CN117460749A - anti-EGFRvIII antibody drug conjugates and uses thereof - Google Patents

Abstract

The present disclosure provides antibody-drug conjugates (ADCs) comprising antibodies conjugated to doxillin that bind to class III variants of EGFR (egfrvlll) and methods of use thereof. As used herein, an antibody or antigen binding fragment thereof, as in certain embodiments, binds human egfrvlll with high affinity. An antibody or antigen-binding fragment thereof as used herein may be a fully human antibody. The ADCs provided herein may be used to treat a variety of cancers.

Description

anti-EGFRvIII antibody drug conjugates and uses thereof

Cross Reference to Related Applications

The present application claims U.S. provisional patent application 63/213,478 filed on 22 th month 6 of 2021; and U.S. provisional patent application 63/242,929 filed on 9 and 10 of 2021; each of these two patent applications is incorporated by reference in its entirety.

Technical Field

The present disclosure relates to antibody-drug conjugates (ADCs) comprising human antibodies and antigen binding fragments of human antibodies, and methods of treatment using these ADCs, which specifically bind to deletion mutants of human Epidermal Growth Factor Receptor (EGFR), particularly class III deletion mutants egfrvlll, wherein the antibodies or antigen binding fragments thereof are conjugated to texilin (tesirine).

Sequence listing

The formal copy of the sequence list is submitted electronically via EFS-Web with the specification in ASCII format, file name 10966WO01_sequence_listing_ST25.TXT, date of creation 2022, 6, 21, size about 49,152 bytes. The sequence listing contained in this ASCII format document is part of the specification and is incorporated herein by reference in its entirety.

Background

Overexpression and/or gene amplification of the Epidermal Growth Factor (EGF) receptor or EGFR has been reported in a variety of human tumors including breast, ovarian, bladder, brain and various squamous cancers (Wong, A.J. et al, 1987, proc. Natl. Acad. Sci. USA,84:6899-6903; harris et al, 1992,Natl.Cancer Inst.Monogr.11:181-187). However, targeting EGFR as an anti-neoplastic therapeutic approach has been problematic because many normal tissues also express this receptor and can be targeted along with the neoplastic target. Meanwhile, many glioblastomas with EGFR gene amplification are reported to often contain gene rearrangements (Ekstrand, A.J. et al, 1992, proc.Natl. Acad. Sci. USA,89:4309-4313; wong A.J. et al, 1992, proc.Natl. Acad. Sci. USA, 89:2965-2969). In one study, 17 out of 44 glioblastomas were found to have one or more changes in EGFR coding sequence, and all of these cases contained amplified EGFR, while none of 22 cases without gene amplification showed any tumor specific sequence abnormalities (Frederick, L. Et al, 2000,Cancer Res 60:1383-1387). The same study also showed that multiple types of EGFR mutations could be detected in separate tumors.

The class III variants of EGFR (EGFRvIII) are the most common EGFR variants in glioblastomas (Bigner et al, 1990,Cancer Res 50:8017-8022; humphrey et al, 1990,Proc Natl Acad Sci USA 87:4207-4211; yamazaki et al, 1990,Jap J Cancer Res 81:773-779; ekstrand et al, 1992,Proc Natl Acad Sci USA 89:4309-4313; wikstrand et al, 1995,Cancer Res 55:3140-3148; and Frederick et al, 2000,Cancer Res 60:1383-1387). EGFRvIII is characterized by a deletion of exons 2-7 of the EGFR gene, which results in an in-frame deletion of 801 base pairs of the coding region, i.e., a deletion of 6-273 amino acid residues (based on the number of residues of mature EGFR), and new glycine production at the fusion junction (Humphrey et al 1988,Cancer Res 48:2231-2238; yamazaki et al, 1990, supra). EGFRvIII has been shown to have ligand independent, weak, but constitutively active kinase activity and enhanced tumorigenicity (Nishikawa et al 1994,Proc Natl Acad Sci USA 91:7727-7731; and Batra et al 1995,Cell Growth and Differentiation 6:1251-1259). In addition to gliomas, EGFRvIII has also been detected in ductal and intraductal breast cancer (Wikstrand et al, 1995,Cancer Res 55:3140-3148), non-small cell lung cancer (Garcia de Palazzo et al, 1993,Cancer Res 53:3217-3220), ovarian cancer (Moscatelo et al, 1995,Cancer Res 55:5536-5539), prostate cancer (olapad-Olaopa et al, 2000,British JCancer 82:186-194) and squamous cell carcinoma of the head and neck (Tinhofer et al, 2011,Clin Cancer Res17 (15): 5197-5204). In contrast, it was reported from these and other studies that normal tissue does 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 egfrvlll makes it a particularly useful target for the treatment of cancers and tumors that express this molecule.

The amino acid sequence of human EGFR is shown as SEQ ID NO 27, and the amino acid sequence of EGFRvIII is shown as SEQ ID NO 28. Antibodies against egfrvlll are described, for example, in US 5,212,290, US 7,736,644, US 7,589,180 and US 7,767,792.

All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

Disclosure of Invention

The present disclosure provides antibody-drug conjugates (ADCs) comprising an antibody that binds egfrvlll and antigen binding fragments thereof, wherein said antibody and antigen binding fragments thereof are conjugated to doxycycline. Texilin contains pyrrolobenzodiazepine(PBD) payload/warhead SG3199 (Tiberghein et al 2016,ACS Medicinal Chemistry Letters 7 (11): 983-987). The ADC is particularly useful for targeting egfrvlll expressing tumor cells.

Antibodies useful in ADCs provided herein may be full length (e.g., igG1 or IgG4 antibodies) or may comprise only antigen binding portions (e.g., fab, F (ab') ₂ Or scFv fragments) and may be modified to affect function, for example to eliminate residual effector function (Reddy et al, 2000, J.Immunol.164:1925-1933).

Exemplary anti-egfrvlll antibodies useful herein are listed in table 1. Table 1 lists amino acid sequence identifiers for the Heavy Chain Variable Region (HCVR), light Chain Variable Region (LCVR), heavy chain complementarity determining regions (HCDR 1, HCDR2, and HCDR 3), and light chain complementarity determining regions (LCDR 1, LCDR2, and LCDR 3) of exemplary anti-EGFRvIII antibodies. The complete heavy and light chain amino acid sequences of exemplary anti-egfrvlll antibodies are listed in table 2. Table 3 lists the nucleic acid sequence identifiers of HCVR, LCVR, HCDR, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of exemplary anti-EGFRvIII antibodies.

The present disclosure provides ADCs comprising an antibody or antigen binding fragment thereof that specifically binds egfrvlll comprising three complementarity determining regions (HCDR 1, HCDR2 and HCDR3, respectively) within 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.

The present disclosure also provides ADCs comprising an antibody or antigen binding fragment thereof that specifically binds egfrvlll comprising three complementarity determining regions (LCDR 1, 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.

The present disclosure provides ADCs comprising an antibody or antigen binding fragment thereof that specifically binds egfrvlll, said antibody or antigen binding fragment thereof comprising 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.

The present disclosure also provides an ADC comprising an antibody or antigen-binding fragment thereof that specifically binds egfrvlll, said antibody or antigen-binding fragment thereof 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.

The present disclosure also provides an ADC comprising an antibody or antigen-binding fragment thereof that specifically binds egfrvlll, said 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.

The present disclosure also provides an ADC comprising an antibody or antigen-binding fragment thereof that specifically binds egfrvlll, said antibody or antigen-binding fragment thereof comprising a heavy chain CDR1 (HCDR 1), said 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.

The present disclosure also provides an ADC comprising an antibody or antigen-binding fragment thereof that specifically binds egfrvlll, said antibody or antigen-binding fragment thereof comprising a heavy chain CDR2 (HCDR 2), said HCDR1 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.

The present disclosure also provides an ADC comprising an antibody or antigen-binding fragment thereof that specifically binds egfrvlll, said antibody or antigen-binding fragment thereof comprising a heavy chain CDR3 (HCDR 3), said HCDR1 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.

The present disclosure also provides an ADC comprising an antibody or antigen-binding fragment thereof that specifically binds egfrvlll, said antibody or antigen-binding fragment thereof comprising a light chain CDR1 (LCDR 1), said 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.

The present disclosure also provides an ADC comprising an antibody or antigen-binding fragment thereof that specifically binds egfrvlll, said antibody or antigen-binding fragment thereof comprising a light chain CDR2 (LCDR 2), said LCDR1 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.

The present disclosure also provides an ADC comprising an antibody or antigen-binding fragment thereof that specifically binds egfrvlll, said antibody or antigen-binding fragment thereof comprising a light chain CDR3 (LCDR 3), said LCDR1 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.

The present disclosure also provides an ADC comprising an antibody or antigen-binding fragment thereof that specifically binds egfrvlll, said antibody or antigen-binding fragment thereof comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR 3) comprised within the HCVR of SEQ ID NO:2 and the LCVR of SEQ ID NO: 10. In certain embodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence compositions 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.

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 a given HCVR and/or LCVR amino acid sequence disclosed herein. Exemplary conventions that may be used to identify boundaries of CDRs include, for example, kabat definitions, chothia definitions, and AbM definitions. In general, kabat definition is based on sequence differences, chothia definition is based on the position of structural loop regions, and AbM definition is a compromise between Kabat and Chothia methods. 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); martin et al, proc. Natl. Acad. Sci. USA 86:9268-9272 (1989) public databases may also be used to identify CDR sequences in antibodies.

The present disclosure includes ADCs comprising anti-egfrvlll antibodies with modified glycosylation patterns. In certain embodiments, antibodies modified to remove undesired glycosylation sites, or lacking fucose moieties present on oligosaccharide chains, may be used, for example, to increase antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al (2002) JBC 277:26733). In other applications, modifications to galactosylation may be made to alter Complement Dependent Cytotoxicity (CDC). In some embodiments, the antibody or antigen binding fragment thereof is non-glycosylated. Non-glycosylated antibodies are point mutated at appropriate residues to prevent glycosylation. In some aspects, the antibody or antigen binding fragment thereof comprises a heavy chain that is non-glycosylated at, for example, N297 (numbered according to EU index) to increase conjugation efficiency. In a particular embodiment, the N297 is mutated to a glutamine (Q) residue, i.e., the antibody comprises an N297Q mutation.

In another aspect, the invention provides a complex comprising an anti-egfrvlll-doxycycline ADC, wherein said antibody or antigen-binding fragment thereof binds to egfrvlll.

In another aspect, the invention provides a pharmaceutical composition comprising an ADC comprising doxillin and a recombinant human antibody or fragment thereof that specifically binds egfrvlll, and a pharmaceutically acceptable carrier. In a related aspect, the invention features a composition that is a combination of an anti-egfrvlll antibody-texilin and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with an anti-egfrvlll antibody-doxycycline ADC. Exemplary combination therapies and co-formulations comprising an anti-egfrvlll antibody-texilin ADC of the present disclosure are disclosed elsewhere herein.

In yet another aspect, the invention provides a therapeutic method of killing tumor cells or inhibiting or attenuating tumor cell growth using an anti-egfrvlll antibody-doxycycline conjugate or an antigen-binding portion of an antibody conjugated to doxycycline. The method of treatment 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-doxycycline conjugate or an antigen-binding fragment of an antibody conjugated to doxycycline. The disorder treated is any disease or condition ameliorated, alleviated, inhibited or prevented by targeting an ADC to egfrvlll.

Other embodiments will become apparent upon reading the detailed description that follows. Other embodiments will become apparent upon reading the detailed description that follows.

Drawings

FIG. 1 shows that the injection was performed at 0.5X10 on the flank of female SCID mice by subcutaneous injection ⁶ Comparison of anti-egfrvlll-texilin conjugate or anti-egfrvlll-maytansinoid DM1 conjugate with respect to tumor volume and weight 61 days after transplantation of MMT-egfrvlll cells.

Detailed Description

Before describing the present disclosure, it is to be understood that this invention is not limited to particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. 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.).

For purposes of describing and defining the present disclosure, it is noted that relative terms such as "substantially," "generally," "about," and the like are utilized herein to represent the inherent degree of uncertainty that may be attributed to any quantitative comparison, value, measurement, or other representation. These terms are also used herein to indicate that a quantitative representation may vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.

In some examples, the term "substantially" in reference to a given parameter, property, or condition may mean and include the extent to which the given parameter, property, or condition is met and the degree of deviation is small (such as within acceptable manufacturing tolerances) as would be understood by one of ordinary skill in the art. For example, a parameter, property, or condition may be at least 90% satisfied, at least 95% satisfied, at least 99% satisfied, or fully satisfied, depending on the particular parameter, property, or condition that is substantially satisfied.

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

Definition of the definition

As used herein, the term "egfrvlll" refers to a human EGFR class III variant having the amino acid sequence shown as SEQ ID No. 28, or a biologically active fragment thereof, which variant or biologically active fragment thereof exhibits any characteristics specific for egfrvlll, which characteristics are contrary to those common to normally expressed EGFR, unless explicitly stated otherwise. EGFRvIII lacks amino acid residues 6 to 273 (i.e., SEQ ID NO:27 without a signal peptide, i.e., residues 1-24) of mature EGFR and contains a new glycine residue at position 6 between amino acid residues 5 and 274.

All references herein to proteins, polypeptides and protein fragments are intended to refer to the human form of the various proteins, polypeptides or protein fragments unless explicitly indicated to be from a non-human species. Thus, unless indicated as being from a non-human species, e.g., "mouse egfrvlll," "monkey egfrvlll," etc., the expression "egfrvlll" means human egfrvlll.

The expression "cell surface expressed egfrvlll" as used herein refers to one or more egfrvlll proteins or extracellular domains thereof expressed on the cell surface in vitro or in vivo such that at least a portion of the egfrvlll proteins are exposed outside the cell membrane and accessible to the antigen binding portion of an antibody. "cell surface expressed EGFRvIII" may comprise or consist of EGFRvIII protein expressed on the surface of cells normally expressing EGFRvIII protein. Alternatively, "cell surface expressed egfrvlll" may comprise or consist of an egfrvlll protein expressed on the surface of a cell, which typically does not express human egfrvlll on its surface, but has been engineered to express egfrvlll on its surface.

The term "antibody" includes immunoglobulin molecules comprising four polypeptide chains (two heavy (H) chains and two light (L) chains) connected to each other by disulfide bonds, as well as multimers thereof (e.g., igM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or V _H ) And a heavy chain constant region. The heavy chain constant region comprises three domains: c (C) _H 1、C _H 2 and C _H 3. Each light chain comprises a light chainVariable regions (abbreviated herein as LCVR or V _L ) And a light chain constant region. The light chain constant region comprises a domain (C _L 1)。V _H Region and V _L The regions can be further subdivided into regions of high variability, known as Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, known as Framework Regions (FR). Each V _H And V _L Consists 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 various embodiments of the present disclosure, the FR of the anti-egfrvlll antibody (or antigen binding portion thereof) may be identical to the human germline sequence, or may be naturally or artificially modified. Amino acid consensus sequences may be defined based on parallel analysis of two or more CDRs.

As used herein, the term "antigen binding portion" of an antibody, an "antigen binding fragment" of an antibody, and the like, includes any naturally occurring, enzymatically available, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen binding fragments of antibodies can be derived from whole antibody molecules, for example, using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques involving manipulation and expression of DNA encoding antibody variable and optionally antibody constant domains. Such DNA is known and/or can be readily obtained from, for example, 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 biological techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, to create cysteine residues, to modify, add or delete amino acids, and the like.

Non-limiting examples of antigen binding fragments include: (i) Fab fragments; (ii) a F (ab') 2 fragment; (iii) Fd fragment; (iv) Fv fragments; (v) a single chain Fv (scFv) molecule; (vi) a dAb fragment; and (vii) a minimal recognition unit consisting of amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated Complementarity Determining Region (CDR) such as a CDR3 peptide) or a restricted FR3-CDR3-FR4 peptide. As used herein, other engineered molecules (such as domain-specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, trisomy, tetrasomy, microscler, nanobody (e.g., monovalent nanobody, bivalent nanobody, etc.), small Modular Immunopharmaceuticals (SMIPs), and shark variable IgNAR domains) are also encompassed within the expression "antigen-binding fragment".

The antigen binding fragment of an antibody typically comprises at least one variable domain. The variable domain may have any size or amino acid composition and will typically include at least one CDR contiguous to or in-frame with one or more framework sequences. In the presence of V _H Domain and V _L Domain associated antigen binding fragments, V _H And V _L The domains may be positioned in any suitable arrangement relative to each other. For example, the variable region may be a dimer and contain V _H -V _H 、V _H -V _L Or V _L -V _L A dimer. Alternatively, the antigen binding fragment of an antibody may contain monomer V _H Or V _L A domain.

In certain embodiments, the 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 can be found within antigen binding fragments of antibodies of the present disclosure include: (i) V (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)V _L -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, a step of; 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 domainsThe domains may be directly linked to each other or may be linked by full or partial hinge or linker regions. The hinge region may be comprised of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which results in 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 comprise one or more monomers V with each other and/or with any of the variable domain and constant domain configurations listed above _H Or V _L The domains are non-covalently associated homodimers or heterodimers (or other multimers) (e.g., via disulfide bonds).

Antibodies as used herein may act through Complement Dependent Cytotoxicity (CDC) or antibody dependent cell-mediated cytotoxicity (ADCC). "complementary 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 nonspecific cytotoxic cells expressing Fc receptors (FcR), such as Natural Killer (NK) cells, neutrophils, and macrophages, recognize antibodies bound on a target cell and thereby cause lysis of the target cell. CDC and ADCC may be measured using assays 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 for the ability of the antibody to fix complement and mediate cell-dependent cytotoxicity. Thus, the isotype of the antibody may be selected depending on whether antibody-mediated cytotoxicity requires the antibody.

In certain embodiments of the present disclosure, the anti-egfrvlll antibodies used herein are human antibodies. As used herein, the term "human antibody" 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 (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo), e.g., in CDRs and particularly in CDR 3. However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been implanted onto a human framework sequence.

In some embodiments, the antibodies used herein may be recombinant human antibodies. As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, produced, or isolated by recombinant means, such as antibodies expressed using recombinant expression vectors transfected into host cells (described further below), antibodies isolated from recombinant human antibody combinatorial libraries (described further below), antibodies isolated from animals (e.g., mice) transgenic for human immunoglobulin genes (see, e.g., taylor et al, (1992) nucleic acids res.20:6287-6295), or antibodies prepared, expressed, produced, or isolated by any other means that involves splicing human immunoglobulin gene sequences to 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 undergo in vitro mutagenesis (or, when using animals that are transgenic for human Ig sequences, undergo in vivo somatic mutagenesis) and thus V of the recombinant antibodies _H And V _L The amino acid sequence of the region is that which is although derived from human germline V _H And V _L Sequences and related thereto, but may not naturally occur within the germline repertoire of human antibodies in vivo.

Human antibodies can exist in two forms that are associated with hinge heterogeneity. In one form, the immunoglobulin molecule comprises a stable four-chain construct of about 150-160kDa, wherein the dimers are held together by interchain heavy chain disulfide bonds. In the second form, the dimer is not linked via an interchain disulfide linkage, and the molecule of about 75-80kDa consists of covalently coupled light and heavy chains (half antibodies). These forms are extremely difficult to isolate even after affinity purification.

The frequency of occurrence of the second form in the various intact IgG isotypes is due to, but is not limited to, structural differences associated with the hinge region isotype of the antibody. Single amino acid substitutions in the hinge region of human IgG4 hinge can be madeThe appearance of the second form was significantly reduced (Angal et al (1993) Molecular Immunology 30:105) to the level typically observed with human IgG1 hinges. The invention is covered on the hinge, C _H 2 or C _H Antibodies having one or more mutations in region 3, which may be desirable, for example, in production, to increase the yield of the desired antibody form.

The antibody used herein may be an isolated antibody. As used herein, "isolated antibody" means an antibody that has been identified and isolated and/or recovered from at least one component of a natural environment. For example, for purposes of this disclosure, an antibody that has been isolated or removed from at least one component of an organism, or from a tissue or cell in which the antibody naturally occurs or is naturally produced, is an "isolated antibody. Isolated antibodies also include in situ antibodies within recombinant cells. An isolated antibody is an antibody that has undergone at least one purification or isolation step. According to certain embodiments, the isolated antibody may be substantially free of other cellular material and/or chemicals.

The anti-egfrvlll antibodies used herein may comprise 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 as compared to the corresponding germline sequences from which the antibodies were derived. Such mutations can be readily determined by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, 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, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to give the corresponding residues of the germline sequence of the antibody, or the corresponding residues of another human germline sequence, or conservative amino acid substitutions of the corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"). One of ordinary skill in the art, starting from the heavy and light chain variable region sequences disclosed herein, can readily generate a variety of antibodies and antigen-binding fragments comprising one or more individual germline mutations or combinations thereof. In certain embodiments, V _H And _{and/or VL} Within a domain of structure The framed and/or CDR residues are mutated back to the residues in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., mutated residues that are present only within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or mutated residues that are present only within CDR1, CDR2, or CDR 3. In other embodiments, one or more of the framework and/or one or more CDR residues are mutated to one or more corresponding residues of a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antibody was originally derived). Furthermore, antibodies as used herein may contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., wherein certain individual residues are mutated to corresponding residues of a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to corresponding residues of a different germline sequence. After obtaining antibodies and antigen binding fragments containing one or more germline mutations, the antibodies and antigen binding fragments can be readily tested for one or more desired properties, such as improvement in binding specificity, increase in binding affinity, improvement or enhancement of antagonistic or agonistic biological properties (as the case may be), reduction in immunogenicity, and the like. Antibodies and antigen binding fragments obtained in this general manner are encompassed within the present disclosure.

The disclosure also includes anti-egfrvlll antibodies useful herein comprising variants of any one of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein with one or more conservative substitutions. For example, the present disclosure includes anti-egfrvlll antibodies having HCVR, LCVR and/or CDR amino acid sequences having, for example, 10 or less, 8 or less, 6 or less, 4 or less, or the like conservative amino acid substitutions relative to any of the HCVR, LCVR and/or CDR amino acid sequences set forth in table 1 herein.

The term "epitope" refers to an antigenic determinant that interacts with a specific antigen binding site (termed a paratope) in the variable region of an antibody molecule. A single antigen may have more than one epitope. Thus, different antibodies may bind to different regions on an antigen and may have different biological effects. Epitopes may be conformational or linear. Conformational epitopes are produced by the spatial juxtaposition of amino acids from different segments of a linear polypeptide chain. Linear epitopes are produced by adjacent amino acid residues in a polypeptide chain. In some cases, an epitope may include a sugar, a phosphoryl group, or a sulfonyl group moiety on an antigen.

The term "substantial identity" or "substantially identical" when applied to polypeptides means that when two peptide sequences are optimally aligned, such as by the programs GAP or BESTFIT, using default GAP weights, at least 95% sequence identity is shared, even more preferably at least 98% or 99% sequence identity. In some aspects, the different residue positions differ by conservative amino acid substitutions. A "conservative amino acid substitution" is an amino acid substitution in which an amino acid residue is substituted with another amino acid residue of a side chain (R group) that has similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of the protein. In the case where conservative substitutions of two or more amino acid sequences differ from each other, the percent sequence identity or degree of similarity may be adjusted upward to correct the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art. See, for example, pearson (1994) Methods mol. Biol.24:307-331, which is incorporated herein by reference. Examples of groups of amino acids containing 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 chain: lysine, arginine, and histidine; (6) acidic side chain: aspartic acid and glutamic acid, and (7) sulfur-containing side chains, i.e., cysteine and methionine. Preferred groups of conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change with positive values in PAM250 log likelihood matrix disclosed in the following documents: gonnet et al (1992) Science 256:1443-1445, which is incorporated herein by reference. A "moderately conservative" substitution is any change with a non-negative value in the PAM250 log likelihood matrix.

Sequence analysis software is typically used to measure sequence similarity, also known as sequence identity, of polypeptides. Protein analysis software uses similarity metrics with respect to assignments to various substitutions, deletions, and other modifications (including conservative amino acid substitutions) to match similar sequences. 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 organisms of different species), or between wild type proteins and mutant proteins thereof. See, e.g., GCG version 6.1. The polypeptide sequences may also be compared using FASTA (program in GCG version 6.1) using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA 3) provide alignment and percent sequence identity (Pearson (2000) supra) of the optimal overlap region between query and search sequences. Another preferred algorithm when comparing sequences of the present disclosure to a database containing a large number of sequences from different organisms is the computer program BLAST, in particular BLASTP or TBLASTN, using default parameters. See, for example, 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.

The subject is a mammal, preferably a human.

Anti-egfrvlll antibodies comprising Fc variants

According to certain embodiments of the present disclosure, an anti-egfrvlll antibody used herein comprises an Fc domain comprising one or more mutations that enhance or reduce binding of the antibody to FcRn receptor at an acidic pH, e.g., compared to a neutral pH. For example, the disclosure includes ADCs comprising an anti-egfrvlll antibody at C of the Fc domain _H 2 or C _H 3 comprising mutations in region, wherein one or more of the mutations is acidicThe affinity of the Fc domain for FcRn is increased in the environment (e.g., in the endosome at a pH ranging from about 5.5 to about 6.0). Such mutations can result in an increase in serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 Modifications at (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, N F or N434Y)]) The method comprises the steps of carrying out a first treatment on the surface of the Or modifications at positions 250 and/or 428; or a modification at position 307 or 308 (e.g., 308F, V F) or a modification at position 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 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 or 308P). In yet another embodiment, the modification comprises a 265A (e.g., D265A) and/or 297A (e.g., N297A) modification.

For example, the disclosure includes ADCs comprising anti-egfrvlll antibodies having an Fc domain comprising one or more pairs or sets 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); 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 within the antibody variable domains disclosed herein are contemplated as within the scope of the present disclosure.

The disclosure also includes compositions comprising a polypeptide having a chimeric heavy chain constant (C _H ) An ADC of a region of an anti-EGFRvIII antibody, wherein the chimeric C _H The region comprises C derived from more than one immunoglobulin isotype _H A section of the zone. For example, antibodies useful herein can comprise chimeric C _H A region comprising C derived from a human IgG1, human IgG2, or human IgG4 molecule _H 2 domain which is associated with a C derived from a human IgG1, human IgG2 or human IgG4 molecule _H 3 or a combination of all or part of the domains. According to certain embodiments, the antibodies useful herein comprise chimeric C with chimeric hinge regions _H A zone. For example, a chimeric hinge may comprise an "upper hinge" amino acid sequence (amino acid residues according to EU numbering at positions 216-227) derived from a human IgG1, human IgG2, or human IgG4 hinge region in combination with a "lower hinge" sequence (amino acid residues according to EU numbering at positions 228-236) derived from a human IgG1, human IgG2, or human IgG4 hinge region. According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from a hinge on human IgG1 or human IgG4 and amino acid residues derived from a hinge under human IgG 2. Comprising chimeric C as described herein _H Antibodies of the region may exhibit modified Fc effector functions in certain embodiments without adversely affecting the therapeutic or pharmacokinetic properties of the antibody. (see, e.g., U.S. provisional application No. 61/759,578 filed on 1, 2, 2013, the disclosure of which is incorporated herein by reference in its entirety).

In one embodiment of the invention, the Fc is IgG4 with the mutation S108P.

Antibody-drug conjugates (ADC)

Provided herein are antibody-drug conjugates (ADCs) comprising an anti-egfrvlll antibody or antigen binding fragment thereof conjugated to doxillin.

The texilin has the following structure:

texilin is also known as SG3249.

Provided herein are compounds having the following structure:

wherein Ab comprises an anti-egfrvlll antibody or antigen binding fragment thereof and-S-is a sulphur bond at a cysteine residue of said antibody or antigen binding fragment thereof. In a particular embodiment, 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 particular embodiments, ab comprises the HCDR1 amino acid sequence of SEQ ID NO. 4, the HCDR2 amino acid sequence of SEQ ID NO. 6, the HCDR3 amino acid sequence of SEQ ID NO. 8, the LCDR1 amino acid sequence of SEQ ID NO. 12, the LCDR2 amino acid sequence of SEQ ID NO. 14, and the LCDR3 amino acid sequence of SEQ ID NO. 16. In particular embodiments, ab comprises a HCVR amino acid sequence that has at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO. 2, and a LCVR amino acid sequence that has at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO. 10. In particular embodiments, ab comprises the HCVR amino acid sequence of SEQ ID NO. 2 and/or the LCVR amino acid sequence of SEQ ID NO. 10.

Also provided herein are compounds having the following structure:

wherein Ab comprises an anti-egfrvlll antibody or antigen binding fragment thereof and-S-is a sulphur bond at a cysteine residue of said antibody or antigen binding fragment thereof. In certain embodiments, ab is a whole antibody. In a particular embodiment, ab comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO. 18. In a particular embodiment, ab comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO. 20. In a particular embodiment, ab comprises a heavy chain and a light chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO. 22. In a particular embodiment, 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 a particular embodiment, 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.

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

The synthesis of doxillin can be carried out, for example, using the procedure described by Tibergheien et al (ACS Medicinal Chemistry Letters2016,7 (11): 983-987). Texilin contains pyrrolobenzodiazepineWarhead/payload component SG3199, having the following structure:

epitope mapping and related techniques

An epitope to which an antibody as used herein binds may consist of a single contiguous sequence of amino acids of 3 or more (e.g., 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) egfrvlll proteins. Alternatively, an epitope may consist of a plurality of non-contiguous amino acids (or amino acid sequences) of egfrvlll. In some embodiments, the epitope is located on or near the ligand binding domain of egfrvlll. In other embodiments, the epitope is located outside the ligand binding domain of egfrvlll, e.g., at a location on the surface of egfrvlll where binding of the ligand to egfrvlll is not interfered with when the antibody binds to such epitope.

According to certain embodiments, antibodies and antigen-binding fragments thereof used herein include anti-EGFRvIII antibodies that specifically bind EGFRvIII (and do not bind EGFR), wherein the antibodies recognize an EGFRvIII connecting peptide (e.g., SEQ ID NO: 23). Such antibodies may be referred to herein as "linker peptide conjugates," "egfrvlll peptide binding antibodies," and the like. According to other embodiments, an anti-EGFRvIII antibody used herein specifically binds EGFRvIII (and does not bind EGFR), wherein the antibody does not recognize an EGFRvIII linker peptide (e.g., does not recognize the linker peptide of SEQ ID NO:23, and/or does not recognize the peptide of SEQ ID NO: 24). Such antibodies may be referred to herein as "conformational binders", "egfrvlll conformational epitope binders", and the like.

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

Various techniques known to those of ordinary skill in the art may be used to determine whether an antibody or antigen-binding fragment thereof "interacts with one or more amino acids" within a polypeptide or protein. Exemplary techniques include, for example, conventional cross-blocking assays (such asAntibodiesDescribed by Harlow and Lane (Cold Spring Harbor Press, cold Spring Harb., N.Y.), alanine scanning mutagenesis analysis, peptide blotting analysis (Reineke, 2004,Methods Mol Biol 248:443-463) and peptide cleavage analysis. In addition, methods such as epitope excision, epitope extraction and chemical modification of the antigen can be employed (Tomer, 2000,Protein Science 9:487-496). Another method that may be used to identify amino acids within polypeptides that interact with antibodies is hydrogen/deuterium exchange detected by mass spectrometry. In general, the hydrogen/deuterium exchange method involves deuterium labeling a protein of interest, and then binding the antibody to the deuterium labeled protein. The protein/antibody complex is then transferred to water to allow hydrogen-deuterium exchange to occur at all residues except the antibody protected residues (still deuterium labeled). After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry to reveal deuterium labeled residues corresponding to the particular amino acid that interacts with the antibody. See For example, ehring (1999) Analytical Biochemistry 267 (2): 252-259; engen and Smith (2001) Anal chem.73:256A-265A.

The disclosure also includes ADCs comprising anti-egfrvlll antibodies (e.g., antibodies comprising any of the amino acid sequences listed in table 1 herein) that bind to the same epitope as any of the specific exemplary antibodies described herein. Also, the disclosure also includes ADCs comprising anti-egfrvlll antibodies that compete with any of the specific exemplary antibodies described herein (e.g., antibodies comprising any of the amino acid sequences listed in table 1 herein) for binding to egfrvlll.

Whether an antibody binds to the same epitope as a reference anti-egfrvlll antibody or competes for binding with a reference anti-egfrvlll antibody can be readily determined by using conventional methods known in the art and exemplified herein. For example, to determine whether a test antibody binds to the same epitope as a reference anti-egfrvlll antibody of the present disclosure, the reference antibody is allowed to bind to egfrvlll protein. The ability of the test antibodies to bind to the egfrvlll molecules was then assessed. If the test antibody is capable of binding to egfrvlll after saturation binding to the reference anti-egfrvlll antibody, the following can be concluded: the test antibody binds to a different epitope than the reference anti-egfrvlll antibody. In another aspect, if the test antibody is unable to bind to the egfrvlll molecule after saturation binding to the reference anti-egfrvlll antibody, the test antibody may bind to the same epitope as the epitope to which the reference anti-egfrvlll antibody of the present disclosure binds. Additional routine experiments (e.g., peptide mutation and binding assays) can then be performed to confirm whether the observed lack of binding of the test antibody is actually due to the same epitope as the reference antibody binding or whether it is a steric block (or another phenomenon) resulting in the observed lack of binding. Such experiments 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 said to bind to the same epitope if substantially all of the amino acid mutations in an antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody. Two antibodies are considered to have an "overlapping epitope" if only a subset of the amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other antibody.

To determine whether an antibody competes for binding (or cross-competes for binding) with a reference anti-egfrvlll antibody, the above binding method was performed in two directions: in a first direction, the reference antibody is allowed to bind to the egfrvlll protein under saturated conditions, and then the binding of the test antibody to the egfrvlll molecule is assessed. In the second direction, the test antibody is allowed to bind to the egfrvlll molecule under saturated conditions, and then the binding of the reference antibody to the egfrvlll molecule is evaluated. If only the first (saturated) antibody is able to bind to the egfrvlll molecule in both directions, the following can be concluded: the test antibody and the reference antibody compete for binding to egfrvlll. As will be appreciated by one of ordinary skill in the art, an antibody that competes for binding with a reference antibody may not necessarily bind to the same epitope as the reference antibody, but may spatially block binding of the reference antibody by binding overlapping or adjacent epitopes.

Biological characteristics of anti-EGFRvIII ADC

The invention includes anti-egfrvlll-doxycycline ADCs that specifically bind to egfrvlll. In some aspects, the ADC comprises an anti-egfrvlll antibody or antigen binding fragment thereof, which does not bind to: (i) a linker peptide of SEQ ID NO. 23; nor (ii) the peptide of SEQ ID NO. 24. In some aspects, the ADC comprises an anti-egfrvlll antibody or antigen binding fragment thereof that exhibits an equilibrium dissociation constant (K _D ) About 500nM, as measured by surface plasmon resonance assay at 37 ℃. In some aspects, the ADC comprises an anti-egfrvlll antibody or antigen binding fragment thereof, said anti-egfrvlllThe antibody or antigen-binding fragment thereof exhibits an equilibrium dissociation constant (K) for human egfrvlll dimer _D ) About 10nM or less, as measured by surface plasmon resonance assay at 37 ℃. In some aspects, the ADC comprises an anti-egfrvlll antibody or antigen binding fragment thereof that does not bind EGFR dimer at a level detectable by a surface plasmon resonance assay.

In some embodiments, the anti-egfrvlll-texilin ADC exhibits one or more of the following characteristics: (a) In egfrvlll expressing cells, exhibiting reduced in vivo viability; (b) Exhibiting in vivo bystander cytotoxicity against cells not expressing egfrvlll co-cultured with cells expressing egfrvlll; (c) In mice with intracranial glioblastoma multiforme expressing egfrvlll, an increase in survival was demonstrated; (d) In the absence of treatment-related weight loss, an anti-tumor effect was exhibited in mice with tumors expressing egfrvlll; (e) Tumor regression was shown in mice with patient-derived glioblastoma multiforme; (f) Demonstrating a stronger tumor killing effect at lower doses relative to the comparison antibody conjugated to MMAF; and/or (g) exhibits greater anti-tumor efficacy in tumor-bearing mice than anti-egfrvlll-maytansinoid ADC.

Preparation of human antibodies

The anti-egfrvlll antibody or antigen binding fragment thereof used herein may be a fully human antibody. Methods for producing monoclonal antibodies, including fully human monoclonal antibodies, are known in the art. Any such known methods may be used in the context of the present disclosure to prepare human antibodies that specifically bind to human egfrvlll.

Using, for example, VELOCIMUNE ^TM The technique, or any other similar known method for producing fully human monoclonal antibodies, initially isolated a high affinity chimeric antibody against egfrvlll having a human variable region and a mouse constant region. Desirable characteristics of antibodies, including affinity, ligand blocking activity, selectivity, epitopes, and the like, are characterized and selected as described in the experimental section below. If desired, with the desired human constant region (e.g., wild-typeOr modified IgG1 or IgG 4) to replace the mouse constant region to produce a fully human anti-egfrvlll antibody. While the constant region selected may vary depending on the particular application, high affinity antigen binding and target-specific features are present in the variable region. In some cases, fully human anti-egfrvlll antibodies are isolated directly from antigen positive B cells.

The present invention includes a method for preparing an ADC comprising an antibody of the invention or an antigen-binding fragment thereof that specifically binds egfrvlll, said method comprising culturing a host cell comprising a polynucleotide encoding such an immunoglobulin in a culture medium under conditions conducive for expression of the polynucleotide: an immunoglobulin comprising an HCVR of said antibody or fragment, and an immunoglobulin comprising an LCVR of said antibody or fragment. One or more of the immunoglobulins of the antibodies or fragments so produced may then be conjugated to a texilin, for example, by reduction (e.g., in the presence of dithiothreitol) of the immunoglobulin chain, and incubating the texilin with the reduced immunoglobulin chain. Host cells that can be expressed in such antibodies or fragments are eukaryotic or prokaryotic host cells, e.g., mammalian cells. Such host cells are well known in the art and many are available from the American type culture Collection (American Type Culture Collection; ATCC). These host cells include, inter alia, chinese Hamster Ovary (CHO) cells, NSO, SP2 cells, heLa 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 a variety of other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse, and hamster cells. Other cell lines that may be used are insect cell lines (e.g., spodoptera frugiperda (Spodoptera frugiperda) or spodoptera frugiperda (Trichoplusia ni)), amphibian cells, bacterial cells, plant cells and fungal cells. Fungal cells include yeast and filamentous fungal cells, including, for example, pichia (Pichia), pichia pastoris (Pichia pastoris), pichia finland (Pichia finlandica), pichia pastoris (Pichia trehalophila), pichia kochia (Pichia koalae), pichia membranaefaciens (Pichia membranaefaciens), pichia pastoris (Pichia minuta) (Ogatae minuta), 3995 red yeast (Pichia lindineri)), pichia pastoris (Pichia opuntia) and Pichia thermotolerans (Pichia thermotolerans), liu Bichi yeast (Pichia salictaria), pichia kudzuvine (Pichia guercuum), pi Jiepu Pichia pastoris (Pichia Pichia pastoris), pichia stiptis (methanol (4), pichia species (Pichia sp.), saccharomyces cerevisiae (Saccharomyces cerevisiae), saccharomyces cerevisiae (Saccharomyces sp)), candida sp), hansenula (Hansenula polymorpha), kluyveromyces (Kluyveromyces), aspergillus niger (3295), fusarcandidum sp (4632), fusarium sp (Pichia pastoris), pichia pastoris (3795), fusarium sp (Pichia pastoris), pichia pastoris (Pichia pastoris), and other species (P.sp.sp.sp.sp.cerevisiae (3795) Physcomitrella patens (Physcomitrella patens) and Neurospora crassa (Neurospora crassa). Part of the invention is to produce an ADC by this method.

Bioequivalence

Anti-egfrvlll antibodies and antibody fragments useful herein encompass proteins having an amino acid sequence different from the antibody but retaining the ability to bind human egfrvlll. Such variant antibodies and antibody fragments comprise one or more additions, deletions or substitutions of amino acids when compared to the parent sequence, but exhibit biological activity substantially equivalent to that of the antibody. Likewise, the DNA sequences encoding anti-egfrvlll antibodies of such antibodies encompass sequences comprising one or more nucleotide additions, deletions or substitutions as compared to the disclosed sequences, but encoding anti-egfrvlll antibodies or antibody fragments that are substantially bioequivalent to the anti-egfrvlll antibodies or antibody fragments of the present disclosure. Examples of such variant amino acid and DNA sequences are discussed above.

Two antigen binding proteins or antibodies are considered bioequivalent if, for example, they are drug equivalents or drug substitutes that do not exhibit a significant difference in rate and extent of absorption when administered in a single dose or multiple doses of the same molar dose under similar experimental conditions. If some antibodies are to the same extent absorbed but differ in the rate of absorption, they may be regarded as equivalents or drug substitutes, but may still be regarded as bioequivalent, since such differences in the rate of absorption are intentional and reflected in the specification, are not necessary for the achievement of an effective in vivo drug concentration (e.g. in long term use), and are regarded as medically insignificant for the particular drug product under investigation.

In one embodiment, two antigen binding proteins are bioequivalent if there are no clinically significant differences in the safety, purity, and potency of the two antigen binding proteins.

In one embodiment, two antigen binding proteins are bioequivalent if a patient can perform such a switch one or more times without an expected increase in risk of adverse reactions, including clinically significant changes in immunogenicity, or reduced effectiveness, as compared to a continuous therapy without a switch between a reference product and a biologic product.

In one embodiment, two antigen binding proteins are bioequivalent if they both function by one or more coaction mechanisms for one or more conditions of use to the extent that such mechanisms are known.

Bioequivalence can be demonstrated by in vivo and in vitro methods. Bioequivalence measures include, for example, (a) in vivo tests in humans or other mammals in which the concentration of antibodies or their metabolites in blood, plasma, serum or other biological fluids is measured over time; (b) In vitro tests associated with and reasonably predictive of human bioavailability data; (c) In vivo tests in humans or other mammals, in which appropriate acute pharmacological effects of the antibody (or target thereof) over time are measured; and (d) establishing a clinical trial of a good control of antibody safety, efficacy, or bioavailability or bioequivalence.

Bioequivalent variants of anti-egfrvlll antibodies as used herein can be constructed, for example, by various substitutions of residues or sequences or deletion of terminal or internal residues or sequences that are not required for biological activity. For example, cysteine residues that are not necessary for biological activity may be deleted or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation. In other cases, bioequivalent antibodies may include anti-egfrvlll antibody variants comprising amino acid changes that modify the glycosylation characteristics of the antibody, e.g., mutations that eliminate or remove glycosylation.

Species selectivity and species cross-reactivity

According to certain embodiments, the present disclosure provides anti-egfrvlll antibodies used herein that bind to human egfrvlll but not to egfrvlll from other species. The disclosure also includes anti-egfrvlll antibodies that bind to human egfrvlll and egfrvlll from one or more non-human species. For example, an anti-egfrvlll antibody useful herein may bind to human egfrvlll and may or may not 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 egfrvlll, as the case may be. According to certain exemplary embodiments, anti-egfrvlll antibodies that specifically bind to human egfrvlll and to egfrvlll of cynomolgus macaque (e.g., macawafascicularis) are provided. Other anti-egfrvlll antibodies of the present disclosure bind to human egfrvlll, but do not bind or only weakly bind to cynomolgus monkey egfrvlll.

Therapeutic formulations and administration

The present disclosure provides pharmaceutical compositions comprising an anti-egfrvlll antibody-textilene Lin Zhuige (i.e., an anti-egfrvlll antibody-textilene ADC). Formulation of the present disclosure with suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolerability, and the likeA pharmaceutical composition. Numerous suitable formulations can be found in all prescription sets known to pharmaceutical chemists: remington's Pharmaceutical Sciences, mack Publishing Company, easton, PA. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid-containing (cationic or anionic) vesicles (such as LIPOFECTIN) ^TM Life Technologies, carlsbad, CA), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsion carbowaxes (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowaxes. See also Powell et al, "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:52:238-311.

The dosage of ADC administered to a patient may vary depending on the age and size of the patient, the disease of interest, the condition, the route of administration, and the like. The preferred dosage is typically calculated from body weight or body surface area. In adult patients, it may be advantageous to administer the antibodies of the present disclosure intravenously, typically in a single dose of about 0.001 to about 20mg/kg body weight, more preferably about 0.002 to about 7, about 0.003 to about 5, or about 0.005 to about 3mg/kg body weight. Exemplary dosages include 1 μg/kg, 3.5 μg/kg, 7 μg/kg, and 10 μg/kg. The frequency and duration of treatment may be adjusted depending on the severity of the condition. Effective dosages and schedules for administration of anti-egfrvlll antibody conjugates can be determined empirically; for example, patient progress may be monitored by periodic assessment and the dose adjusted accordingly. In addition, dose inter-species scaling may be performed using methods well known in the art (e.g., mordinti et al, 1991, pharmacut. Res. 8:1351).

Various delivery systems are known and may be used to administer the pharmaceutical compositions of the present disclosure, e.g., encapsulated in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, e.g., 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 composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through the epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered with other bioactive agents. Administration may be systemic or local. Accordingly, provided herein are methods of administering an anti-egfrvlll antibody-texilin ADC to a subject, the method comprising injecting the ADC into the subject. In some aspects, the ADC is subcutaneously injected into the subject. In some aspects, the ADC is injected intravenously into the subject. In some aspects, the ADC is injected intramuscularly into the subject.

The pharmaceutical compositions of the present disclosure may be provided in a container. The pharmaceutical compositions of the present disclosure may be provided in an injection device. The pharmaceutical composition may be delivered subcutaneously or intravenously using standard needles and syringes. Furthermore, for subcutaneous delivery, pen-type delivery devices are readily applicable for delivering the pharmaceutical compositions of the present disclosure. Such pen delivery devices may be reusable or disposable. Reusable pen delivery devices typically utilize replaceable cartridges containing a pharmaceutical composition. Once all of the pharmaceutical composition within 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 may then be reused. In disposable pen delivery devices, there is no replaceable cartridge. In practice, disposable pen delivery devices are prefilled with a pharmaceutical composition in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

A variety of reusable pen-type delivery devices and auto-injector delivery devices are used to subcutaneously deliver the pharmaceutical compositions of the present disclosure. Examples include, but are not limited to, AUTOPEN ^TM (Owen Mumford,Inc.,Woodstock,UK)、DISETRONIC ^TM Pen (Disetronic Medical Systems, bergdorf, switzerland), HUMALOG MIX 75/25 ^TM Pen and HUMALOG ^TM Pen, HUMALIN 70/30 ^TM Pen (Eli Lilly and Co., indianapolis, ind.), NOVOPEN ^TM I. II and III (Novo Nordisk, copenhagen, denmark), NOVOPEN JUNIOR ^TM (Novo Nordisk,Copenhagen,Denmark)、BD ^TM Pen (Becton Dickinson, franklin Lakes, NJ), OPTIPEN ^TM 、OPTIPEN PRO ^TM 、OPTIPEN STARLET ^TM And OPTICLIK ^TM (Sanofi-Aventis, frankfurt, germany) and the like. Examples of disposable pen delivery devices useful for subcutaneous delivery of the pharmaceutical compositions of the present disclosure include, but are not limited to, SOLOSTAR ^TM Pen (Sanofi-Aventis), FLEXPEN ^TM (Novo Nordisk), and KWIKPEN ^TM (Eli Lilly)、SURECLICK ^TM Autoinjector (Amgen, thonsand Oaks, calif.), PENLET ^TM (Haselmeier, stuttgart, germany), EPIPEN (Dey, L.P.), and HUMIRA ^TM Pen (Abbott Labs, abbott Park IL), and the like.

In some cases, the pharmaceutical composition may be delivered in a controlled release system. In one embodiment, a pump (see Langer, supra; sefton,1987,CRC Crit.Ref.Biomed.Eng.14:201) may be used. In another embodiment, a polymeric material may be used; see Medical Applications of Controlled Release, langer and Wise (editions), 1974, crc Pres., boca Raton, florida. In yet another embodiment, the controlled release system may be placed in proximity to the composition target, thus requiring only a small portion of the systemic dose (see, e.g., goodson,1984, supra, medical Applications of Controlled Release, volume 2, pages 115-138). Other controlled release systems are discussed in the review by Langer,1990, science 249:1527-1533.

Injectable formulations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injection, drip infusion, and the like. These injectable formulations can be prepared by well known methods. For example, injectable formulations can be prepared, for example, by dissolving, suspending or emulsifying the antibodies or salts thereof described above in sterile aqueous or oily media conventionally used for injection. As the aqueous medium for injection, there are, for example, physiological saline, isotonic solution containing glucose and other auxiliary agents and the like, which may be used in combination with a suitable solubilizing agent such as alcohol (e.g., ethanol), polyol (e.g., propylene glycol, polyethylene glycol), nonionic surfactant [ e.g., polysorbate 80, HCO-50 (hydrogenated castor oil polyoxyethylene (50 mol) adduct) ] and the like. As the oily medium, for example, sesame oil, soybean oil, etc., are used, and they may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in a suitable ampoule.

Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared in dosage forms suitable for unit doses conforming to the dosage of the active ingredient. Such dosage forms in unit dosage form include, for example, tablets, pills, capsules, injectable solutions (ampoules), suppositories and the like. The amount of the aforementioned antibodies contained is typically from about 5 to about 500mg per dosage form in a unit dose; especially in the injectable form, it is preferred for other dosage forms to contain the aforementioned antibodies in an amount of about 5 to about 100mg and about 10 to about 250 mg.

Thus, the invention includes a method of administering an ADC of the invention to a subject (e.g., wherein the subject has cancer), comprising the step of introducing the ADC into the subject, e.g., by injection or any of the methods discussed herein.

The invention also includes a container (e.g., a glass or plastic vial; or a bag, such as an iv bag) or any such device comprising an ADC of the invention, e.g., a syringe comprising a barrel, plunger, and needle.

Therapeutic uses of anti-EGFRvIII antibody conjugates

The present disclosure includes such methods: the method comprises administering to a subject in need thereof a therapeutic composition comprising an antibody-drug conjugate comprising an anti-egfrvlll antibody conjugated to doxillin (e.g., an anti-egfrvlll antibody or ADC comprising any one of the HCVR/LCVR or CDR sequences listed in table 1 herein). The therapeutic composition may comprise any one of an anti-egfrvlll antibody or antigen binding fragment thereof conjugated to doxillin, and a pharmaceutically acceptable carrier or diluent.

The ADCs of the present disclosure are particularly useful for treating, preventing and/or ameliorating any disease or disorder associated with or mediated by egfrvlll expression or activity or overexpression, or which may be treated by blocking the interaction between egfrvlll and EGFR ligands or inhibiting egfrvlll activity and/or signaling, and/or promoting receptor internalization and/or reducing the number of cell surface receptors. For example, the ADCs of the present disclosure may be used to treat tumors that express egfrvlll 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 genital tract, muscle, bone, skin and appendages, connective tissue, spleen, immune system, hematopoietic cells and bone marrow, liver and urinary tract, and special sensory organs such as the eyes. In certain embodiments, the ADC of the present disclosure is used to treat one or more of the following cancers: glioblastoma, renal cell carcinoma, pancreatic cancer, head and neck cancer, prostate cancer, glioblastoma, 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.

In the context of the methods of treatment described herein, the anti-egfrvlll antibody-texti Lin Zhuige may be administered as monotherapy (i.e., as the sole therapeutic agent) or in combination with one or more additional therapeutic agents (examples of which are described elsewhere herein).

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

Combination therapy and formulation

The present disclosure includes compositions and therapeutic formulations comprising any of the anti-egfrvlll antibody-doxillin conjugates described herein in combination with one or more additional therapeutically active components, as well as methods of treatment comprising administering such combinations to a subject in need thereof.

The anti-egfrvlll antibody-doxycycline conjugate used herein can be co-formulated and/or administered in combination with one or more additional therapeutically active components selected from the group consisting of: PRLR antagonists (e.g., anti-PRLR antibodies or small molecule inhibitors of PRLR), EGFR antagonists (e.g., anti-EGFR antibodies [ e.g., cetuximab ] or panitumumab (panitumumab)]Or small molecule inhibitors of EGFR [ e.g., gefitinib or erlotinib]) Antagonists of another EGFR family member such as Her2/ErbB2, erbB3 or ErbB4 (e.g., anti-ErbB 2[ e.g., trastuzumab) or T-DM1anti-ErbB 3 or anti-ErbB 4 antibodies or small molecule inhibitors of ErbB2, erbB3 or ErbB4 activity), cMET antagonists (e.g., anti-cMET antibodies), IGF1R antagonists (e.g., anti-IGF 1R antibodies), B-raf inhibitors (e.g., vorofil) Nylon (vemurafenib), sorafenib (sorafenib), GDC-0879, PLX-4720), PDGFR-alpha inhibitors (e.g., anti-PDGFR-alpha antibodies), PDGFR-beta inhibitors (e.g., anti-PDGFR-beta antibodies or small molecule kinase inhibitors, e.g., imatinib mesylate or sunitinib malate), PDGF ligand inhibitors (e.g., anti-PDGF-A, PDGF-B, PDGF-C or PDGF-D antibodies, aptamers, sirnas, etc.), VEGF antagonists (e.g., VEGF-Trap, such as aflibercept (aflibercept), see, e.g., US 7,087,411 (also referred to herein as "VEGF inhibitory fusion protein"), anti-VEGF antibodies (e.g., bevacizumab)), small molecule kinase inhibitors of VEGF receptors (e.g., sunitinib, sorafenib, or pazopanib), DLL4 antagonists (e.g., anti-DLL 4 antibodies disclosed in US2009/0142354, such as REGN 421), ang2 antagonists (e.g., anti-Ang 2 antibodies disclosed in US2011/0027286, such as H1H 685P), FOLH1 antagonists (e.g., anti-FOLH 1 antibodies), STEAP1 or STEAP2 antagonists (e.g., anti-STEAP 1 antibodies or anti-STEAP 2 antibodies), TMPRSS2 antagonists (e.g., anti-TMPRSS 2 antibodies), MSLN antagonists (e.g., anti-MSLN antibodies), CA9 antagonists (e.g., anti-CA 9 antibodies), and urolysin antagonists (e.g., anti-urolysin [ e.g., anti-UPK 3A) ]Antibodies), MUC16 antagonists (e.g., anti-MUC 16 antibodies), tn antigen antagonists (e.g., anti-Tn antibodies), CLEC12A antagonists (e.g., anti-CLEC 12A antibodies), TNFRSF17 antagonists (e.g., anti-TNFRSF 17 antibodies), LGR5 antagonists (e.g., anti-LGR 5 antibodies), monovalent CD20 antagonists (e.g., monovalent anti-CD 20 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-egfrvlll antibody-doxycycline 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.

The present disclosure includes compositions and therapeutic formulations comprising any of the anti-egfrvlll antibody conjugates described herein with one or moreCombinations of multiple chemotherapeutic agents. Examples of chemotherapeutic agents include: alkylating agents, e.g. thiotepa and cyclophosphamide (Cytoxan) ^TM ) The method comprises the steps of carrying out a first treatment on the surface of the Alkyl sulfonates such as busulfan (busulfan), imperoshu (imposulfan) and piposulfan (piposulfan); aziridines such as benzotepa (benzodopa), carboquinone (carboquone), mettussidine (meturedopa) and uratepa (uredopa); ethyleneimines and methyltriamines, including altretamine, triamcinolone acetoamide, triethylenephosphoramide, triethylenethiophosphamide and trimethylol melamine; nitrogen mustards, such as chlorambucil (chlorrambucil), napthalene mustards (chlormaphazine), cholesteryl phosphoramide (chlorophosphoramide), estramustine (estramustine), ifosfamide (ifosfamide), mechlorethamine (mechlorethamine), chlorambucil hydrochloride (mechlorethamine oxide hydrochloride), melphalan (melphalan), novenchin (novembrane), bennethol (phenaterine), prednisolone (prednisone), triamcinolone (trofosfamide), uracil mustards (uracils mustards); nitroureas, for example carmustine (carmustine), chloroureptin (chlorozotocin), fotemustine (fotemustine), lomustine (lomustine), nimustine (nimustine), ramustine (ranimustine); antibiotics, for example, aclacinomycin (aclacinomycin), actinomycin (actinomycin), anthramycin (authamycin), azoserine (azaserine), bleomycin (bleomycin), actinomycin C (cactinomycin), calicheamicin (calicheamicin), casrubicin (carabin), carminomycin (carminomycin), acidophilicin (carzinophilin), chromomycins (chromomycins), actinomycin D (dactinomycin), daunorubicin (daunorubicin), dithimycin (deoxyubicin), 6-diazo-5-oxo-L-norubicin, doxorubicin (doxorubin), epirubicin (epirubicin), epoubicin (escarubicin), idarubicin (idarubicin), hemacycin (mitomycin), mitomycin (35 mycin), dactinomycin (vancomycin), novobicin (nucomycin), nucomycin (pomycins), nuomicin (pomycins), and nuomicin (pomycins) (puromycin), tri-iron doxorubicin (queamycin), rodobicin (rodorubicin), streptozocin (streptozocin), tuberculin (tubercidin), ubenimex (ubenimex), zinostatin (zinostatin), zorubicin (zorubicin); antimetabolites, for example, methotrexate (methotrexate) and 5-fluorouracil (5-FU); folic acid analogs such as, for example, dimethyl folic acid (denopterin), methotrexate, pteroyltri-glutamic acid (pteroplegin), trimetric sand (trimetricate); purine analogs, e.g., fludarabine (fludarabine), 6-mercaptopurine, thioazane (thiamiprine), thioguanine (thioguanine); pyrimidine analogs, for example, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, fluorouridine; androgens, for example, card Lu Gaotong (calasterone), droxidone propionate (dromostanolone propionate), epithioandrosterol (epiostanol), melandrane (mepistostane), testolactone (testolactone); anti-adrenal classes, such as aminoglutethimide (aminoglutethimide), mitotane (mitotane), trilostane (trilostane); folic acid supplements, for example, folinic acid (folinic acid); acetoglucurolactone (aceglatone); aldehyde phosphoramidate glycoside (aldophosphamide glycoside); aminolevulinic acid (aminolevulinic acid); amsacrine (amacrine); betabacib (bestabuic); bisantrene (bisantrene); edatraxate (edatraxate); ground phosphoramide (defofame); dimecoxine (demecolcine); deaquinone (diaziquone); eformitine (elfomithin); ammonium elide (elliptinium acetate); etodolac (etoglucid); gallium nitrate; hydroxyurea; lentinan (lentinan); lonidamine (lonidamine); mitoguazone (mitoguazone); mitoxantrone (mitoxantrone); mo Pai darol (mopidamol); diamine nitroacridine (nitroane); penstatin (penstatin); egg ammonia nitrogen mustard (phenol); pirarubicin (pirarubicin); podophylloic acid (podophyllinic acid); 2-ethyl hydrazide; procarbazine (procarbazine); PSK (phase shift keying) ^TM The method comprises the steps of carrying out a first treatment on the surface of the Raschig (razoxane); sisofilan (silzofuran); spiral germanium (spiral); alternaria alternata (L.) KuntzeKetoacid (tenuazonic acid); triiminoquinone (triaziquone); 2,2',2 "-trichlorotriethylamine; uratam (urethan); vindesine (vindeline); dacarbazine (dacarbazine); mannomustine (mannomustine); dibromomannitol (mitobronitol); dibromodulcitol (mitolactol); pipobromine (pipobroman); metropolicine (tetracytine); cytarabine (arabinoside) ("Ara-C"); cyclophosphamide; thiotepa; taxanes, e.g. Taxol (Taxol) ^TM Bristol-Myers Squibb Oncology, prencton, N.J.) and docetaxel (Taxote) ^TM The method comprises the steps of carrying out a first treatment on the surface of the Aventis antonyl, france); chlorambucil (chloranil); gemcitabine (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin (cispratin) and carboplatin (carboplatin); vinblastine (vinblastine); platinum (platinum); etoposide (VP-16); ifosfamide (ifosfamide); mitomycin C (mitomycin C); mitoxantrone (mitoxantrone); vincristine (vincristine); vinorelbine (vinorelbine); novelline (naveldine); norubin (novantrone); teniposide (teniposide); daunomycin (daunomycin); aminopterin (aminopterin); hilded (xeloda); ibandronate (ibandronate); CPT-11; topoisomerase inhibitor RFS2000; difluoromethyl ornithine (DMFO); retinoic acid (retinoic acid); epothilones (esperamicins); capecitabine (capecitabine); and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to modulate or inhibit the effect of hormones on tumors, such as antiestrogens including, for example, tamoxifen, raloxifene, (raloxifene), aromatase-inhibiting 4 (5) -imidazole, 4-hydroxy tamoxifen, troxifene (trioxifene), raloxifene hydrochloride (keoxifene), LY 117018, onapristone (onapristone) and toremifene (toremifene) (farston) and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprorelin (leuprorelin) and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

The anti-egfrvlll antibody conjugates of the present disclosure may also be administered and/or co-formulated in combination with an antiviral agent, an antibiotic, an analgesic, a corticosteroid, a steroid, oxygen, an antioxidant, a COX inhibitor, a cardioprotectant, a metal chelator, IFN- γ, and/or an NSAID.

One or more additional therapeutically active components, such as any of the agents listed above or derivatives thereof, may be administered prior to, concurrent with, or immediately after the administration of the anti-egfrvlll antibody-texilin conjugate of the present disclosure; (for the purposes of this disclosure, such an administration regimen is considered to be "combined" administration of the anti-egfrvlll antibody-doxycycline conjugate with additional therapeutically active components). The present disclosure includes pharmaceutical compositions wherein the anti-egfrvlll antibody-doxycycline conjugates of the present disclosure are co-formulated with additional therapeutically active components as described elsewhere herein.

Administration protocol

According to certain embodiments of the present disclosure, multiple doses of an anti-egfrvlll antibody-textilene Lin Zhuige (or a pharmaceutical composition comprising a combination of an anti-egfrvlll antibody-textilene conjugate and any of the additional therapeutically active agents mentioned herein) may be administered to a subject over a defined time frame. Methods according to this aspect of the disclosure include sequentially administering multiple doses of an anti-egfrvlll antibody-doxycycline conjugate of the disclosure to a subject. As used herein, "sequentially administered" means that each dose of anti-egfrvlll antibody is administered to a subject at a different point in time, e.g., on different days separated by a predetermined interval (e.g., hour, day, week, or month). The present disclosure includes such methods: the method comprises sequentially administering a single initial dose of an anti-egfrvlll antibody-textilene Lin Zhuige, followed by one or more second doses of an anti-egfrvlll antibody-textilene Lin Zhuige, and optionally followed by one or more third doses of an anti-egfrvlll antibody-textilene conjugate to the patient.

The terms "initial dose", "second dose" and "third dose" refer to the temporal order of administration of the anti-egfrvlll antibody-doxycycline conjugate of the present disclosure. Thus, an "initial dose" is the dose administered at the beginning of a treatment regimen (also referred to as a "baseline amount"); a "second dose" is a dose administered after the initial dose; and a "tertiary dose" is a dose administered after the second dose. The initial dose, the second dose and the third dose may all contain the same amount of anti-egfrvlll antibody-texti Lin Zhuige but may generally differ from each other in the frequency of administration. However, in certain embodiments, the amount of anti-egfrvlll antibody-doxycycline conjugate contained in the initial dose, the second dose, and/or the third dose is different from one another (e.g., up-or down-regulated as appropriate) during the course of treatment. In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered as "loading doses" at the beginning of a treatment regimen, followed by subsequent doses (e.g., a "maintenance dose") at a lower frequency.

In certain exemplary embodiments of the present disclosure, each second and/or third dose is 1 to 26 (e.g., 1 ¹ / ₂ 、2、2 ¹ / ₂ 、3、3 ¹ / ₂ 、4、4 ¹ / ₂ 、5、5 ¹ / ₂ 、6、6 ¹ / ₂ 、7、7 ¹ / ₂ 、8、8 ¹ / ₂ 、9、9 ¹ / ₂ 、10、10 ¹ / ₂ 、11、11 ¹ / ₂ 、12、12 ¹ / ₂ 、13、13 ¹ / ₂ 、14、14 ¹ / ₂ 、15、15 ¹ / ₂ 、16、16 ¹ / ₂ 、17、17 ¹ / ₂ 、18、18 ¹ / ₂ 、19、19 ¹ / ₂ 、20、20 ¹ / ₂ 、21、21 ¹ / ₂ 、22、22 ¹ / ₂ 、23、23 ¹ / ₂ 、24、24 ¹ / ₂ 、25、25 ¹ / ₂ 、26、26 ¹ / ₂ Or more) weekly. As used herein, the phrase "immediately preceding dose" refers to the dose of the anti-egfrvlll antibody-doxillin conjugate being administered to a patient in a sequence of multiple administrations immediately prior to the administration of the next dose in the sequence, without an intermediate dose.

Methods according to this aspect of the disclosure may include administering any number of second and/or third doses of the anti-egfrvlll antibody-texti Lin Zhuige to the patient. For example, in certain embodiments, only a single second dose is administered to the patient. In other embodiments, the patient is administered the second dose two or more times (e.g., 2, 3, 4, 5, 6, 7, 8, or more times). Also, in certain embodiments, only a single third dose is administered to the patient. In other embodiments, the patient is administered the third dose two or more times (e.g., 2, 3, 4, 5, 6, 7, 8, or more times). The administration regimen may be performed indefinitely throughout the lifetime of the particular subject, or until such treatment is no longer therapeutically desirable or advantageous.

In embodiments involving multiple second doses, each second dose may be administered at the same frequency as the other second doses. For example, each second dose may be administered to the patient 1 to 2 weeks or 1 to 2 months after the immediately preceding dose. Similarly, in embodiments involving multiple third doses, each third dose may be administered at the same frequency as the other third doses. For example, each third dose may be administered to the patient 2 to 12 weeks after the immediately preceding dose. In certain embodiments of the present disclosure, the frequency of the second and/or third doses administered to the patient may vary during the course of the treatment regimen. The doctor can also adjust the frequency of administration during the course of treatment according to the needs of the individual patient after the clinical examination.

The present disclosure includes an administration regimen wherein 2 to 6 loading doses are administered to a patient at a first frequency (e.g., once weekly, once biweekly, once every three weeks, once monthly, once every two months, etc.), and then two or more maintenance doses are administered to the patient at a lower frequency. For example, if the loading dose is administered at a frequency of once a month, the maintenance dose may be administered to the patient every six weeks, every two months, every three months, etc., in accordance with this aspect of the disclosure.

Examples

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the present disclosure, 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 numbers used, but some experimental errors and deviations should be accounted for. Unless otherwise indicated, molecular weight is average molecular weight, temperature is in degrees celsius and pressure is at or near atmospheric pressure.

Example 1 production of anti-EGFRvIII antibodies

By using immunogens comprising the extracellular domain of EGFRvIIIMice (i.e., engineered mice comprising DNA encoding human immunoglobulin heavy and kappa light chain variable regions) were immunized to obtain anti-egfrvlll antibodies.

Antibody immune responses were monitored by egfrvlll specific immunoassay. When the desired immune response is reached, spleen cells are harvested and fused with mouse myeloma cells to maintain their viability and form hybridoma cell lines. Hybridoma cell lines were screened and selected to identify cell lines that produced egfrvlll specific antibodies. Using this technique, an exemplary H1H1863N2 anti-egfrvlll chimeric antibody (i.e., having a human variable domain and a mouse constant domain) was obtained. The variable domain sequence of this antibody was originally disclosed in U.S.9,475,875. This antibody is referred to herein as REGN1076. The non-glycosylated form of the antibody is referred to herein as REGN3124, wherein asparagine (N) at residue 297 of the heavy chain of REGN1076 antibody is mutated to glutamine (Q) as measured by EU index numbering (i.e., H1H1863N 2-N297Q). Variable region sequences and complete heavy and light chain sequences are provided below.

Separately, reduced fucosylation REGN1076[ "REGN1076 (Fuc-) ] was prepared in the CHO host cell line described as" 8088 "in U.S. patent application 2010/0304436A1, which is incorporated by reference in its entirety. Mass spectrometry analysis of the resulting (Fuc-) antibodies confirmed that core fucose was removed relative to the original antibody.

Table 1 lists amino acid sequence identifiers for the heavy and light chain variable regions and CDRs of exemplary anti-egfrvlll antibodies used 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 listed in Table 3.

Table 1: sequence identifiers of the variable region amino acid sequences of REGN1076 and REGN3124

Table 2: sequence identifiers of complete heavy and light chain amino acid sequences of REGN1076 and REGN3124

Table 3: sequence identifiers of the variable region nucleic acid sequences of REGN1076 and REGN3124

As will be appreciated by those of ordinary skill in the art, antibodies with a particular Fc isotype can be converted to antibodies with a different Fc isotype (e.g., antibodies with mouse IgG1Fc can be converted to antibodies with human IgG4, etc.), but in any event the variable domains (including CDRs) (represented by the numerical identifiers shown in table 1) will remain the same and the binding characteristics are expected to be the same or substantially similar, regardless of the nature of the Fc domain.

Antibodies were found to internalize rapidly into egfrvlll positive tumor cells. Some additional biological properties of exemplary anti-egfrvlll antibodies produced according to the methods of the present embodiments are described in detail in the examples shown below.

Control and comparative constructs used in the examples below

For comparison purposes, control constructs were included in the following experiments: the comparison antibody, referred to herein as COMP, is a humanized anti-egfrvlll antibody (hIgG 1) 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 U.S. patent application publication 2010/0056762. This antibody is also known as ABT-414. The "hu806" antibody is known to bind to residues 311-326 (SEQ ID NO: 24) or residues 44-59 of EGFR vIII (SEQ ID NO: 28) of amplified or overexpressed EGFR (SEQ ID NO: 27). COMP-MMAF refers to ABT-414 antibody conjugated to monomethyl auristatin F (MMAF) through a non-cleavable linker.

Control 1932 and control 3892 are isotype control antibodies. Control 1932 had no Fc modification and control 3892 had N297Q modification.

EXAMPLE 2 conjugation and characterization of Texilin-antibody

Antibodies REGN1076 and REGN3124 and isotype control antibodies control 1932 (without Fc modification) and control 3892 (with N297Q modification) (10 mg/mL each) were treated with 1mM dithiothreitol at 37 ℃ for 30 min in 50mM HEPES or PBS, 150mM NaCl, pH 7.5. After gel filtration (G-25, pH 4.5 sodium acetate), maleimide linker payload (also known as SG3249, as 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 reducing antibody and the mixture was adjusted to pH 7.0 with 1M HEPES (pH 7.4). The conjugate was purified by size exclusion chromatography and sterile filtered. The protein concentration was determined by UV and the payload to antibody ratio was determined by mass spectrometry. Size exclusion HPLC determined that all conjugates used were >95% monomer and LC-MS determined that <0.5% unconjugated linker payload was present. The ratio of payload to antibody is shown in table 4.

To determine the loading of the antibody with doxycycline, the conjugate was deglycosylated, reduced and analyzed by LC-MS.

For the assay, 50 μg of conjugate was diluted to a final concentration of 1mg/mL using mil-Q water. mu.L of PNG enzyme F solution [ PNG enzyme F solution was prepared by: add 150. Mu.L PNG enzyme F stock (New England Biolabs, catalogue #P0704L) and 850. Mu.L mil-Q water and mix thoroughly]Added to the diluted conjugate solution and then incubated overnight at 37 °c. 2.4. Mu.L of 0.5M TCEP was added to the sample to give a final TCEP concentration of 20mM for the resulting material, which was then incubated at 50℃for 30 minutes. mu.L of each sample was injected into LC-MS (Waters Syntat G2-Si) and eluted with a gradient of 0.1mL/min mobile phase 20% -40% for 25 min (mobile phase A:0.1% v/v FA in H) ₂ O is as follows; mobile phase B:0.1% v/v FA in acetonitrile). LC separation was performed on a Waters Acquity BEH C18 column (1.0X10 mM, 1.7. Mu.M).

Deconvolution of the mass spectrum was performed, and the identified light and heavy chain peaks represent the light chain (L) with linker payloads values=0 and 1 and the heavy chain (H) with linker payloads values=0, 1, 2 and 3. From the intensity values of each species, the drug-to-antibody ratio (DAR) of the homodimer antibody conjugate was calculated using the following equation 1. DAR for each conjugate is provided in table 4.

Equation 1:

table 4: yield percentage and ratio of payload to antibody

Antibodies to	Yield (%)	DAR(MS)
			REGN 1076-Texilin	60	2.4-2.6
REGN 3124-Texilin	80	3.4
			Control 3892-Texilin	60	2.8

EXAMPLE 3 Biacore binding kinetics of EGFRvIII monoclonal antibody

Equilibrium dissociation constant (K) for binding of egfrvlll to PDB conjugate of anti-egfrvlll antibody _D Value) was measured on a Biacore 2000 or 3000 instrument using a real-time surface plasmon resonance biosensor assay. The Biacore sensor surface was derivatized by coupling of amine to a monoclonal mouse anti-human Fc antibody (GE Healthcare, #br-1008-39) to capture anti-egfrvlll antibody drug conjugate and unmodified parent antibody expressed with human constant regions. 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 the human EGFRvIII extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (hEGFRvIII-MMH; SEQ ID NO: 29) (ranging from 600nM to 22.2 nM) prepared in HBS-EP running buffer were injected onto the surface of anti-EGFRvIII antibody drug conjugate or antibody capture at a flow rate of 50. Mu.L/min. The hEGFRvIII-MMH was monitored for association with each of the captured antibody drug conjugate and monoclonal antibody for 4 minutes. Subsequently, the hEGFRvIII-MMH dissociation in HBS-EP running buffer was monitored for 6-8 minutes. By injection of 20mM H ₃ PO ₄ To regenerate the anti-human Fc surface. All binding kinetics experiments were performed at 25 ℃. Kinetic association (k) was determined by fitting a real-time sensorgram to a 1:1 binding model using a scanner 2.0c curve fitting software _a ) Dissociation (k) _d ) A rate constant. All sensorgrams were double referenced by subtracting the buffer injection sensorgram signal from the corresponding analyte sensorgram, thereby eliminating artifacts resulting from dissociation of antibodies from the capture surface. Binding dissociation equilibrium constant (K) _D ) Dissociation half-life (t 1/2) is calculated from kinetic rate constants, formulaThe following are provided:

K _D (M)＝k _d /k _a and t1/2 (min) =ln2/(60 xk) _d )

The binding kinetics parameters for hEGFRvIII-MMH binding to anti-egfrvlll antibody drug conjugates and antibodies at 25 ℃ are shown in table 5. As shown, the parent antibodies and their corresponding antibody drug conjugates exhibited similar binding K to hEGFRvIII-MMH under the conditions tested _D Values.

Table 5: biacore kinetics of binding of human EGFRvIII-MMH to anti-EGFRvIII conjugates and unmodified parent antibodies

Example 4 cell killing Activity of anti-EGFRvIII antibody-Texilin ADC

To determine the relative cell killing efficacy of the anti-egfrvlll antibody drug conjugate of the present invention, a cell killing assay was performed on cell lines expressing human egfrvlll. To develop a cell line, lipofectamine LTX and Plus Reagent were used to generate U251 cells (Sigma, # 9063001) (referred to herein as U251 MG/hEGFRvIII) expressing human EGFRvIII (hEGFRvIII; amino acids 1 to 380 of accession number NP-005219.2, where amino acids 30 to 297 are deleted, resulting in a linked glycine residue, SEQ ID NO: 25). The U251 cell line was maintained in complete growth medium (MEM early's Salts) +10% FBS+1% L-glutamine/penicillin/streptomycin+1% nonessential amino acids+sodium pyruvate).

To measure in vitro cytotoxicity of anti-egfrvlll antibody drug conjugate, nuclear counts after 6 days of treatment with antibody drug conjugate were assessed. U251MG and U251/hEGFRvIII cells in complete growth medium were seeded at 3000 cells/well in 96 well plates (Perkinelmer, # 6055308) and at 5% CO ₂ Is grown overnight at 37 ℃. For the cell viability curve, serial dilutions of antibody drug conjugate and payload were added to cells at final concentrations of 100nM to 1.5pM (based on toxin concentration) and then at 5% CO ₂ At 37 DEG CAnd 6 days of cultivation. The last well in each dilution series (untreated well) was taken as a blank containing either medium Alone (ADC) or medium+0.2% DMSO (payload) and plotted as a 3-fold serial dilution continuation. Cells were then treated with 3 μg/mL Hoechst 33342 nuclear stain (thermo Fisher, # H2 3570) while fixed with 4% formaldehyde (thermo Fisher, # 28908) and images were acquired on an Opera Phenix (Perkinelmer). Nuclear counts were determined by Harmony image analysis software (Perkinelmer) and cell viability was expressed as a percentage of untreated (100% viability) cells. Determination of IC using four parameter logistic equation on 10 point dose response curve (GraphPad Prism) ₅₀ Values. The percent maximum kill for each test article was determined as follows: 100-minimum percent viability. IC of each test article ₅₀ The values and percent maximum kill are shown in table 6.

As shown in Table 6, the anti-EGFRvIII antibody-drug conjugates REGN 3124-and REGN 1076-Texilin (glycosylated form of REGN 3124) reduced cell viability, REGN 3124-Texilin IC in U251MG/hEGFRvIII cells ₅₀ The value was 33pM and REGN 1076-texilin was 84pM. REGN 3124-Texilin and REGN 1076-Texi Lin Shasi parent U251MG cells, IC of REGN 3124-Texilin ₅₀ The value was 2.6nM and REGN1076-Texilin was 4.9nM. Similarly conjugated isotype control antibody control 3892-texilin reduced cell viability, IC in U251MG/hEGFRvIII cells ₅₀ The value was 3.9nM, 1.8nM in the U251MG parent cell. The free payload of terxillin (SG 3199) killed U251MG/hEGFRvIII cells, IC ₅₀ A value of 10pM, and U251MG parent cell, IC ₅₀ The value was 2pM.

Cytotoxicity of REGN1076 (REGN 1076-MMAF) conjugated to the comparative MMAF payload was also tested. Similar to other tested anti-EGFRvIII ADCs, REGN1076-MMAF killed U251MG/hEGFRvIII cells, IC ₅₀ The value was 47pM. Anti EGFRvIII ADC REGN1076-MMAF has weak cytotoxicity in parent U251MG cells, IC ₅₀ The value was 52nM. Non-binding isotype control antibodies (control 1932-MMAF) similarly conjugated to MMAF had weak cytotoxicity, IC, in all cell lines tested ₅₀ Greater than 100nM.

Table 6: cell viability of U251/hEGFRvIII and parental cell lines

ADC can undergo bystander killing when a cytotoxic payload is released from a target cell and then taken up by surrounding antigen negative (bystander) cells. To evaluate potential bystander killing effects of REGN 3124-and REGN 1076-texilens, cellTrace was used ^TM Far infrared (thermo fisher, #c 34564) pre-labels U251MG/hEGFRvIII cells. A 1:1 co-culture of 1500 cells/well far infrared labeled U251MG/hEGFRvIII cells and 1500 cells/well unlabeled U251MG cells was incubated with a range of concentrations (100 nM to 1.5 pM) of ADC or free payload M31 for 6 days. Cells were then treated with 3. Mu.g/mL Hoechst 33342 nuclear stain (ThermoFisher, # H270) while fixed with 4% formaldehyde (ThermoFisher, # 28908). Images were collected on an Opera Phoenix microscope (PerkinELmer). All cells were identified by Hoechst-labeled nuclei and cell counts were separated into far infrared positive U251MG/hEGFRvIII cells (U251 in co-culture) and far infrared parental U251MG cell populations by Harmony image analysis software (PerkinELmer). Cell viability, expressed as a percentage of untreated (100% viability), was determined for each cell population separately. IC (integrated circuit) ₅₀ And the percent maximum kill values were determined as described above and summarized in table 6.

REGN 3124-and REGN 1076-Texilin killed U251MG/hEGFRvIII cells, IC of the co-culture ₅₀ The values were 43pM and 82pM, respectively, and the killing effect was similar to that observed in U251MG/hEGFRvIII single cultures. REGN 3124-and REGN 1076-Texilin also kills U251MG parent cells, IC of the co-culture ₅₀ The values were 28pM and 59pM, respectively, indicating that these ADCs have bystander killing activity. Non-binding ADC control 3892-Texi Lin Shasi U251MG/hEGFRvIII and U251MG parental cells, IC ₅₀ The values were 4.0nM and 2.4nM, respectively.

Bystander activity of REGN1076 (REGN 1076-MMAF) conjugated to the comparative MMAF payload was also tested. REGN1076-MMAF demonstrated potent cytotoxicity against U251MG/hEGFRvIII cells of co-culture, IC ₅₀ The value was 15pM. In contrast to the textilene conjugate, REGN1076-MMAF has weak cytotoxicity, IC, in the U251 parental cells of the co-culture ₅₀ The value was 28nM. Non-binding ADCs conjugated to MMAF (control 1932-MMAF) were poorly cytotoxic, IC in the co-culture assay ₅₀ Value of>100nM。

Example 5 epitope mapping of Hydrogen/deuterium (H/D) exchanged anti-EGFRvIII antibodies to human epidermal growth factor receptor variants (hEGFRvIII)

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) was performed to determine the amino acid residue of EGFR receptor variant 3 (hEGFRvIII ECD (L25-A380). MmH (SEQ ID NO: 29), amino acid sequence see appendix) interacting with REGN 3124. A general description of the HDX-MS method is described, for example, in Ehring (1999) Analytical Biochemistry 267 (2): 252-259; and Engen and Smith (2001) Anal. Chem. 73:256A-265A.

HDX-MS experiments were performed on an integrated HDX/MS platform consisting of leapetec HDX PAL system for deuterium labeling and quenching, waters Acquity M-Class for sample digestion and loading, waters Acquity M-Class for analysis of gradients (μbinary solvent manager), and Thermo Q Exactive HF mass spectrometer for peptide mass measurement.

Preparation of the labeling solution at pD7.0 as D ₂ PBS buffer in O (10 mM phosphate buffer, 140mM NaCl and 3mM KCl, corresponding to pH7.4 at 25 ℃). For deuterium labeling, 10 μl of egfrvlll (egfrvlll extracellular domain with myc histidine tag (L25-a 380), SEQ ID NO 29, 66 μΜ) or egfrvlll (Ag-Ab complex) premixed with REGN3124 at a molar ratio of 1:0.6 was labeled with 90 μ L D at different time points at 20 ℃ (e.g. non-deuterated control = 0 seconds; deuterium labeling for 5 min, 20 min and 80 min) ₂ O-labelling solution incubation. For each time point, experiments were performed in duplicate. By adding 100. Mu.L of pre-chilled quench buffer (0.5M TCEP-HCl, 8M urea and 1% formic acid)To 100 μl of sample was added to quench the deuteration reaction. The mixed samples were incubated at 20℃for 5 minutes. The quenched samples were then injected into a Waters HDX manager for online pepsin/protease XIII digestion. Digested peptides were captured on a 1.0mm x 50mm C8 chromatographic column (novabiosays) and separated by a 13 min gradient of 10% -32% B (mobile phase a:0.5% formic acid in water, mobile phase B:0.1% formic acid in acetonitrile). The isolated peptides were analyzed by Q exact HF mass spectrometry in LC-MS/MS or LC-MS mode.

LC-MS/MS data of non-deuterated egfrvlll samples were searched for a database containing egfrvlll and its random sequences using a Byonic search engine (Protein Metrics) and default parameters of non-specific enzyme digestion. A list of common human glycans is defined as potential variable modifications. The identified peptide list was then imported into HDX Workbench software (version 3.3) along with LC-MS data for all deuterated samples to calculate deuterium uptake levels for individual peptides in each repetition at 3 HDX time points.

For a given peptide, the centroid mass (intensity weighted average mass) of the spectrum of the non-deuterated (0 second) control is first calculated. The average centroid mass of the control of the antigen and Ag-Ab complex non-deuterated was considered to be the mass of 0% deuterium incorporation percentage (mass 0%D). For each deuterated sample, the absolute D absorbance is defined as the mass difference of the mass center mass of the deuterated sample and the mass of 0%D. The percent deuterium incorporation (%d) was determined by comparing the mass of the centroid with the masses of 0 and 100%d (maximum deuterium uptake mass shift, defined as 80% of the mass difference between the N-2 deuterium atoms and the N-2 hydrogen atoms, where N equals the number of non-proline amino acids in the peptide).

For each peptide, absolute deuterium uptake and% D values were calculated for the two replicates of each HDX time point, respectively. For each HDX time point, the duplicate absolute deuterium uptake and% D values for antigen and Ag-Ab complexes were averaged. The average of the% D values at the 5-minute and 20-minute HDX time points is then expressed as the individual% D values of the antigen or Ag-Ab complex, which values are defined as "antigen% D" or "Ag-Ab% D". The difference between% antigen D and% Ag-Ab D is defined as delta% D (delta%), which represents the overall change in deuterium incorporation for a given peptide compared to antigen and Ag-Ab complexes.

200 peptides from hEGFRvIII were identified in total from hEGFRvIII alone and hEGFRvIII complexed with REGN3124 samples, representing 84% sequence coverage of hEGFRvIII. Any peptide that shows a greater than 5% reduction in deuterium uptake is defined as significantly protected (delta% D < -5%). The peptide corresponding to amino acids 64-82GPCRKVCNGIGIGEFKDSL on hEGFRvIII (SEQ ID NO: 26) was significantly protected by REGN 3124.

Table 7: hEGFRvIII peptide with significant protection when forming the hEGFRvIII-REGN3124 complex as compared to hGFRvIII alone

EGFRvlll ECD (L25-A380). MmH (mmH label underlined)

LEEKKGNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIAEQKLISEEDLGGEQKL ISEEDLHHHHHH(SEQ ID NO:29)

Example 6 anti-EGFRvIII antibody-Texilin ADC demonstrated significant anti-tumor efficacy against EGFRvIII transfected glioblastoma multiforme cell line xenografts

Antitumor efficacy of REGN 1076-and REGN 3124-texilin ADCs was initially assessed in glioblastoma cell line xenograft models transfected to express egfrvlll because of the internal targeting after in vitro cultureLoss of primary expression. The model first evaluated was U251/EGFRvIII, where tumors passed 10X 10 ⁶ Subcutaneous transplantation of 1:1 mixtures of individual cells with matrigel on the right flank of male SCID mice was established. Approximately 30 days after implantation, tumors were grown to approximately 130mm ³ Treatment is then started. The efficacy of the ADC was also evaluated in U87/egfrvlll, where tumors were generated by 3 x 10 on the right flank of male SCID mice ⁶ Subcutaneous transplantation of individual cells. Approximately 25 days after implantation, the U87/EGFvIII tumor was grown to approximately 190mm ³ Treatment is then started. Mice were randomized into groups of 7-8 each and treated with single dose test or control ADC. Tumor growth was monitored 60-70 days after treatment.

Experimental results:

preliminary studies in mice carrying U251/egfrvlll xenografts assessed the activity of REGN 1076-and REGN 3124-texiln against egfrvlll ADC after a single dose designed to deliver 2.5 or 5 μg/kg PBD payload (table 8). The growth of xenografts treated with control-texilin or control-N297Q-texilin ADC was not significantly delayed relative to vehicle control treated tumors. However, during the course of the study, a significant delay in tumor growth was observed in tumors treated with REGN 1076-or REGN 3124-texilin ADC at a payload dose of 2.5 μg/kg. Higher ADC doses delivering 5 μg/kg PBD payload had greater anti-tumor effect relative to control treatment. At equal dose levels, REGN 3124-texilin ADC produces a more durable anti-tumor effect relative to REGN 1076-texilin. Overall, all anti-egfrvlll treatment groups survived to completion of the study around 60 days post-dose. Treatment was independent of body weight, and an approximately 10% -15% increase in body weight was observed in all groups over the course of the study.

The activity of REGN 1076-and REGN 3124-texiln ADCs was also assessed in the U87/egfrvlll tumor xenograft model (table 9). Here, single doses of REGN 1076-and REGN 3124-Texilin ADCs were compared at a dose delivering 2.5 μg/kg PBD payload. The model showed very rapid growth and animals treated with vehicle control were euthanized 10 days post-dose as the tumor reached the end of the study. Control-doxillin or control-N297Q-doxillin ADC mediated a certain delay in tumor growth, but all tumors grew and animals were euthanized 24 days after dosing as tumors reached the study endpoint. REGN 1076-and REGN 3124-both deliver 2.5 μg/kg PBD payload, mediating significant and durable regression of tumor xenografts. All animals treated with anti-egfrvlll survived until completion of the study 70 days after dosing. At the end of the study, individual tumors in REGN 1076-texilin showed regrowth. All tumors treated with REGN 3124-texilin were inhibited. Treatment was independent of body weight, and an approximately 5% increase in body weight was observed in all groups over the course of the study.

Table 8: relative to the control, anti-egfrvlll-texiln PBD ADC mediated regression of U251/egfrvlll xenografts (day 36 post treatment).

Table 9: relative to the control, anti-egfrvlll-texti Lin Zhuige mediated regression of the U87/egfrvlll xenograft (day 10 post treatment).

Example 7 anti-EGFRvIII antibody-Texilin ADC demonstrated significant anti-tumor efficacy against in situ placed EGFRvIII positive glioblastoma multiforme patient-derived xenografts

To assess efficacy of anti-egfrvlll ADC against GBM tumors placed in situ in the brain, the tumor was treated by 3 x 10 ⁵ Injection of individual PDX cells to establish intracranial GBM6 (highly and homoplasmic egfrvlll expression) or GBM59 (moderately and heteroplasmically egfrvlll expression) patient-derived xenograft (PDX) tumors. Intracranial injection was performed 1mm anterior and 2mm lateral to bregma, 3mm deep. All in situ GBM PDX studies were performed by Translational Drug Development inc. Allowing in situ GBM6PDX was established for 14+ -1 days, GBM59 PDX was allowed to establish for 25+ -1 days, and mice were then randomized into 7-8 groups and treated with single dose test or control ADCs. Mice were monitored for signs of morbidity for about 90 days and euthanized before reaching a moribund state.

Experimental results:

in a preliminary study of in situ GBM6 PDX tumor-bearing mice (study a), mice treated with vehicle exhibited rapidly worsening clinical signs, and 7/8 mice were euthanized within 30 days of treatment (table 10). Isotype control ADCs did not produce any clinical effect, and mice in this group were rapidly euthanized due to tumor-induced morbidity. Treatment with a 7 μg/kg payload dose of REGN 3124-texilin (DAR 3.4) significantly prolonged survival compared to the shorter 25-day and 26.5-day median survival observed in the control group. The median survival of the anti-egfrvlll-texel ADC group has not been reached because 5/8 of the mice survived to the final point of observation 94 days post-dose.

The second study (study B) was initiated in mice bearing in situ placed GBM6 PDX tumors (table 11). In addition, the survival of isotype control ADC-mediated mice was not prolonged relative to vehicle-treated mice, and the median survival of both groups was near 20 days with no survival. REGN 3124-texilin (DAR 3.4) extended survival at 3.5 and 7 μg/kg payload dose levels, but higher doses allowed more mice (5/8) to survive until day 95 post-treatment study completion. REGN 3124-Texilin (DAR 1.9) has a similar effect as REGN 3124-Texilin (DAR 3.4), with a median survival of 77 days post-treatment, 4/8 mice survived to the end of the study. Rapid deterioration of animal body weight was observed in mice with tumor-induced morbidity. In contrast, animals treated with REGN 3124-texilin showed long-term survival, with an associated weight gain of 10% -15% during post-treatment observations.

The efficacy of REGN 3124-texilin was also assessed against GBM59 PDX tumors placed in situ (table 12). In this study (study C), all 8 mice treated with vehicle died from tumor burden within 30 days. Isotype control ADC mediated a partial extension of survival relative to vehicle control, but euthanized all mice due to morbidity 42 days post-treatment, resulting in a median survival of 32.5 days. For the GBM6 model, a significant prolongation of survival was observed in mice receiving a single 7 μg/kg payload dose of REGN 3124-Texilin. REGN 3124-texilin with DAR 1.9 and DAR 3.4 survived 7/8 mice until the 94 th day post-treatment study was completed, and thus, the median survival for these groups was not achieved. In this study, less weight gain was observed in mice treated with DAR 1.9REGN3124-texilin relative to DAR 3.4 ADC. The brains of mice from study C were collected at various time points, particularly when mice were euthanized due to overt disease, weight loss, or other clinical criteria, or when the study was completed at day 94 post-treatment. Histological analysis was performed. GBM59 cells were not observed in any of the brains of mice treated with REGN 3124-texilin. Similar results were also observed in GBM6 PDX study (study a). Subsequent immunohistochemistry showed that GMB59 PDX showed moderate and heterogeneous egfrvlll expression.

Table 10: anti-EGFRvIII-TexiLin ADC significantly prolonged survival of intracranial GBM6 GBM PDX mice (study A)

Table 11: anti-EGFRvIII-TexiLin ADC significantly prolonged survival of intracranial GBM6 GBM PDX mice (study B)

Table 12: anti-EGFRvIII-TexiLin ADC significantly prolonged survival of intracranial GBM59 GBM PDX mice (study C)

Example 8 REGN 1076-and REGN 3124-texilin ADCs demonstrate significant anti-tumor efficacy against egfrvlll positive glioblastoma multiforme patient-derived xenografts

Patient-derived xenografts with endogenous expression of egfrvlll (representing the tumor biology of glioblastoma multiforme) were used to further investigate the efficacy of REGN 1076-and REGN 3124-texilin ADCs. GBM PDX studies were performed by Translational Drug Development inc. Subcutaneous tumors of GBM6 or GBM59 PDX were established by transplanting about 50mg of PDX fragments into the flank of nude mice. Once the tumor volume reached about 125mm 16-18 days post-implantation ³ The mice can be randomized into groups of 7-8 and treated with a single dose of test or control ADC delivering 3.5 or 7 μg/kg of pyrrolobenzodiazepineA dose of (PBD) payload dose. Tumor growth was monitored 60 days after treatment.

Experimental results:

in GBM6 PDX tumor-bearing mice treated with vehicle, rapid growth of the tumor was observed, and the tumor reached the endpoint of the study 18 days after treatment. Isotype control-texilin ADC only mediated a slight delay in tumor growth, with tumors reaching the study endpoint on day 22 post-treatment. Anti-egfrvlll ADC mediated very significant and durable tumor regression compared to vehicle and control treated tumors (table 13). In the GBM6 model, 3.5 μg/kg payload dose of REGN 1076-or REGN 3124-Texilin generally had equivalent antitumor efficacy, and 5/8 and 4/8 animals were tumor-free at study completion on day 60 post-treatment. REGN 1076-or REGN 3124-texilin treatment delivering 7 μg/kg PBD payload produced greater and longer lasting efficacy, 7/8 and 8/8 tumor free at 60 days post treatment study completion. No treatment-related weight loss was observed, with an approximately 15% increase in animal weight over the course of the study.

Table 13: anti-egfrvlll-doxycycline ADC mediates regression of GBM6 PDX tumors relative to control (day 18 post-treatment).

The relative effects of REGN 3124-texilin ADC with antibody ratios (DAR) of 1.9 and 3.4 were evaluated in GBM59 tumor-bearing mice. Again, rapid tumor growth was observed in mice treated with vehicle and control ADC, both groups reached the study endpoint 19 days after treatment (table 14). At a 3.5 μg/kg PBD dose, REGN 3124-texilin (DAR 3.4) mediated moderate antitumor effects, tumors reached the end of the study at day 30 post-treatment. REGN 3124-texilin (DAR 1.9) was also active and tumors reached the endpoint of the study on day 51 after treatment with this drug. Consistent with other studies, REGN 3124-texilin ADC treatment delivering a 7 μg/kg payload dose produced greater and more durable inhibition of GBM59 tumor growth. At this dose, REGN 3124-Texilin (DAR 1.9) caused 3/7 tumors to be less than 50mm at day 60 completion of the study ³ While REGN 3124-Texilin (DAR 3.4) caused 5/7 tumors to be less than 50mm at the completion of the study ³ . No treatment-related weight loss was observed, with an approximately 10% increase in animal weight over the course of the study.

Table 14: anti-egfrvlll-texti Lin Zhuige mediates regression of GBM59 PDX tumors relative to control (day 19 post-treatment).

Example 9 evaluation of fractionated dose regimen of regn 3124-texilin ADC in egfrvlll positive GBM59 PDX tumor bearing mice.

A study was performed using the subcutaneous GBM59 PDX tumor model to assess the effect of different dosage regimens of REGN 3124-texilin conjugate on antitumor efficacy. This study was also performed by Translational Drug Development inc. Subcutaneous tumor GBM59 PDX was established by transplanting about 50mg of PDX fragment into the flank of nude mice. Once the tumor volume reached about 125mm 13 days after implantation ³ Mice can be randomized into 7 groups and treated with test or control ADCs. Isotype control ADC was dosed in a single dose equal to 7 μg/kg PBD payloadAnd (3) application. Animals in the low dose group received 1.75 μg/kg of ADC REGN 3124-texti Lin Zhuige per dose on day 0 and day 4 post-treatment, resulting in a cumulative PBD dose of 3.5 μg/kg. The other group received REGN 3124-Texilin, and the cumulative dose delivered was 7 μg/kg. The dose was divided into individual doses of 3X 2.33. Mu.g/kg, 2X 3.5. Mu.g/kg or 1X 7. Mu.g/kg and delivered on days 0, 4 and 8 (2.33. Mu.g/kg), 0 and 4 (3.5. Mu.g/kg) or 0 (7. Mu.g/kg). Tumor growth was monitored 60 days after treatment.

Experimental results:

in this study, isotype control ADC did not cause any tumor growth delay relative to vehicle control, and both groups were euthanized at day 19 post-treatment as the mean tumor volume had reached the study endpoint (table 15). In animals receiving 2×1.75 μg/kg REGN 3124-texilin, significant inhibition of tumor volume was observed, with all mice surviving until 60 days post-treatment study completion. All dose regimens given a cumulative PBD payload dose of 7 μg/kg produced further and very pronounced antitumor effects. In mice given REGN 3124-texilin at a PBD dose of 3X 2.33. Mu.g/kg, 3/7 was tumor-free at the completion of the study, with an average tumor volume of less than 90mm ³ . In the group receiving REGN 3124-Texilin (delivering 2X 3.5. Mu.g/kg and 1X 7. Mu.g/kg PBD dose), 2/7 and 3/7 mice were tumor-free at study completion and the average tumor volume of both groups was less than 5mm ³ This suggests a significant and durable efficacy of REGN 3124-texilin in this study. No treatment-related weight loss was observed, with an approximately 10% increase in animal weight over the course of the study.

Table 15: anti-egfrvlll-texti Lin Zhuige mediates regression of GBM6 PDX tumors relative to control (day 18 post-treatment).

Example 10 comparison of anti-EGFRvIII-Texillin ADC with anti-EGFRvIII-maytansinoid DM1 ADC

To establish tumors, 0.5X10 ⁶ Subcutaneous injection of MMT-EGFRvIII cellsIn the flank of female SCID mice. Once the tumor volume reaches about 140mm ³ (day 8) mice can be randomized into 7 groups and treated with test and control ADCs with either doxillin or DM1 payload. The agent was administered for 3 days over a period of 17 days. Tumor growth was monitored 61 days after implantation.

The antitumor efficacy of the various egfrvlll ADCs was then assessed over time (fig. 1). In mice treated with 1mg/kg REGN 1076-texilin ADC, complete eradication of the tumor was observed during the study period. Control-texilin ADC mediated a transient anti-tumor effect, but all tumors eventually showed rapid progression to the protocol endpoint tumor volume. REGN1076-DM1 administered at 1 or 15mg/kg induced only moderate delay in tumor growth and all tumors progressed rapidly to the protocol endpoint, compared to the significant efficacy observed following REGN 1076-texilin administration. Control DM1 ADC had no effect relative to vehicle control. The results for anti-egfrvlll at a dose level of 1mg/kg indicate greater potency for anti-egfrvlll-texel conjugate relative to anti-egfrvlll-maytansinoid DM1 conjugate. Treatment in this study did not induce severe weight loss.

Example 11 evaluation of anti-EGFRvIII-Texilin conjugate and COMP-MMAF ADC in EGFRvIII positive tumor model.

The activity of REGN 3124-Texilin ADC was evaluated simultaneously with the comparative ADC (i.e., COMP-MMAF) (monomethyl auristatin F, 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) in U251/EGFRvIII tumor xenograft model and intracranial in situ GBM59 PDX model. For initial xenograft studies, U251/EGFRvIII tumors passed 10X 10 ⁶ Subcutaneous transplantation of 1:1 mixtures of individual cells with matrigel on the right flank of male SCID mice was established. Approximately 30 days after implantation, tumors were grown to approximately 175mm ³ Treatment is then started. Mice were randomized into 8 groups and treated with single dose test or control ADC. Tumor growth was monitored 71 days after treatment.

To evaluate REGN3124Efficacy of the texillin and COMP-MMAF ADC against GBM tumors placed in situ in the brain by 3 x 10 ⁵ Injection of individual PDX cells to establish intracranial GBM59 PDX tumors. Intracranial injection was performed 1mm anterior and 2mm lateral to bregma, 3mm deep. All in situ GBM PDX studies were performed by Translational Drug Development inc. In situ GBM59 PDX was allowed to build for 25 days, then mice were randomized into 8 groups each and treated with a single dose of test or control ADC. Mice were monitored for signs of morbidity for 94 days and euthanized before reaching a moribund state.

Experimental results:

studies in mice carrying U251/egfrvlll xenografts evaluate the activity of REGN 3124-texilin designed to deliver 7 μg/kg of PBD payload, and COMP-MMAF ADC activity, at ADC doses of 1, 2.5 and 5mg/kg (table 16). All dose levels included isotype controls in this study, but only moderate antitumor effects were observed for these control agents. REGN 3124-Texilin showed significant anti-tumor activity in this study, as a single dose of 0.53mg/kg ADC (7 μg/kg PBD payload dose) was able to induce sustained regression of xenografts. No persistent regression was observed for tumors treated with 1mg/kg COMP-MMAF. Anti-tumor activity was observed with 2.5mg/kg COMP-MMAF, but 5mg/kg COMP-MMAF was required to achieve sustained activity similar to that produced by treatment with REGN 3124-texilin at the completion of the study at day 71 post-dose. All animals in this study had a 10% increase in body weight during the post-treatment observation period.

Table 16: the anti-egfrvlll-doxycycline conjugate and COMP-MMAF conjugate mediate regression of the U251/egfrvlll xenograft relative to the control (day 36 post-treatment).

The efficacy of REGN 3124-texilin (DAR 1.9) and COMP-MMAF ADC was also assessed against in situ placed GBM59 PDX tumors (table 17). In this study, all 8 mice treated with vehicle died from tumor burden within 25 days. The survival of isotype control-texilin and control MMAF ADC was not prolonged relative to vehicle control. Very significant survival prolongation of REGN 3124-textilene Lin Jiedao compared to the control group, in which 5/8 of the mice survived until the study was completed on day 95 post-dose. 1mg/kg COMP-MMAF ADC did not induce a significant increase in survival, as the median survival of this group was identical to the 5mg/kg control-MMAF. Higher 5mg/kg COMP-MMAF treatment did induce some increase in survival relative to MMAF control, but all mice died of tumor burden within 35 days of treatment, resulting in a median survival of 23.5 days.

Table 17: activity of REGN 3124-Texilin conjugate and COMP-MMAF conjugate in mice with in situ placed GBM59 GBM PDX tumor.

Informal sequence listing

An informal sequence listing of the sequences disclosed herein is provided below.

Table 18: sequence identifier and corresponding nucleic acid and amino acid sequences

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

Sequence listing

<110> Ruizhen pharmaceutical Co (Regeneron Pharmaceuticals, inc.)

F.De Lfeinuo

M.Kaili

Jecica-keshina

T-Nitoli

G.S. Stokes

<120> anti-EGFRvIII antibody drug conjugates and uses thereof

<130> 10966WO01

<140> TBA

<141> 2022-06-21

<150> 63/213,478

<151> 2021-06-22

<150> 63/242,929

<151> 2021-09-10

<160> 29

<170> patent in version 3.5

<210> 1

<211> 366

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 1

gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctctggatt ccccttcagt agctacgaca tgcactgggt ccgccaagct 120

acaggaaaag gtctggagtg ggtctcagct attggtactg ctggtgccac atactatcca 180

ggctccgtga agggccgatt caccatctcc agagaaaatg ccaagaactc cttgtatctt 240

caaatgaaca gcctgagagc cggggacacg gctgtgtatt actgtgcaag aggggattac 300

gtttggggga cttatcgtcc cctctttgac tactggggcc agggaaccct ggtcaccgtc 360

tcctca 366

<210> 2

<211> 122

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 2

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Ser Ser Tyr

20 25 30

Asp Met His Trp Val Arg Gln Ala Thr Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Gly Thr Ala Gly Ala Thr Tyr Tyr Pro Gly Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gly Asp Tyr Val Trp Gly Thr Tyr Arg Pro Leu Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 3

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 3

ggattcccct tcagtagcta cgac 24

<210> 4

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 4

Gly Phe Pro Phe Ser Ser Tyr Asp

1 5

<210> 5

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 5

attggtactg ctggtgccac a 21

<210> 6

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 6

Ile Gly Thr Ala Gly Ala Thr

1 5

<210> 7

<211> 48

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 7

gcaagagggg attacgtttg ggggacttat cgtcccctct ttgactac 48

<210> 8

<211> 16

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 8

Ala Arg Gly Asp Tyr Val Trp Gly Thr Tyr Arg Pro Leu Phe Asp Tyr

1 5 10 15

<210> 9

<211> 321

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 9

gacatccagt tgacccagtc tccatccttc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgct gggccagtca gggcattaac aattatttag cctggtatca acaaaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccactt tgcaaactgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240

gaagattttg caacttatta ctgtcagcag cttaatagtt acccgctcac tttcggcgga 300

gggaccaagg tggagatcaa a 321

<210> 10

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 10

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Trp Ala Ser Gln Gly Ile Asn Asn Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asn Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 11

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 11

cagggcatta acaattat 18

<210> 12

<211> 6

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 12

Gln Gly Ile Asn Asn Tyr

1 5

<210> 13

<211> 9

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 13

gctgcatcc 9

<210> 14

<211> 3

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 14

Ala Ala Ser

1

<210> 15

<211> 27

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 15

cagcagctta atagttaccc gctcact 27

<210> 16

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 16

Gln Gln Leu Asn Ser Tyr Pro Leu Thr

1 5

<210> 17

<211> 1359

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 17

gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctctggatt ccccttcagt agctacgaca tgcactgggt ccgccaagct 120

acaggaaaag gtctggagtg ggtctcagct attggtactg ctggtgccac atactatcca 180

ggctccgtga agggccgatt caccatctcc agagaaaatg ccaagaactc cttgtatctt 240

caaatgaaca gcctgagagc cggggacacg gctgtgtatt actgtgcaag aggggattac 300

gtttggggga cttatcgtcc cctctttgac tactggggcc agggaaccct ggtcaccgtc 360

tcctcagcct ccaccaaggg cccatcggtc ttccccctgg caccctcctc caagagcacc 420

tctgggggca cagcggccct gggctgcctg gtcaaggact acttccccga accggtgacg 480

gtgtcgtgga actcaggcgc cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag 540

tcctcaggac tctactccct cagcagcgtg gtgaccgtgc cctccagcag cttgggcacc 600

cagacctaca tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga caagaaagtt 660

gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg 720

gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 780

acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 840

aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 900

tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 960

ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 1020

atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 1080

gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 1140

gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 1200

cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 1260

aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 1320

tacacgcaga agtccctctc cctgtctccg ggtaaatga 1359

<210> 18

<211> 452

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 18

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Ser Ser Tyr

20 25 30

Asp Met His Trp Val Arg Gln Ala Thr Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Gly Thr Ala Gly Ala Thr Tyr Tyr Pro Gly Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gly Asp Tyr Val Trp Gly Thr Tyr Arg Pro Leu Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 19

<211> 1359

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 19

gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctctggatt ccccttcagt agctacgaca tgcactgggt ccgccaagct 120

acaggaaaag gtctggagtg ggtctcagct attggtactg ctggtgccac atactatcca 180

ggctccgtga agggccgatt caccatctcc agagaaaatg ccaagaactc cttgtatctt 240

caaatgaaca gcctgagagc cggggacacg gctgtgtatt actgtgcaag aggggattac 300

gtttggggga cttatcgtcc cctctttgac tactggggcc agggaaccct ggtcaccgtc 360

tcctcagcct ccaccaaggg cccatcggtc ttccccctgg caccctcctc caagagcacc 420

tctgggggca cagcggccct gggctgcctg gtcaaggact acttccccga accggtgacg 480

gtgtcgtgga actcaggcgc cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag 540

tcctcaggac tctactccct cagcagcgtg gtgaccgtgc cctccagcag cttgggcacc 600

cagacctaca tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga caagaaagtt 660

gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg 720

gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 780

acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 840

aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 900

taccaaagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 960

ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 1020

atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 1080

gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 1140

gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 1200

cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 1260

aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 1320

tacacgcaga agtccctctc cctgtctccg ggtaaatga 1359

<210> 20

<211> 452

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 20

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Ser Ser Tyr

20 25 30

Asp Met His Trp Val Arg Gln Ala Thr Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Gly Thr Ala Gly Ala Thr Tyr Tyr Pro Gly Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gly Asp Tyr Val Trp Gly Thr Tyr Arg Pro Leu Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Gln Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 21

<211> 645

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 21

gacatccagt tgacccagtc tccatccttc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgct gggccagtca gggcattaac aattatttag cctggtatca acaaaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccactt tgcaaactgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240

gaagattttg caacttatta ctgtcagcag cttaatagtt acccgctcac tttcggcgga 300

gggaccaagg tggagatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 360

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420

cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480

gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540

ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600

ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag 645

<210> 22

<211> 214

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 22

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Trp Ala Ser Gln Gly Ile Asn Asn Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asn Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 23

<211> 13

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 23

Leu Glu Glu Lys Lys Gly Asn Tyr Val Val Thr Asp His

1 5 10

<210> 24

<211> 16

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 24

Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu Asp Gly Val Arg Lys Cys

1 5 10 15

<210> 25

<211> 356

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 25

Leu Glu Glu Lys Lys Gly Asn Tyr Val Val Thr Asp His Gly Ser Cys

1 5 10 15

Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu Asp Gly Val

20 25 30

Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val Cys Asn Gly

35 40 45

Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn

50 55 60

Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile

65 70 75 80

Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu

85 90 95

Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly

100 105 110

Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala

115 120 125

Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln

130 135 140

Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg

145 150 155 160

Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys

165 170 175

Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr

180 185 190

Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys

195 200 205

Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys

210 215 220

Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg

225 230 235 240

Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg

245 250 255

Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu

260 265 270

Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys

275 280 285

Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys

290 295 300

Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala

305 310 315 320

Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly

325 330 335

Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile

340 345 350

Pro Ser Ile Ala

355

<210> 26

<211> 19

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 26

Gly Pro Cys Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys

1 5 10 15

Asp Ser Leu

<210> 27

<211> 1210

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 27

Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala

1 5 10 15

Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln

20 25 30

Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe

35 40 45

Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn

50 55 60

Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys

65 70 75 80

Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val

85 90 95

Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr

100 105 110

Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn

115 120 125

Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu

130 135 140

His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu

145 150 155 160

Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met

165 170 175

Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro

180 185 190

Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln

195 200 205

Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg

210 215 220

Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys

225 230 235 240

Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp

245 250 255

Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro

260 265 270

Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly

275 280 285

Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His

290 295 300

Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu

305 310 315 320

Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val

325 330 335

Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn

340 345 350

Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp

355 360 365

Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr

370 375 380

Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu

385 390 395 400

Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp

405 410 415

Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln

420 425 430

His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu

435 440 445

Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser

450 455 460

Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu

465 470 475 480

Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu

485 490 495

Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro

500 505 510

Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn

515 520 525

Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly

530 535 540

Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro

545 550 555 560

Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro

565 570 575

Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val

580 585 590

Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp

595 600 605

Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys

610 615 620

Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly

625 630 635 640

Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu

645 650 655

Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met Arg Arg Arg His

660 665 670

Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg Glu Leu

675 680 685

Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala Leu Leu

690 695 700

Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu Gly Ser

705 710 715 720

Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile Pro Glu Gly Glu

725 730 735

Lys Val Lys Ile Pro Val Ala Ile Lys Glu Leu Arg Glu Ala Thr Ser

740 745 750

Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr Val Met Ala Ser

755 760 765

Val Asp Asn Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu Thr Ser

770 775 780

Thr Val Gln Leu Ile Thr Gln Leu Met Pro Phe Gly Cys Leu Leu Asp

785 790 795 800

Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln Tyr Leu Leu Asn

805 810 815

Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Asp Arg Arg

820 825 830

Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Thr Pro

835 840 845

Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu Gly Ala

850 855 860

Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val Pro Ile Lys Trp

865 870 875 880

Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr His Gln Ser Asp

885 890 895

Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Ser

900 905 910

Lys Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ser Ser Ile Leu Glu

915 920 925

Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr

930 935 940

Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg Pro Lys

945 950 955 960

Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys Met Ala Arg Asp Pro Gln

965 970 975

Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met His Leu Pro Ser Pro

980 985 990

Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp Glu Glu Asp Met Asp

995 1000 1005

Asp Val Val Asp Ala Asp Glu Tyr Leu Ile Pro Gln Gln Gly Phe

1010 1015 1020

Phe Ser Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser Ser Leu

1025 1030 1035

Ser Ala Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp Arg Asn

1040 1045 1050

Gly Leu Gln Ser Cys Pro Ile Lys Glu Asp Ser Phe Leu Gln Arg

1055 1060 1065

Tyr Ser Ser Asp Pro Thr Gly Ala Leu Thr Glu Asp Ser Ile Asp

1070 1075 1080

Asp Thr Phe Leu Pro Val Pro Glu Tyr Ile Asn Gln Ser Val Pro

1085 1090 1095

Lys Arg Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn Gln

1100 1105 1110

Pro Leu Asn Pro Ala Pro Ser Arg Asp Pro His Tyr Gln Asp Pro

1115 1120 1125

His Ser Thr Ala Val Gly Asn Pro Glu Tyr Leu Asn Thr Val Gln

1130 1135 1140

Pro Thr Cys Val Asn Ser Thr Phe Asp Ser Pro Ala His Trp Ala

1145 1150 1155

Gln Lys Gly Ser His Gln Ile Ser Leu Asp Asn Pro Asp Tyr Gln

1160 1165 1170

Gln Asp Phe Phe Pro Lys Glu Ala Lys Pro Asn Gly Ile Phe Lys

1175 1180 1185

Gly Ser Thr Ala Glu Asn Ala Glu Tyr Leu Arg Val Ala Pro Gln

1190 1195 1200

Ser Ser Glu Phe Ile Gly Ala

1205 1210

<210> 28

<211> 943

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 28

Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala

1 5 10 15

Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Gly Asn Tyr

20 25 30

Val Val Thr Asp His Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser

35 40 45

Tyr Glu Met Glu Glu Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly

50 55 60

Pro Cys Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp

65 70 75 80

Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr

85 90 95

Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp

100 105 110

Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu

115 120 125

Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro

130 135 140

Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg

145 150 155 160

Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu

165 170 175

Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly

180 185 190

Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile

195 200 205

Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile

210 215 220

Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His

225 230 235 240

Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys

245 250 255

Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys

260 265 270

Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys

275 280 285

Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys

290 295 300

Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp

305 310 315 320

Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn

325 330 335

Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu

340 345 350

Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly

355 360 365

Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val

370 375 380

Gly Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe

385 390 395 400

Met Arg Arg Arg His Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu

405 410 415

Gln Glu Arg Glu Leu Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro

420 425 430

Asn Gln Ala Leu Leu Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile

435 440 445

Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp

450 455 460

Ile Pro Glu Gly Glu Lys Val Lys Ile Pro Val Ala Ile Lys Glu Leu

465 470 475 480

Arg Glu Ala Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala

485 490 495

Tyr Val Met Ala Ser Val Asp Asn Pro His Val Cys Arg Leu Leu Gly

500 505 510

Ile Cys Leu Thr Ser Thr Val Gln Leu Ile Thr Gln Leu Met Pro Phe

515 520 525

Gly Cys Leu Leu Asp Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser

530 535 540

Gln Tyr Leu Leu Asn Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr

545 550 555 560

Leu Glu Asp Arg Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn Val

565 570 575

Leu Val Lys Thr Pro Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala

580 585 590

Lys Leu Leu Gly Ala Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys

595 600 605

Val Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr

610 615 620

Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu

625 630 635 640

Met Thr Phe Gly Ser Lys Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile

645 650 655

Ser Ser Ile Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys

660 665 670

Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met Ile Asp Ala

675 680 685

Asp Ser Arg Pro Lys Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys Met

690 695 700

Ala Arg Asp Pro Gln Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met

705 710 715 720

His Leu Pro Ser Pro Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp

725 730 735

Glu Glu Asp Met Asp Asp Val Val Asp Ala Asp Glu Tyr Leu Ile Pro

740 745 750

Gln Gln Gly Phe Phe Ser Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu

755 760 765

Ser Ser Leu Ser Ala Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp

770 775 780

Arg Asn Gly Leu Gln Ser Cys Pro Ile Lys Glu Asp Ser Phe Leu Gln

785 790 795 800

Arg Tyr Ser Ser Asp Pro Thr Gly Ala Leu Thr Glu Asp Ser Ile Asp

805 810 815

Asp Thr Phe Leu Pro Val Pro Glu Tyr Ile Asn Gln Ser Val Pro Lys

820 825 830

Arg Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn Gln Pro Leu

835 840 845

Asn Pro Ala Pro Ser Arg Asp Pro His Tyr Gln Asp Pro His Ser Thr

850 855 860

Ala Val Gly Asn Pro Glu Tyr Leu Asn Thr Val Gln Pro Thr Cys Val

865 870 875 880

Asn Ser Thr Phe Asp Ser Pro Ala His Trp Ala Gln Lys Gly Ser His

885 890 895

Gln Ile Ser Leu Asp Asn Pro Asp Tyr Gln Gln Asp Phe Phe Pro Lys

900 905 910

Glu Ala Lys Pro Asn Gly Ile Phe Lys Gly Ser Thr Ala Glu Asn Ala

915 920 925

Glu Tyr Leu Arg Val Ala Pro Gln Ser Ser Glu Phe Ile Gly Ala

930 935 940

<210> 29

<211> 384

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 29

Leu Glu Glu Lys Lys Gly Asn Tyr Val Val Thr Asp His Gly Ser Cys

1 5 10 15

Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu Asp Gly Val

20 25 30

Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val Cys Asn Gly

35 40 45

Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn

50 55 60

Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile

65 70 75 80

Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu

85 90 95

Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly

100 105 110

Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala

115 120 125

Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln

130 135 140

Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg

145 150 155 160

Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys

165 170 175

Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr

180 185 190

Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys

195 200 205

Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys

210 215 220

Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg

225 230 235 240

Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg

245 250 255

Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu

260 265 270

Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys

275 280 285

Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys

290 295 300

Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala

305 310 315 320

Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly

325 330 335

Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile

340 345 350

Pro Ser Ile Ala Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Gly Gly

355 360 365

Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu His His His His His His

370 375 380

Claims (37)

1. An antibody-drug conjugate (ADC) comprising an antibody or antigen-binding fragment thereof that specifically binds egfrvlll, wherein said antibody or antigen-binding fragment thereof comprises:

a Heavy Chain Variable Region (HCVR) comprising three heavy chain complementarity determining regions (HCDR 1, HCDR2, and HCDR 3) within the Heavy Chain Variable Region (HCVR), the HCVR comprising the amino acid sequence of SEQ ID NO: 2; and

a Light Chain Variable Region (LCVR) comprising three light chain complementarity determining regions (LCDR 1, LCDR2, and LCDR 3) within the Light Chain Variable Region (LCVR), the LCVR comprising the amino acid sequence of SEQ ID NO: 10;

and wherein the antibody is conjugated to doxycycline.

2. The ADC of claim 1, wherein the anti-egfrvlll antibody or antigen binding fragment thereof neither binds:

(i) A linker peptide of SEQ ID NO. 23; nor is combined with

(ii) The peptide of SEQ ID NO. 24.

3. The ADC of claim 1 or claim 2, wherein the antibody or antigen-binding fragment thereof exhibits an equilibrium dissociation constant (K _D ) About 500nM, as measured by surface plasmon resonance assay at 37 ℃.

4. The ADC of any one of claims 1-3, wherein the antibody or antigen-binding fragment thereof exhibits an equilibrium dissociation constant (K _D ) About 10nM or less, as measured by surface plasmon resonance assay at 37 ℃.

5. The ADC of any one of claims 1-4, wherein the antibody or antigen binding fragment thereof does not bind EGFR dimer at a level detectable by a surface plasmon resonance assay.

6. The ADC of any one of claims 1-5, wherein the antibody or antigen-binding fragment thereof comprises:

the HCVR, which includes,

HCDR1 comprising the amino acid sequence of SEQ ID NO. 4,

HCDR2 comprising the amino acid sequence of SEQ ID NO. 6, an

HCDR3 comprising the amino acid sequence of SEQ ID NO. 8,

and an LCVR, which includes,

LCDR1 comprising the amino acid sequence of SEQ ID NO. 12,

LCDR2 comprising the amino acid sequence of SEQ ID NO. 14, an

LCDR3 comprising the amino acid sequence of SEQ ID NO. 16.

7. The ADC of any one of claims 1-6, wherein the antibody or antigen-binding fragment thereof comprises:

HCVR comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID No. 2; and

LCVR comprising an amino acid sequence that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO. 10.

8. The ADC of any one of claims 1-7, wherein the antibody or antigen-binding fragment thereof comprises:

HCVR comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID No. 2; and

LCVR comprising an amino acid sequence that has at least 98% sequence identity to the amino acid sequence of SEQ ID NO. 10.

9. The ADC of any one of claims 1-8, wherein the antibody or antigen-binding fragment thereof comprises:

HCVR comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID No. 2; and

LCVR comprising an amino acid sequence that has at least 99% sequence identity to the amino acid sequence of SEQ ID NO. 10.

10. The ADC of any one of claims 1-9, wherein the antibody or antigen-binding fragment thereof comprises

HCVR comprising the amino acid sequence of SEQ ID No. 2; and

LCVR comprising the amino acid sequence of SEQ ID NO. 10.

11. The ADC of any one of claims 1-10, wherein the antibody or antigen-binding fragment thereof is a whole antibody.

12. The ADC of any one of claims 1-11, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID No. 18 or SEQ ID No. 20.

13. The ADC of any one of claims 1-12, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the light chain comprises the amino acid sequence of SEQ ID No. 22.

14. The ADC of any one of claims 1-11, 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.

15. The ADC of any one of claims 1-11, 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.

16. The ADC of any one of claims 1 to 15, wherein the drug-to-antibody ratio (DAR) is about 1 to about 4.

17. The ADC of any one of claims 1-16, wherein the antibody is non-glycosylated at N297.

18. The ADC of any one of claims 1-11, wherein the antibody comprises an N297Q mutation in hig 1 Fc, as determined by EU index numbering.

19. The ADC of any one of claims 1-11, wherein the antibody or antigen-binding fragment thereof comprises:

HCDR1 comprising the amino acid sequence of SEQ ID NO. 4,

HCDR2 comprising the amino acid sequence of SEQ ID NO. 6,

HCDR3 comprising the amino acid sequence of SEQ ID NO. 8,

LCDR1 comprising the amino acid sequence of SEQ ID NO. 12,

LCDR2 comprising the amino acid sequence of SEQ ID NO. 14, an

LCDR3 comprising the amino acid sequence of SEQ ID NO. 16;

wherein the heavy chain of the antibody or fragment is non-glycosylated and comprises the N297Q mutation, and wherein the antibody or fragment is conjugated to doxillin.

20. An ADC according to any one of claims 1 to 19, wherein the antibody or antigen binding fragment thereof interacts with at least one residue within the amino acid sequence of SEQ ID No. 26.

21. The ADC of any one of claims 1 to 20, wherein the ADC has one or more of the following features:

(a) In egfrvlll expressing cells, exhibiting reduced in vivo viability;

(b) Exhibiting in vivo bystander cytotoxicity against cells not expressing egfrvlll co-cultured with cells expressing egfrvlll;

(c) In mice with intracranial glioblastoma multiforme expressing egfrvlll, an increase in survival was demonstrated;

(d) In the absence of treatment-related weight loss, an anti-tumor effect was exhibited in mice with tumors expressing egfrvlll;

(e) Tumor regression was shown in mice with patient-derived glioblastoma multiforme;

(f) Demonstrating a stronger tumor killing effect at lower doses relative to the comparison antibody conjugated to MMAF; and

(g) In tumor-bearing mice, exhibit greater anti-tumor efficacy than anti-egfrvlll-maytansinoid ADC.

22. A complex comprising the ADC of any one of claims 1-20, wherein the antibody or antigen-binding fragment thereof binds to egfrvlll.

23. A container or injection device comprising an ADC as claimed in any one of claims 1 to 21.

24. A pharmaceutical composition comprising an ADC of any one of claims 1 to 21 and a pharmaceutically acceptable carrier or diluent.

25. The pharmaceutical composition of claim 24, further comprising one or more additional therapeutic agents selected from the group consisting of: chemotherapeutic agents, anti-inflammatory agents, and analgesic agents.

26. A method of treating cancer in a subject in need thereof having a tumor that expresses egfrvlll comprising administering to the subject a therapeutically effective amount of the ADC of any one of claims 1 to 21 or the pharmaceutical composition of claim 24.

27. A method for treating cancer or tumor in a subject in need thereof, or reducing tumor growth in a subject in need thereof, and/or causing tumor regression in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the ADC of any one of claims 1-21, or the pharmaceutical composition of claim 24.

28. The method of claim 26, wherein the cancer or tumor is selected from the group consisting of: glioblastoma, ductal or ductal breast cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, and squamous cell carcinoma of the head and neck.

29. The method of claim 26, further comprising administering one or more additional therapeutic agents selected from the group consisting of: chemotherapeutic agents, anti-inflammatory agents, and analgesic agents.

30. The method of claim 26, further comprising administering a second ADC comprising an antibody or antigen-binding fragment thereof and a cytotoxin, wherein the antibody or antigen-binding fragment thereof of the second ADC specifically binds egfrvlll and further binds to the connecting peptide of SEQ ID No. 23 and/or the peptide of SEQ ID No. 24.

31. A method for administering the ADC of any one of claims 1 to 21 into a subject, comprising injecting the ADC into the subject.

32. The method of claim 31, wherein the ADC is injected subcutaneously, intravenously, or intramuscularly into the subject.

33. A method for preparing an ADC of any one of claims 1-21, comprising culturing a host cell comprising the polynucleotide encoding such an immunoglobulin in a medium under conditions conducive for expression of the polynucleotide: an immunoglobulin comprising an HCVR of said ADC and an immunoglobulin comprising an LCVR of said ADC.

34. The method of claim 33, further comprising passing texti Lin Zhuige to one or more of the immunoglobulins.

35. The method of claim 34, wherein the conjugation is performed by: reducing the immunoglobulin chain in the presence of a reducing agent, and incubating the texillin with the reduced immunoglobulin chain.

36. The method of claim 35, wherein the reducing agent is dithiothreitol.

37. An ADC which is a product as claimed in any one of claims 33 to 36.