + 0.2% DMSO (payload) and were plotted as a succession of 3-fold serial dilutions. Cells were then treated with 3ug / mL Hoechst 33342 nuclear stain (ThermoFisher, #H3570) while fixed with 4% formaldehyde (ThermoFisher, #28908) and images were acquired with Opera Phenix (PerkinELmer). Nuclei counts were determined via Harmony image analysis software (PerkinELmer) and cell viability was expressed as a percentage of untreated (100% viable) cells. IC 50 Values ​​were determined using a four-parameter logistic equation over a 10-point dose-response curve (GraphPad Prism). Maximum % kill was determined for each test article as follows: 100-minimum viability. IC of each test article 50 The values ​​and maximum % kill are shown in Table 6.

[0135] As summarized in Table 6, the anti-EGFRvIII antibody-drug conjugates REGN3124-tesirin and REGN1076-tesirin (a glycosylated version of REGN3124) reduced cell viability and IC 50The IC values ​​were 33 pM for REGN3124-Tesilin and 84 pM for REGN1076-Tesilin in U251MG / hEGFRvIII cells. REGN3124-Tesilin and REGN1076-Tesilin killed parental U251MG cells and 50 The IC values ​​were 2.6 nM for REGN3124-Tesilin and 4.9 nM for REGN1076-Tesilin. A similarly conjugated isotype control antibody, 3892-Tesilin, reduced cell viability and 50 The values ​​were 3.9 nM in U251MG / hEGFRvIII cells and 1.8 nM in U251MG parental cells. The free payload of Tesilin (SG3199) had an IC of 10 pM. 50 Killed U251MG / hEGFRvIII cells at IC value of 2 pM 50 value killed parental U251MG cells.

[0136] REGN1076 conjugated to a control MMAF payload (REGN1076-MMAF) was also tested for cytotoxicity. Similar to the other tested anti-EGFRvIII ADCs, REGN1076-MMAF had an IC 50 The anti-EGFRvIII ADC REGN1076-MMAF killed U251MG / hEGFRvIII cells with an IC value of 52 nM in parental U251MG cells. 50 A non-binding, similarly conjugated isotype control antibody against MMAF (control 1932-MMAF) showed weak cytotoxicity with IC values ​​above 100 nM. 50 and showed weak cytotoxicity in all strains tested.

[0137] Table 6. Cell viability in U251 / hEGFRvIII and parental cell lines TIFF2024527235000012.tif77142

[0138] Bystander killing by ADCs can occur when the cytotoxic payload is released from the target cell and then taken up by surrounding antigen-negative (bystander) cells. To assess potential bystander killing by REGN3124-Tesilin and REGN1076-Tesilin, U251MG / hEGFRvIII cells were pre-labeled with CellTrace™ Far red (Thermo Fisher, #C34564). 1:1 co-cultures of 1500 cells / well far-red labeled U251MG / hEGFRvIII cells and 1500 cells / well unlabeled U251MG cells were incubated with either ADC or free payload M31 at a range of concentrations (100 nM-1.5 pM) for 6 days. Cells were then treated with 3ug / mL Hoechst 33342 nuclear stain (ThermoFisher, #H3570) while fixed with 4% formaldehyde (ThermoFisher, #28908). Images were acquired with an Opera Phenix Microscope (PerkinELmer). All cells were identified by Hoechst-labeled nuclei and cell numbers were separated into far-red positive U251MG / hEGFRvIII cells (U251 in co-culture) and far-red negative parental U251MG cell populations via Harmony image analysis software (PerkinELmer). Cell viability was determined separately for each cell population and expressed as a percentage of untreated (100% viable) cells. IC 50 and % maximum kill values ​​were determined as described above and are summarized in Table 6.

[0139] REGN3124-tesirin and REGN1076-tesirin had IC of 43 pM and 82 pM, respectively. 50 REGN3124-tesirin and REGN1076-tesirin also killed U251MG / hEGFRvIII cells from the co-culture with IC values ​​similar to those observed in U251MG / hEGFRvIII monocultures. REGN3124-tesirin and REGN1076-tesirin also killed U251MG / hEGFRvIII cells from the co-culture with IC values ​​of 28 pM and 59 pM, respectively. 50Both ADCs killed parental U251MG cells from the co-culture with IC values ​​of 4.0 nM and 2.4 nM, suggesting bystander killing activity by these ADCs. The non-binding ADC control 3892-tesirin had IC values ​​of 4.0 nM and 2.4 nM, respectively. 50 At these values, U251MG / hEGFRvIII and parental U251MG cells were killed.

[0140] REGN1076 conjugated to a control MMAF payload (REGN1076-MMAF) was also tested for bystander activity. REGN1076-MMAF had an IC of 15 pM. 50 In contrast to the tesirin conjugate, REGN1076-MMAF showed potent cytotoxicity against U251MG / hEGFRvIII cells from the co-culture with an IC value of 28 nM. 50 The nonbinding ADC conjugated to MMAF (control 1932-MMAF) had an IC value of less than 100 nM in the coculture assay. 50 The cytotoxicity was weak at the above values.

[0141] Example 5. Hydrogen / Deuterium (H / D) Exchange-Based Epitope Mapping of Anti-EGFRvIII Antibodies on Human Epidermal Growth Factor Receptor Variant (hEGFRvIII) Hydrogen-deuterium exchange mass spectrometry (HDX-MS) was performed to determine the amino acid residues of epidermal growth factor receptor variant 3 (hEGFRvIII ECD(L25-A380).mmH (SEQ ID NO: 29), amino acid sequence in the appendix) that interact with REGN3124. A general description of the HDX-MS methodology is described, for example, in Ehring (1999) Analytical Biochemistry 267(2):252-259, and Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0142] HDX-MS experiments were performed on an integrated HDX / MS platform consisting of a Leaptec HDX PAL system for deuterium labeling and quenching, a Waters Acquity M-Class (Auxiliary solvent manager) for sample digestion and loading, a Waters Acquity M-Class (μBinary solvent manager) for analytical gradients, and a Thermo Q Exactive HF mass analyzer for peptide mass measurement.

[0143] Labeling solutions were prepared as PBS buffer (10 mM phosphate buffer, 140 mM NaCl, and 3 mM KCl, equivalent to pH 7.4 at 25° C.) in DO at pD 7.0. For deuterium labeling, 10 μL of EGFRvIII (EGFRvIII extracellular domain with myc histidine tag (L25-A380), SEQ ID NO: 29, 66 μM) or EGFRvIII premixed with REGN3124 at a molar ratio of 1:0.6 (Ag-Ab complex) was incubated with 90 μL of DO labeling solution at 20° C. for various time points (e.g., 0 seconds for non-deuterated control, 5 minutes, 20 minutes, and 80 minutes for deuterium labeling). For each time point, experiments were performed in duplicate. The deuteration reaction was quenched by adding 100 μL of pre-chilled quench buffer (0.5 M TCEP-HCl, 8 M urea, and 1% formic acid) to 100 μL of sample. The mixed sample was incubated at 20 °C for 5 min. The quenched sample was then injected into a Waters HDX Manager for online pepsin / protease XIII digestion. Digested peptides were captured on a 1.0 mm × 50 mm C8 column (NovaBioassays) and separated by a 13 min gradient separation from 10% to 32% B (mobile phase A: 0.5% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile). The separated peptides were analyzed by Q Exactive HF mass spectrometry in LC-MS / MS or LC-MS mode.

[0144] The LC-MS / MS data of the non-deuterated EGFRvIII samples were searched against a database containing EGFRvIII and its randomized sequence using the Byonic search engine (Protein Index) with default parameters for non-specific enzymatic digestion. A list of common human glycans was defined as potential variable modifications. The list of identified peptides, together with the LC-MS data from all deuterated samples, was then imported into HDX Workbench software (version 3.3) to calculate the deuterium incorporation of individual peptides in each replicate of the three HDX time points.

[0145] For a given peptide, the centroid mass (intensity weighted average mass) of the spectrum is first calculated for the non-deuterated (0 s) control. The average centroid mass of the non-deuterated controls of antigen and Ag-Ab complex is considered as the mass of 0% deuterium incorporation (mass of 0% D). For each deuterated sample, the absolute D incorporation is defined as the mass difference between the centroid mass of the deuterated sample and the mass of 0% D. The deuterium incorporation percentage (% D) is determined by comparing the centroid mass with the masses of 0 and 100% D (maximum D incorporation mass shift defined as 80% of the mass difference between N-2 deuterium atoms and N-2 hydrogen atoms, where N is equal to the number of non-proline amino acids in the peptide). TIFF2024527235000013.tif34156

[0146] For each peptide, absolute D incorporation and %D values ​​were calculated for the two replicates of each HDX time point separately. For each HDX time point, the duplicate absolute D incorporation and %D values ​​were averaged for the antigen and Ag-Ab complex. The average of the %D values ​​at the 5 min and 20 min HDX time points is then presented as a single %D value for the antigen or Ag-Ab complex, defined as "antigen %D" or "Ag-Ab %D". The difference between antigen %D and Ag-Ab %D is defined as delta %D (Δ%), which represents the overall change in deuterium incorporation for a given peptide, comparing antigen and Ag-Ab complex.

[0147] A total of 200 peptides derived from hEGFRvIII were identified from both hEGFRvIII alone and hEGFRvIII in complex with REGN3124 samples, representing 84% sequence coverage of hEGFRvIII. Any peptide that exhibited a greater than 5% decrease in percent deuterium incorporation was defined as significantly protected (Δ%D<-5%). The peptide corresponding to amino acids 64-82 GPCRKVCNGIGIGEFKDSL (SEQ ID NO:26) on hEGFRvIII was significantly protected by REGN3124.

[0148] Table 7. hEGFRvIII peptides with significant protection upon formation of hEGFRvIII-REGN3124 complex compared to hEGFRvIII alone TIFF2024527235000014.tif61151

[0149] EGFRvlll ECD(L25-A380).mmH (mmH tag is underlined) TIFF2024527235000015.tif42156

[0150] Example 6. Anti-EGFRvIII antibody-Tesilin ADC shows significant anti-tumor efficacy against EGFRvIII-transfected glioblastoma multiforme cell line xenografts The antitumor efficacy of the REGN1076-Tesilin and REGN3124-Tesilin ADCs was first evaluated in glioblastoma cell line xenograft models transfected to express EGFRvIII due to loss of endogenous expression of the target following in vitro culture. The first model evaluated was U251 / EGFRvIII, where tumors were cultured at 10×10 6 The cells were mixed 1:1 with Matrigel and implanted subcutaneously onto the right flank of male SCID mice. Tumors grew to approximately 130 mm before treatment began and approximately 30 days after implantation. 3 The efficacy of the ADC in U87 / EGFRvIII was also evaluated, with tumors grown to 3×10 6Cells were established by subcutaneous implantation onto the right flank of male SCID mice. U87 / EGFvIII tumors grew to approximately 190 mm before treatment began and approximately 25 days after implantation. 3 Mice were randomized into groups of 7-8 and treated with a single dose of test or control ADC. Tumor growth was monitored for 60-70 days after treatment.

[0151] Test Results: Initial studies in U251 / EGFRvIII xenograft-bearing mice evaluated the activity of REGN1076-Tesilin and REGN3124-Tesilin anti-EGFRvIII ADCs after a single dose designed to deliver 2.5 or 5ug / kg PBD payload (Table 8). Growth of xenografts treated with control-Tesilin or control-N297Q-Tesilin ADCs was not significantly delayed compared to vehicle control treated tumors. However, significant tumor growth delay was observed in tumors treated with REGN1076-Tesilin or REGN3124-Tesilin ADCs at a payload dose of 2.5ug / kg over the course of the study. Higher ADC doses that delivered a PBD payload of 5ug / kg had even greater antitumor effects compared to control treatment. REGN3124-Tesilin ADCs provided more sustained antitumor effects compared to REGN1076-Tesilin at comparable dose levels. Overall, all anti-EGFRvIII-treated groups survived until study completion at approximately day 60 post-dose. No treatment related weight gains were observed, with all groups experiencing approximately a 10-15% increase in body weight over the course of the study.

[0152] The activity of REGN1076-Tesilin ADC and REGN3124-Tesilin ADC was also evaluated in the U87 / EGFRvIII tumor xenograft model (Table 9). Here, a single dose of REGN1076-Tesilin and REGN3124-Tesilin ADC was compared at a dose that delivered a PBD payload of 2.5ug / kg. This model showed very rapid growth and animals treated with vehicle control were euthanized 10 days after dosing as tumors reached the study endpoint. Control-Tesilin or control-N297Q-Tesilin ADC mediated some delay in tumor growth, but all tumors grew and animals were euthanized 24 days after dosing as tumors reached the study endpoint. Both REGN1076-Tesilin and REGN3124-Tesilin delivered a PBD payload of 2.5ug / kg mediated significant and sustained regression of tumor xenografts. All anti-EGFRvIII treated animals survived until study completion at 70 days post-dosing. A single tumor in REGN1076-Tesilin showed regrowth toward the end of the study. All tumors treated with REGN3124-Tesilin remained in inhibition. No treatment related weight gain was observed, with all groups observing approximately a 5% increase in body weight over the course of the study.

[0153] Table 8. Anti-EGFRvIII-Tesilin PBD ADC mediated regression of U251 / EGFRvIII xenografts compared to controls (day 36 post-treatment) TIFF2024527235000016.tif81154

[0154] Table 9. Anti-EGFRvIII-Tesilin conjugate mediated regression of U87 / EGFRvIII xenografts compared to controls (day 10 post-treatment) TIFF2024527235000017.tif63154

[0155] Example 7. Anti-EGFRvIII antibody-tesirin ADC shows significant anti-tumor efficacy against orthotopically placed EGFRvIII-positive glioblastoma multiforme patient-derived xenografts To evaluate the efficacy of anti-EGFRvIII ADCs against GBM tumors orthotopically placed in the brain, intracranial GBM6 (high and homogeneous EGFRvIII expression) or GBM59 (moderate and heterogeneous EGFRvIII expression) patient-derived xenograft (PDX) tumors were cultured at 3×10 5 PDX cells were injected into the graft. Intracranial injections were performed 1 mm anterior and 2 mm lateral to the bregma at a depth of 3 mm. All orthotopic GBM PDX studies were performed by Translational Drug Development Inc. Orthotopic GBM6 PDXs were allowed to settle for 14+-1 days and GBM59 PDXs were allowed to settle for 25+-1 days, after which mice were randomized into groups of 7-8 and treated with a single dose of test or control ADC. Mice were monitored for signs of peri-morbidity for approximately 90 days and euthanized before reaching a moribund state.

[0156] Test Results: In an initial study (Study A) with orthotopic GBM6 PDX tumor-bearing mice, vehicle-treated mice showed rapidly worsening clinical signs, with 7 of 8 mice being euthanized within 30 days of treatment (Table 10). The isotype control ADC did not produce any clinical benefit, and mice in this group were rapidly euthanized due to tumor-induced per-morbidity. In contrast to the short median survival of 25 and 26.5 days observed in the control groups, treatment with REGN3124-Tesilin (DAR 3.4) at a 7ug / kg payload dose resulted in a highly significant extension of survival. The median survival of the anti-EGFRvIII-Tesilin ADC group was not obtained, as 5 of 8 mice survived to the last observation time point of 94 days after administration.

[0157] A second study (Study B) was initiated in mice bearing orthotopically placed GBM6 PDX tumors (Table 11). Again, the mediated isotype control ADC did not extend survival compared to vehicle-treated mice, with median survival for both groups approaching 20 days, with no mice surviving. REGN3124-Tesilin (DAR 3.4) extended survival at both 3.5 and 7ug / kg payload dose levels, but the higher dose resulted in more mice (5 of 8) surviving to study completion 95 days after treatment. REGN3124-Tesilin (DAR 1.9) was similarly effective to REGN3124-Tesilin (DAR 3.4), with median survival of 77 days after treatment, with 4 of 8 mice surviving to study completion. Rapid deterioration of animal weight was observed in mice exhibiting tumor-induced peri-morbidity. In contrast, animals treated with REGN3124-tesirin that demonstrated long-term survival showed an associated 10-15% increase in body weight over the course of the post-treatment observation period.

[0158] The efficacy of REGN3124-Tesilin was also evaluated against orthotopically placed GBM59 PDX tumors (Table 12). In this study (Study C), all eight mice treated with vehicle died of tumor burden at 30 days. Although the isotype control ADC mediated a partial extension of survival compared to vehicle control, all mice were euthanized due to peri-morbidity at 42 days after treatment, resulting in a median survival of 32.5 days. For the GBM6 model, highly significant extension of survival was observed in mice receiving a single 7ug / kg payload dose of REGN3124-Tesilin. REGN3124-Tesilin with DAR 1.9 and DAR 3.4 resulted in 7 of 8 mice surviving until study completion at 94 days after treatment, therefore median survival in these groups was not obtained. In this study, less robust weight gain was observed in mice treated with DAR 1.9 REGN3124-Tesilin compared to the DAR 3.4 ADC. Brains from mice in Study C were harvested at various time points, specifically when mice were euthanized due to obvious disease, weight loss, or other clinical measures, or at the completion of the study 94 days after treatment. Histological analysis was performed. No GBM59 cells were found in any of the brains from mice treated with REGN3124-Tesilin. Similar results were seen in the GBM6 PDX study (Study A). Subsequent immunohistochemistry showed that GMB59 PDXs showed moderate and heterogeneous expression of EGFRvIII.

[0159] Table 10. Anti-EGFRvIII-Tesilin ADC significantly extended survival of mice bearing intracranial GBM6 GBM PDX (Study A) TIFF2024527235000018.tif45154

[0160] Table 11. Anti-EGFRvIII-Tesilin ADC significantly extended survival of mice bearing intracranial GBM6 GBM PDX (Study B) TIFF2024527235000019.tif63154

[0161] Table 12. Anti-EGFRvIII-Tesilin ADC significantly extended survival of mice bearing intracranial GBM59 GBM PDX (Study C) TIFF2024527235000020.tif54154

[0162] Example 8. REGN1076-Tesilin and REGN3124-Tesilin ADCs Show Significant Antitumor Efficacy Against EGFRvIII-Positive Glioblastoma Multiforme Patient-Derived Xenografts The efficacy of the REGN1076-Tesilin and REGN3124-Tesilin ADCs was further investigated using patient-derived xenografts with endogenous expression of EGFRvIII, representative of glioblastoma multiforme tumor biology. GBM PDX studies were conducted by Translational Drug Development Inc. Subcutaneous tumors of GBM6 or GBM59 PDX were established by implanting approximately 50 mg of PDX fragments into the flanks of nude mice. Tumor volumes reached approximately 125 mm 16-18 days after implantation. 3 Once tumor size reached 100%, mice were randomized into groups of 7-8 and treated with a single dose of test or control ADC delivering a dose equivalent to either a 3.5 or 7 ug / kg pyrrolobenzodiazepine (PBD) payload dose. Tumor growth was monitored for 60 days following treatment.

[0163] Test Results: In vehicle-treated GBM6 PDX tumor-bearing mice, rapid tumor growth was observed, with tumors reaching the study endpoint 18 days after treatment. The isotype control-Tesilin ADC mediated only a slight delay in tumor growth, with tumors reaching the study endpoint 22 days after treatment. In contrast to vehicle and control-treated tumors, the anti-EGFRvIII ADC mediated highly significant and durable tumor regression (Table 13). In the GBM6 model, the 3.5ug / kg payload dose from REGN1076-Tesilin or REGN3124-Tesilin generally had comparable antitumor efficacy, with 5 of 8 and 4 of 8 animals being tumor-free at the completion of the study 60 days after treatment. REGN1076-Tesilin or REGN3124-Tesilin treatment delivered a 7ug / kg PBD payload resulting in greater and sustained efficacy, with 7 of 8 and 8 of 8 mice being tumor-free at the completion of the study 60 days after treatment. No treatment-related weight loss was observed, and animals gained weight by approximately 15% over the course of the study.

[0164] Table 13. Anti-EGFRvIII-Tesilin ADC mediated GBM6 PDX tumor regression compared to control (day 18 post-treatment) TIFF2024527235000021.tif81154

[0165] The relative efficacy of REGN3124-Tesilin ADCs with drug:antibody ratios (DAR) of 1.9 and 3.4 was evaluated in GBM59 tumor-bearing mice. Rapid tumor growth in mice treated with vehicle and control ADCs was again observed, with both groups reaching the study endpoint 19 days after treatment (Table 14). At a PBD dose of 3.5ug / kg, REGN3124-Tesilin (DAR 3.4) mediated a moderate antitumor effect, with tumors reaching the study endpoint 30 days after treatment. REGN3124-Tesilin (DAR 1.9) was also active, with tumors reaching the study endpoint 51 days after treatment with this agent. Consistent with other studies, REGN3124-Tesilin ADC treatment delivering a payload dose of 7ug / kg resulted in greater and more sustained inhibition of GBM59 tumor growth. At this dose, REGN3124-Tesilin (DAR 1.9) demonstrated that 3 of 7 tumors were 50 mm at the completion of the study on day 60. 3 REGN3124-tesirin (DAR 3.4) resulted in 5 of 7 tumors measuring 50 mm at study completion. 3 No treatment-related weight loss was observed, and animals gained approximately 10% weight over the course of the study.

[0166] Table 14. Anti-EGFRvIII-Tesilin conjugate mediated GBM59 PDX tumor regression compared to control (day 19 post-treatment) TIFF2024527235000022.tif72154

[0167] Example 9. Evaluation of a fractionated dose schedule of REGN3124-Tesilin ADC in EGFRvIII-positive GBM59 PDX tumor-bearing mice To evaluate the effect of different dose schedules of REGN3124-Tesilin conjugate on antitumor efficacy, a study was conducted using a subcutaneous GBM59 PDX tumor model. This study was also conducted by Translational Drug Development Inc. Subcutaneous GBM59 PDX tumors were established by implanting approximately 50 mg of PDX fragments into the flanks of nude mice. The tumor volume was approximately 125 mm on day 13 after implantation. 3 Upon reaching PBD, mice were randomized into groups of 7 and treated with test or control ADC. The isotype control ADC was administered in a single dose equivalent to a PBD payload of 7ug / kg. Animals in the low dose group received the ADC REGN3124-Tesirin conjugate at 1.75ug / kg per dose on days 0 and 4 post-treatment, resulting in a cumulative PBD dose of 3.5ug / kg. Additional groups received REGN3124-Tesirin delivering a cumulative 7ug / kg dose. This was split into individual doses of 3x2.33ug / kg, 2x3.5ug / kg, or 1x7ug / kg delivered on days 0, 4, and 8 (2.33ug / kg), 0 and 4 (3.5ug / kg, or day 0 (7ug / kg). Tumor growth was monitored for 60 days post-treatment.

[0168] Test Results: In this study, the isotype control ADC did not cause any delay in tumor growth compared to the vehicle control, and both groups were euthanized on day 19 after treatment because the mean tumor volume reached the study endpoint (Table 15). In animals receiving REGN3124-Tesilin at 2×1.75 ug / kg, significant inhibition of tumor volume was observed, and all mice survived until study completion 60 days after treatment. All dose schedules resulting in a cumulative PBD payload dose of 7 ug / kg resulted in additional and highly significant antitumor effects. In mice administered REGN3124-Tesilin at 3×2.33 ug / kg PBD dose, 3 of 7 mice were tumor-free at study completion, with a mean tumor volume of 90 mm 3In the groups receiving REGN3124-tesirin delivered at 2 x 3.5ug / kg and 1 x 7ug / kg PBD doses, 2 of 7 and 3 of 7 mice were tumor free at the completion of the study, with mean tumor volumes in both groups of 5 mm 3 The mean mean body weight was less than 1.5 mg / kg / day, indicating significant and sustained efficacy of REGN3124-tesirin in this study. No treatment-related weight loss was observed, and animals gained weight by approximately 10% over the course of the study.

[0169] Table 15. Anti-EGFRvIII-Tesilin conjugate mediated GBM6 PDX tumor regression compared to control (day 18 post-treatment) TIFF2024527235000023.tif72154

[0170] Example 10. Comparison of anti-EGFRvIII-tesirin ADC with anti-EGFRvIII-maytansinoid DM1 ADC To establish tumors, 0.5 x 10 6 MMT-EGFRvIII cells were injected subcutaneously into the flank of female SCID mice. Tumor volumes were approximately 140 mm 3 Upon reaching tumor mass (day 8), mice were randomized into groups of 7 and treated with test and control ADCs using either the Tecilin or DM1 payload. Drugs were administered 3 days over a 17-day period. Tumor growth was monitored for 61 days after implantation.

[0171] The antitumor efficacy of each EGFRvIII ADC was then evaluated over time (Figure 1). Complete tumor eradication was observed in mice treated with 1 mg / kg REGN1076-Tesirin ADC for the duration of the study. The control-Tesirin ADC mediated a transient antitumor effect, but all tumors ultimately showed rapid progression toward the protocol endpoint tumor volume. In contrast to the significant efficacy observed after administration of REGN1076-Tesirin, REGN1076-DM1 administered at either 1 or 15 mg / kg induced only a moderate delay in tumor growth, with all tumors rapidly progressing toward the protocol endpoint. The control DM1 ADC was ineffective compared to the vehicle control. The anti-EGFRvIII results at the 1 mg / kg dose level indicate greater potency of the anti-EGFRvIII-Tesirin conjugate compared to the anti-EGFRvIII-maytansinoid DM1 conjugate. None of the treatments induced severe weight loss in this study.

[0172] Example 11. Evaluation of anti-EGFRvIII-Tesilin conjugates and COMP-MMAF ADC in EGFRvIII-positive tumor models The activity of the REGN3124-Tesilin ADC was evaluated in U251 / EGFRvIII tumor xenograft models and intracranial orthotopic GBM59 PDX models in parallel with the activity of a comparator ADC, COMP-MMAF (monomethylauristatin F, an auristatin-based ADC prepared according to the procedure described in Phillips et al., 2016, Mol Cancer Ther. 15(4):661-669; see also Doronina et al., 2006, Bioconjugate Chem. 17:114-124). For initial xenograft studies, U251 / EGFRvIII tumors were cultured at 10×10 6 The cells were mixed 1:1 with Matrigel and implanted subcutaneously onto the right flank of male SCID mice. Tumors grew to approximately 175 mm before treatment began and approximately 30 days after implantation. 3Mice were randomized into groups of 8 and treated with a single dose of test or control ADC. Tumor growth was monitored for 71 days after treatment.

[0173] To evaluate the efficacy of REGN3124-tesirin and COMP-MMAF ADCs against orthotopically placed GBM tumors in the brain, intracranial GBM59 PDX tumors were cultured at 3×10 5 PDX cells were injected into the graft. Intracranial injections were performed at a depth of 3 mm, 1 mm anterior and 2 mm lateral to the bregma. All orthotopic GBM PDX studies were performed by Translational Drug Development Inc. After allowing orthotopic GBM59 PDXs to establish for 25 days, mice were randomized into groups of 8 and treated with a single dose of test or control ADC. Mice were monitored for signs of peri-morbidity for 94 days and euthanized before reaching a moribund state.

[0174] Test Results: Studies in U251 / EGFRvIII xenograft-bearing mice evaluated the activity of REGN3124-Tesilin, designed to deliver a PBD payload of 7ug / kg, as well as the activity of the COMP-MMAF ADC at ADC doses of 1, 2.5, and 5mg / kg (Table 16). Isotype controls were included for all dose levels in the study, but only modest antitumor effects were observed with these control agents. REGN3124-Tesilin demonstrated clear antitumor activity in this study, as a single dose of 0.53mg / kg ADC (7ug / kg PBD payload dose) was able to induce sustained regression of the xenografts. No sustained regression was observed in tumors treated with COMP-MMAF at 1mg / kg. Activity against tumors was seen using 2.5 mg / kg COMP-MMAF, but 5 mg / kg COMP-MMAF was required to achieve sustained activity similar to that observed with treatment with REGN3124-tesirin at the completion of the study 71 days after dosing. All animals in this study showed a 10% increase in body weight over the post-treatment observation period.

[0175] Table 16. Regression of U251 / EGFRvIII xenografts mediated by anti-EGFRvIII-tesirin conjugate and COMP-MMAF conjugate compared to controls (day 36 post-treatment) TIFF2024527235000024.tif73154

[0176] The efficacy of REGN3124-Tesilin (DAR 1.9) and COMP-MMAF ADC was also evaluated against orthotopically placed GBM59 PDX tumors (Table 17). In this study, all eight mice treated with vehicle died of tumor burden at 25 days. Isotype control-Tesilin and control MMAF ADC did not extend survival compared to the vehicle control group. In contrast to the control, REGN3124-Tesilin mediated a highly significant extension of survival, with five of eight mice in this group surviving to completion of the study 95 days after dosing. COMP-MMAF ADC at 1 mg / kg did not induce a significant increase in survival, as the median survival of this group was the same as control-MMAF at 5 mg / kg. Although the higher 5 mg / kg COMP-MMAF treatment induced an increased degree of survival compared to the MMAF control, all mice died of tumor burden within 35 days of treatment, resulting in a median survival of 23.5 days.

[0177] Table 17. Activity of REGN3124-Tesilin conjugates and COMP-MMAF conjugates in mice bearing orthotopically placed GBM59 GBM PDX tumors TIFF2024527235000025.tif72154

[0178] Unofficial sequence listing An informal sequence listing listing the sequences disclosed herein is provided below.

[0179] Table 18: Sequence identifiers and corresponding nucleic acid and amino acid sequences TIFF2024527235000026.tif43162TIFF2024527235000027.tif239162TIFF20245272350 00028.tif240162TIFF2024527235000029.tif242162TIFF2024527235000030.tif170162

[0180] The present disclosure is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the present disclosure, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to be within the scope of the appended claims.

Claims

1. An antibody-drug conjugate (ADC) comprising an antibody or an antigen-binding fragment thereof that specifically binds to EGFRvIII, wherein the antibody or the antigen-binding fragment thereof comprises a heavy-chain variable region (HCVR) containing three heavy-chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) within the amino acid sequence of SEQ ID NO: 2, and a light-chain variable region (LCVR) containing three light-chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) within the amino acid sequence of SEQ ID NO: 10 and the antibody or the antigen-binding fragment thereof is conjugated to tesirine, the antibody-drug conjugate (ADC).

2. The anti-EGFRvIII antibody or the antigen-binding fragment thereof (i) does not bind to the linker peptide of SEQ ID NO: 23 (ii) nor to the peptide of SEQ ID NO: 24 The ADC according to claim 1.

3. When the antibody or antigen-binding fragment thereof is measured by surface plasmon resonance assay at 37° C., an equilibrium dissociation constant (K Ｄ ), for the human EGFRvIII monomer of about 500 nM, of the ADC according to claim 1.

4. When the antibody or antigen-binding fragment thereof is measured by surface plasmon resonance assay at 37° C., an equilibrium dissociation constant (K Ｄ ) for human EGFRvIII dimer of about 10 nM or less, the ADC according to claim 1.

5. The ADC according to claim 1, wherein the antibody or the antigen-binding fragment thereof does not bind to the EGFR dimer at a detectable level by surface plasmon resonance assay.

6. The antibody or the antigen-binding fragment thereof comprises an HCVR containing HCDR1 with the amino acid sequence of SEQ ID NO: 4, HCDR2 with the amino acid sequence of SEQ ID NO: 6, and HCDR3 with the amino acid sequence of SEQ ID NO: 8 and an LCVR containing LCDR1 with the amino acid sequence of SEQ ID NO: 12, LCDR2 with the amino acid sequence of SEQ ID NO: 14, and LCDR3 with the amino acid sequence of SEQ ID NO: 16 The ADC according to claim 1.

7. The ADC according to claim 1, wherein the antibody or the antigen-binding fragment thereof comprises an HCVR containing an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2, and an LCVR containing an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 10 The ADC according to claim 1.

8. The ADC according to claim 1, wherein the antibody or the antigen-binding fragment thereof comprises an HCVR containing an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 2, and an LCVR containing an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 10 The ADC according to claim 1.

9. The ADC according to claim 1, wherein the antibody or the antigen-binding fragment thereof comprises an HCVR containing an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 2, and an LCVR containing an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 10 An LCVR comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 10 The ADC according to claim 1, comprising the same. **Claim 10** The antibody or antigen-binding fragment thereof is an HCV R comprising the amino acid sequence of SEQ ID NO: 2, an LCVR comprising the amino acid sequence of SEQ ID NO: 10 The ADC according to claim 1, comprising the same. **Claim 11** The ADC according to claim 1, wherein the antibody or antigen-binding fragment thereof is a full antibody. **Claim 12** The ADC according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, and the heavy chain comprises the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO:

20. **Claim 13** The ADC according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, and the light chain comprises the amino acid sequence of SEQ ID NO:

22. **Claim 14** The ADC according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 18 and a light chain comprising the amino acid sequence of SEQ ID NO:

22. **Claim 15** The ADC according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 20 and a light chain comprising the amino acid sequence of SEQ ID NO:

22. **Claim 16** The ADC according to claim 1, wherein the drug-to-antibody ratio (DAR) is from about 1 to about 4. **Claim 17** The ADC according to claim 1, wherein the antibody or antigen-binding fragment thereof is aglycosylated at N297. **Claim 18** The ADC according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises an N297Q mutation determined by EU indexing in hIgG1 Fc. **Claim 19** The antibody or antigen-binding fragment thereof is an HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 6, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 8, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 12, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 14, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 16 The ADC according to claim 1, comprising the same, wherein the heavy chain of the antibody or antigen-binding fragment thereof is aglycosylated and comprises an N297Q mutation, and the antibody or antigen-binding fragment thereof is conjugated to tesirine. **Claim 20** The ADC according to claim 1, wherein the antibody or antigen-binding fragment thereof interacts with at least one residue within the amino acid sequence of SEQ ID NO:

26. **Claim 21** The following features: (a) showing reduced in vivo survival rate in EGFRvIII-expressing cells (b) showing in vivo bystander cytotoxicity against non-EGFRvIII-expressing cells co-cultured with EGFRvIII-expressing cells (c) showing extended survival in mice having intracranial glioblastoma multiforme expressing EGFRvIII (d) showing antitumor effect in mice having EGFRvIII-expressing tumors in the absence of treatment-related weight loss (e) showing tumor regression in mice having patient-derived glioblastoma multiforme (f) showing greater tumoricidal activity at a lower dosage as compared to a control antibody conjugated to MMAF, and (g) showing greater antitumor efficacy than anti-EGFRvIII-maytansinoid ADC in tumor-bearing mice The ADC according to claim 1, having one or more of the above. **Claim 22** A complex comprising the ADC according to claim 1, wherein the antibody or antigen-binding fragment thereof binds to EGFRvIII. **Claim 23** A container or injection device comprising the ADC according to claim 1. **Claim 24** A pharmaceutical composition comprising the ADC according to claim 1 and a pharmaceutically acceptable carrier or diluent. **Claim 25** The pharmaceutical composition according to claim 24, further comprising one or more additional therapeutic agents selected from the group consisting of chemotherapeutic agents, anti-inflammatory agents, and analgesics. **Claim 26** A composition comprising the ADC according to any one of claims 1 to 21 or the pharmaceutical composition according to claim 24 for use in a method of treating cancer in a subject in need thereof, wherein the subject is suffering from an EGFRvIII-expressing tumor, said composition. **Claim 27** A composition comprising the ADC according to any one of claims 1 to 21 or the pharmaceutical composition according to claim 24 for use in a method of treating cancer or a tumor, or reducing tumor growth, and / or inducing tumor regression, in a subject in need thereof. **Claim 28** The method according to claim 26, wherein the cancer or tumor is selected from the group consisting of glioblastoma, ductal carcinoma or intraductal carcinoma of the breast, non-small cell lung cancer, ovarian cancer, prostate cancer, and squamous cell carcinoma of the head and neck. **Claim 29** The method according to claim 26, further comprising administering one or more additional therapeutic agents selected from the group consisting of chemotherapeutic agents, anti-inflammatory agents, and analgesics. **Claim 30**: The composition according to claim 26, wherein the method further comprises administering a second ADC comprising an antibody or an antigen-binding fragment thereof and a cytotoxin, and the antibody or the antigen-binding fragment thereof of the second ADC specifically binds to EGFRvIII and also binds to the linker peptide of SEQ ID NO: 23 and / or the peptide of SEQ ID NO:

24. **Claim 31** A composition for use in a method for administering an ADC as claimed in any one of claims 1 to 21 to a subject's body, the method comprising injecting the ADC into the subject's body, the composition comprising the ADC. **Claim 32** The composition according to claim 31, wherein the ADC is injected subcutaneously, intravenously, or intramuscularly into the subject's body. **Claim 33** A method for producing an ADC as claimed in any one of claims 1 to 21, comprising culturing a host cell comprising a polynucleotide encoding an immunoglobulin comprising the HCVR of the ADC and an immunoglobulin comprising the LCVR of the ADC in a culture medium under conditions favorable for the expression of the polynucleotide. **Claim 34** The method according to claim 33, further comprising conjugating tesirine to one or more of the immunoglobulins. **Claim 35** The method according to claim 34, wherein the conjugation is carried out by reducing the immunoglobulin chain in the presence of a reducing agent and incubating the reduced immunoglobulin chain with the tesirine. **Claim 36** The method according to claim 35, wherein the reducing agent is dithiothreitol. **Claim 37** An ADC which is the product according to claim 33.

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