Anti-EGFRvIII antibody drug conjugates and their use

Abstract

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

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the interests of U.S. Provisional Patent Application No. 63 / 213,478, filed on 22 June 2021, and U.S. Provisional Patent Application No. 63 / 242,929, filed on 10 September 2021, each of which is incorporated herein by reference in whole.

[0002] Areas of disclosure This disclosure relates to an antibody-drug conjugate (ADC) (the antibody or its antigen-binding fragment is conjugated to tecilin) ​​that specifically binds to a human epidermal growth factor receptor (EGFR) deletion variant, specifically the class III deletion variant EGFRvIII, and an antigen-binding fragment of the human antibody, and to a therapeutic method using such ADC.

[0003] Sequence List An official copy of the sequence listing has been submitted electronically via EFS-Web at the same time as this Specified, as an ASCII format sequence listing file named "10966WO01_Sequence_Listing_ST25.TXT", created on June 21, 2022, and approximately 49,152 bytes in size. The sequence listing contained in this ASCII format document is part of this Specified, and the entire document is incorporated herein by reference. [Background technology]

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

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

[0006] The amino acid sequence of human EGFR is shown in SEQ ID NO: 27, and the amino acid sequence of EGFRvIII is shown in SEQ ID NO: 28. Antibodies against EGFRvIII are described, for example, in US5,212,290 (Patent Document 1), US7,736,644 (Patent Document 2), US7,589,180 (Patent Document 3), and US7,767,792 (Patent Document 4).

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

[0008]

Patent Document 1

Patent document 2

Patent Document 3

Patent document 4

Non-licensed literature

[0009] [Non-licensed document 1] Wong,AJet al.,1987,Proc.Natl.Acad.Sci.USA,84:6899-6903 [Non-licensed document 2] Harris et al.,1992,Natl.Cancer Inst.Monogr.11:181-187 [Non-licensed document 3] Ekstrand, AJ et al., 1992, Proc. Natl. Acad. Sci. USA, 89: 4309-4313

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

[0010] Summary of the Disclosure This disclosure provides an antibody-drug conjugate (ADC) comprising an antibody conjugating to EGFRvIII and its antigen-binding fragment, wherein the antibody and its antigen-binding fragment are conjugated to tecilin. Tecilin contains a pyrrolobenzodiazepine (PBD) payload / warhead, SG3199 (Tiberghien et al., 2016, ACS Medicinal Chemistry Letters 7(11):983-987). The ADC is particularly useful for targeting tumor cells expressing EGFRvIII.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0026] This disclosure includes an ADC comprising an anti-EGFRvIII antibody having a modified glycosylation pattern. In some embodiments, for example, modified antibodies that remove undesirable glycosylation sites or that lack a fucose portion present on the oligosaccharide chain may be useful to enhance antibody-dependent cell-mediated cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, galactosylation modifications can be made to modify complement-dependent cell-mediated cytotoxicity (CDC) activity. In some embodiments, the antibody or its antigen-binding fragment is aglycosylated. The aglycosylated antibody is point-mutated with a suitable residue to prevent glycosylation. In some embodiments, the antibody or its antigen-binding fragment contains a heavy chain aglycosylated at, for example, N297 (according to EU index numbering) to improve conjugation efficiency. In certain embodiments, N297 is mutated with a glutamine (Q) residue, i.e., the antibody contains the N297Q mutation.

[0027] In another embodiment, the present invention provides a complex comprising an anti-EGFRvIII-tesirin ADC, wherein the antibody or its antigen-binding fragment is conjugated to EGFRvIII.

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

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

[0030] [Invention 1001] An antibody-drug conjugate (ADC) comprising an antibody or its antigen-binding fragment that specifically binds to EGFRvIII, wherein the antibody or its antigen-binding fragment is The heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO: 2 contains three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3), The light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 10 contains three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) and The antibody-drug conjugate (ADC) comprises the antibody conjugated to tesirin. [Invention 1002] Anti-EGFRvIII antibody or its antigen-binding fragment, (i) Also in the conjugation peptide of SEQ ID NO: 23 (ii) Also in the peptide of SEQ ID NO. 24 A non-bonding ADC of the present invention 1001. [Invention 1003] When the antibody or its antigen-binding fragment is measured by a surface plasmon resonance assay at 37°C, the equilibrium dissociation constant (K) for human EGFRvIII monomers is approximately 500 nM. D An ADC according to the present invention 1001 or 1002, which shows ). [Invention 1004] When the antibody or its antigen-binding fragment is measured by a surface plasmon resonance assay at 37°C, the equilibrium dissociation constant (K) for human EGFRvIII dimers is approximately 10 nM or less. D An ADC according to any of the present invention 1001 to 1003, which shows ). [Invention 1005] An ADC according to any one of the present invention 1001 to 1004, wherein the antibody or its antigen-binding fragment does not bind to the EGFR dimer at a level detectable by surface plasmon resonance assay. [Invention 1006] The antibody or its antigen-binding fragment HCDR1 containing the amino acid sequence of SEQ ID NO: 4, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, and HCDR3 containing the amino acid sequence of SEQ ID NO: 8 HCVR including, LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence of SEQ ID NO: 14, and LCDR3 containing the amino acid sequence of SEQ ID NO: 16 LCVR including An ADC according to any of invention 1001 to 1005, including the present invention. [Invention 1007] The antibody or its antigen-binding fragment HCVR containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 2, LCVR containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 10 An ADC according to any of the inventions 1001 to 1006, including the present invention. [Invention 1008] The antibody or its antigen-binding fragment HCVR containing an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 2, LCVR containing an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 10 An ADC according to any of the present invention 1001 to 1007, including the present invention. [Invention 1009] The antibody or its antigen-binding fragment HCVR containing an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 2, LCVR containing an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 10 An ADC according to any of the inventions 1001 to 1008, including the present invention. [Invention 1010] The antibody or its antigen-binding fragment HCVR containing the amino acid sequence of SEQ ID NO: 2, LCVR containing the amino acid sequence of SEQ ID NO: 10 An ADC according to any of the inventions 1001 to 1009, including the present invention. [Invention 1011] An ADC according to any one of the present invention 1001 to 1010, wherein the antibody or its antigen-binding fragment is a complete antibody. [Invention 1012] An ADC according to any one of the present invention 1001 to 1011, wherein the antibody or its antigen-binding fragment comprises a heavy chain and a light chain, and the heavy chain comprises the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 20. [Invention 1013] The ADC according to any one of the present invention 1001 to 1012, wherein the antibody or its antigen-binding fragment comprises a heavy chain and a light chain, and the light chain comprises the amino acid sequence of SEQ ID NO: 22. [Invention 1014] An ADC according to any one of the invention 1001 to 1011, wherein the antibody or its antigen-binding fragment comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 18 and a light chain containing the amino acid sequence of SEQ ID NO: 22. [Invention 1015] An ADC according to any one of the invention 1001 to 1011, wherein the antibody or its antigen-binding fragment comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 20 and a light chain containing the amino acid sequence of SEQ ID NO: 22. [Invention 1016] An ADC according to any of the invention 1001 to 1015, wherein the drug-to-antibody ratio (DAR) is approximately 1 to approximately 4. [Invention 1017] The ADC according to any of the present invention 1001 to 1016, wherein the antibody is aglycosylated at N297. [Invention 1018] The ADC according to any one of the invention 1001 to 1011, wherein the antibody contains the N297Q mutation determined by EU index numbering in hIgG1 Fc. [Invention 1019] The antibody or its antigen-binding fragment HCDR1 containing the amino acid sequence of SEQ ID NO: 4, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, HCDR3 containing the amino acid sequence of SEQ ID NO: 8, LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence of SEQ ID NO: 14, and LCDR3 containing the amino acid sequence of SEQ ID NO: 16 An ADC according to any one of the present invention 1001 to 1011, comprising, wherein the heavy chain of the antibody or fragment is aglycosylated and contains the N297Q mutation, and the antibody or fragment is conjugated to tesirin. [Invention 1020] An ADC according to any one of the present invention 1001 to 1019, wherein the antibody or its antigen-binding fragment interacts with at least one residue in the amino acid sequence of SEQ ID NO: 26. [Invention 1021] The following features: (a) Shows reduced in vivo survival rate in EGFRvIII-expressing cells. (b) Demonstrating in vivo bystander cytotoxicity against non-EGFRvIII-expressing cells co-cultured with EGFRvIII-expressing cells, (c) In mice with intracranial glioblastoma expressing EGFRvIII, prolonged survival was observed. (d) Exhibit antitumor effects in mice with EGFRvIII-expressing tumors in the absence of treatment-related weight loss. (e) In mice with patient-derived glioblastoma multiforme, tumor regression is observed. (f) Compared to a comparative control antibody conjugated to MMAF, it exhibits greater tumor-killing activity at lower doses, and (g) In tumor-bearing mice, it exhibits greater antitumor efficacy than anti-EGFRvIII-maytansinoid ADC. An ADC according to any of the present invention 1001 to 1020, having one or more of the features of the present invention. [Invention 1022] A complex comprising an ADC according to any of the present invention 1001 to 1020, wherein the antibody or its antigen-binding fragment binds to EGFRvIII. [Invention 1023] A container or injection device comprising any ADC according to invention 1001 to 1021. [Invention 1024] A pharmaceutical composition comprising any ADC according to invention 1001 to 1021, and a pharmaceutically acceptable carrier or diluent. [Invention 1025] A pharmaceutical composition of the present invention 1024, further comprising one or more additional therapeutic agents selected from the group consisting of chemotherapeutic agents, anti-inflammatory agents, and analgesics. [Invention 1026] A method for treating cancer in a subject who is in need of treatment and who has an EGFRvIII-expressing tumor, comprising administering a therapeutically effective amount of any ADC of Invention 1001 to 1021 or a pharmaceutical composition of Invention 1024 to the subject. [Invention 1027] A method for treating cancer or a tumor, reducing tumor growth, and / or inducing tumor regression in a subject in need thereof, comprising administering a therapeutically effective amount of any ADC of Invention 1001 to 1021 or a pharmaceutical composition of Invention 1024 to the subject. [Invention 1028] The method of the present invention 1026, wherein the cancer or tumor is selected from the group consisting of glioblastoma, ductal carcinoma or intraductal carcinoma, non-small cell lung cancer, ovarian cancer, prostate cancer, and squamous cell carcinoma of the head and neck. [Invention 1029] The method of the present invention 1026, further comprising administering one or more additional therapeutic agents selected from the group consisting of chemotherapeutic agents, anti-inflammatory agents, and analgesics. [Invention 1030] The method of the present invention 1026, further comprising administering a second ADC comprising an antibody or an antigen-binding fragment thereof and a cytotoxin, wherein the antibody or antigen-binding fragment of the second ADC specifically binds to EGFRvIII and also binds to the conjugation peptide of SEQ ID NO: 23 and / or the peptide of SEQ ID NO: 24. [Invention 1031] A method for administering any ADC according to invention 1001 to 1021 to a target body, the method comprising injecting the ADC into the target body. [Invention 1032] The method of the present invention 1031, wherein the ADC is injected subcutaneously, intravenously, or intramuscularly into the body of the subject. [Invention 1033] A method for producing any ADC according to any of the present invention 1001 to 1021, comprising culturing a host cell containing an immunoglobulin comprising the HCVR of the ADC and a polynucleotide encoding the immunoglobulin comprising the LCVR of the ADC in a culture medium under conditions favorable for the expression of the polynucleotide. [Invention 1034] The method of the present invention 1033, further comprising conjugating tesirin to one or more of the immunoglobulins. [Invention 1035] The method of the present invention 1034, wherein the conjugation is carried out by reducing the immunoglobulin chain in the presence of a reducing agent and incubating the reduced immunoglobulin chain with tesirin. [Invention 1036] The method of the present invention 1035, wherein the reducing agent is dithiothreitol. [Invention 1037] ADC, which is a product of any of invention 1033 to 1036. Other embodiments will become apparent from the following examination of embodiments for carrying out the invention. [Brief explanation of the drawing]

[0031] [Figure 1] This paper compares tumor volume and body weight 61 days after subcutaneous injection of 0.5 × 10⁶ MMT-EGFRvIII cells into the flanks of female SCID mice using either an anti-EGFRvIII-tesillin conjugate or an anti-EGFRvIII-meitansinoid DM1 conjugate. [Modes for carrying out the invention]

[0032] Detailed explanation Before describing this disclosure, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, and therefore the methods and conditions may vary. Since the scope of this disclosure is limited only by the appended claims, it should also be understood that the terms used herein are intended to describe only specific embodiments and are not intended to limit them.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. Where used herein, the term “about” means that, when used in reference to a specific numerical value, the value may vary by up to 1% or less from the value mentioned. For example, where used herein, the expression “about 100” includes 99 and 101, as well as all values ​​in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0034] To describe and define this disclosure, note that this specification uses relative terms such as “substantially,” “generally,” and “approximately” to express the degree of inherent uncertainty that may arise from any quantitative comparison, value, measurement, or other expression. These terms are also used this specification to express the degree to which quantitative expressions may vary from the references given without resulting in a change in the fundamental function of the subject matter in question.

[0035] In some examples, the term “substantially” with respect to a given parameter, characteristic, or condition may mean, and may include, to the extent that a person skilled in the art would understand that the given parameter, characteristic, or condition is met with a small degree of variability, such as within acceptable manufacturing tolerances. For example, depending on a particular parameter, characteristic, or condition that is substantially met, the parameter, characteristic, or condition may be met at least 90%, at least 95%, at least 99%, or fully.

[0036] Any methods and materials similar or equivalent to those described herein may be used in the practice or testing of this disclosure, but exemplary methods and materials are described herein. All patents, patent applications, and non-patent publications referenced herein are incorporated in their entirety by reference.

[0037] definition As used herein, the term "EGFRvIII" refers, unless otherwise specified, to a human EGFR class III variant having the amino acid sequence shown in SEQ ID NO: 28, or a biologically active fragment thereof, exhibiting any features specific to EGFRvIII as opposed to those common to normally expressed EGFR. EGFRvIII lacks amino acid residues 6–273 of mature EGFR (i.e., SEQ ID NO: 27, which lacks the signal peptide (i.e., residues 1–24)) and contains a new glycine residue at position 6 between amino acid residues 5 and 274.

[0038] All references to proteins, polypeptides, and protein fragments herein are intended to refer to the human versions of the respective proteins, polypeptides, or protein fragments unless expressly specified as being of non-human origin. Therefore, the expression "EGFRvIII" means human EGFRvIII unless specifically identified as being of non-human origin, such as "mouse EGFRvIII" or "monkey EGFRvIII."

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

[0040] The term "antibody" encompasses immunoglobulin molecules, including four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or V H H) and a heavy chain constant region. The heavy chain constant region comprises three domains, C H H1, C H H2, and C H H3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or V L L) and a light chain constant region. The light chain constant region comprises one domain (C L L1). The V H H region and the V L L region can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), interspersed with relatively conserved regions called framework regions (FRs). Each V H H and V L L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the present disclosure, the FRs of the anti-EGFRvIII antibody (or antigen-binding portion thereof) may be identical to human germline sequences, or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on parallel analysis of two or more CDRs.

[0041] As used herein, the terms “antigen-binding portion” and “antigen-binding fragment” of an antibody include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies may be derived from a complete antibody molecule using any suitable standard method, such as protein digestion or recombinant genetic engineering techniques, which involve the manipulation and expression of DNA encoding an antibody variable region and optionally a constant domain. Such DNA is known and / or readily available, for example, from commercially available sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. DNA can be sequenced and manipulated by chemical or molecular biological techniques to, for example, arrange one or more variable domains and / or constant domains in a suitable configuration, introduce codons, create cysteine ​​residues, modify, add, or delete amino acids.

[0042] Non-limiting examples of antigen-binding fragments include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single-chain Fv(scFv) molecules, (vi) dAb fragments, and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable region of an antibody (e.g., isolated complementarity-determining regions (CDRs) such as the CDR3 peptide) or the restricted FR3-CDR3-FR4 peptide. Other manipulated molecules such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-implanted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunotherapies (SMIPs), and shark variable IgNAR domains are also included in the expression “antigen-binding fragment” as used herein.

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

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

[0045] The antibodies useful herein may function via complement-dependent cell-mediated cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). "Complement-dependent cell-mediated cytotoxicity" (CDC) refers to the lysis of antigen-expressing cells by the antibodies of this disclosure in the presence of complement. "Antibody-dependent cell-mediated cytotoxicity" (ADCC) refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing the Fc receptor (FcR) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize a bound antibody on a target cell, thereby resulting in the lysis of the target cell. CDC and ADCC may be measured using assays well known and available in the art (see, for example, U.S. Patents 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 crucial in its ability to immobilize complement and mediate cell-dependent cytotoxicity. Therefore, antibody isotypes may be selected based on whether it is desirable for the antibody to mediate cytotoxicity.

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

[0047] In some embodiments, the antibodies useful herein may be recombinant human antibodies. The term “recombinant human antibody,” as used herein, is intended to include all human antibodies prepared, expressed, created, or isolated by genetic recombination means, such as antibodies expressed using a recombinant expression vector transduced into host cells (detailed below), antibodies isolated from recombinants, combinatorial human antibody libraries (detailed below), antibodies isolated from animals transgenic to human immunoglobulin genes (e.g., mice) (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, created, or isolated by any other means, including splicing human immunoglobulin gene sequences 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 are subjected to in vitro mutagenesis (or, if transgenic animals are used for the human Ig sequence, in vivo mutagenesis of somatic cells), so the V of the recombinant antibody H Region and V L The amino acid sequence of the region is human germline V H Array and V L While it is derived from and related to the sequence, it cannot naturally exist in the in vivo human antibody germline repertoire.

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

[0049] The frequency of occurrence of the second form in various intact IgG isotypes is due to, but is not limited to, structural differences related to the hinge region isotype of the antibody. A single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the occurrence of the second form to the level typically observed using the human IgG1 hinge (Angal et al. (1993) Molecular Immunology 30:105). This invention relates to the hinge, C H 2 regions or C H The antibodies include those having one or more mutations in three regions, and these mutations may be desirable, for example, in production, to improve the yield of a desired antibody type.

[0050] Antibodies useful herein may be isolated antibodies. As used herein, “isolated antibody” means an antibody identified, isolated, and / or recovered from at least one component of its natural environment. For example, an antibody isolated or extracted from at least one component of an organism, or from a tissue or cell in which such antibody is naturally present or naturally produced, is an “isolated antibody” for the purposes of this disclosure. Isolated antibodies also include antibodies in situ within recombinant cells. An isolated antibody is an antibody that has undergone at least one purification or isolation step. According to certain embodiments, an isolated antibody may not substantially contain other cellular material and / or chemicals.

[0051] Anti-EGFRvIII antibodies useful herein may include one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR region of the heavy and light chain variable domains compared to the corresponding germline sequence from which the antibody is derived. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from publicly available antibody sequence databases. This disclosure includes an ADC comprising an antibody and its antigen-binding fragment derived from one of the amino acid sequences disclosed herein, wherein one or more amino acids in one or more frameworks and / or CDR regions are mutated to a corresponding residue in the germline sequence from which the antibody is derived, or a corresponding residue in another human germline sequence, or a conserved amino acid substitution of a corresponding germline residue (such sequence changes are collectively referred to herein as “germline mutations”). Those skilled in the art can readily produce many antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof, starting from the heavy chain variable region sequences and light chain variable region sequences disclosed herein. In certain embodiments, V H and / or V LIn other embodiments, all of the framework and / or CDR residues within the domain are mutated back to residues found in the original germline sequence from which the antibody originates. In other embodiments, only certain residues, for example, only mutant residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only mutant residues found within CDR1, CDR2, or CDR3, are mutated back to the original germline sequence. In other embodiments, one or more of the framework and / or CDR residues are mutated to corresponding residues in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody originally originates). Furthermore, antibodies useful herein may contain any combination of two or more germline mutations within the framework and / or CDR region, for example, where certain individual residues are mutated to corresponding residues in a particular germline sequence, while certain other residues, different from the original germline sequence, are maintained or mutated to corresponding residues in a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced (optionally obtained) the biological properties of the antagonist or agonist, or reduced immunogenicity. Antibodies and antigen-binding fragments obtained in this general manner are included in this disclosure.

[0052] This disclosure also includes anti-EGFRvIII antibodies useful herein, comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, this disclosure includes anti-EGFRvIII antibodies having HCVR, LCVR, and / or CDR amino acid sequences having, for example, 10 or fewer, 8 or fewer, 6 or fewer, or 4 or fewer conservative amino acid substitutions to any of the HCVR, LCVR, and / or CDR amino acid sequences listed in Table 1 herein.

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

[0054] When referring to polypeptides, the term “substantial identity” or “substantially identical” means that two peptide sequences share at least 95% sequence identity, and more preferably at least 98% or 99%, when optimally aligned using default gap weights by programmed GAP or BESTFIT, etc. In some embodiments, non-identical residue positions differ only by conserved amino acid substitutions. A “conservative amino acid substitution” is when an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of the protein. If two or more amino acid sequences differ from each other by conservative substitutions, the percentage of sequence identity or degree of similarity may be adjusted upward to compensate for the conservative nature of the substitutions. 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 (incorporated herein by reference). Examples of amino acid groups having side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conserved amino acid substituents are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative permutation is any change that has a positive value in the PAM250 log-likelihood matrix, as disclosed in Gonnet et al. (1992) Science 256:1443-1445, which is incorporated herein by reference. A “moderately conservative” permutation is any change that has a non-negative value in the PAM250 log-likelihood matrix.

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

[0056] The subjects are mammals, preferably humans.

[0057] Anti-EGFRvIII antibody containing Fc variant According to certain embodiments of this disclosure, anti-EGFRvIII antibodies useful herein include an Fc domain containing one or more mutations that enhance or reduce antibody binding to the FcRn receptor at acidic pH compared to neutral pH. For example, this disclosure includes the C of the Fc domain. H 2 or C HThe ADC contains an anti-EGFRvIII antibody with mutations in three regions, where the mutations enhance the affinity of the Fc domain to FcRn in an acidic environment (e.g., in endosomes with a pH in the range of approximately 5.5 to 6.0). Such mutations may prolong the serum half-life of the antibody when administered to animals. Non-limiting examples of such Fc modifications include, for example, modifications at position 250 (e.g., E or Q), positions 250 and 428 (e.g., L or F), position 252 (e.g., L / Y / F / W or T), position 254 (e.g., S or T), and position 256 (e.g., S / R / Q / E / D, or T), or modifications at position 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., A, W, H, F, or Y [N434A, N434W, N434H, N434F, or N434Y]), or modifications at position 250 and / or 428, or modifications at position 307 or 308 (e.g., 308F, V308F), and position 434. In one embodiment, modifications include modifications of 428L (e.g., M428L) and 434S (e.g., N434S), modifications of 428L, 259I (e.g., V259I), and 308F (e.g., V308F), modifications of 433K (e.g., H433K) and 434 (e.g., 434Y), modifications of 252, 254, and 256 (e.g., 252Y, 254T, and 256E), modifications of 250Q and 428L (e.g., T250Q and M428L), and modifications of 307 and / or 308 (e.g., 308F and / or 308P). In yet another embodiment, modifications include modifications of 265A (e.g., D265A) and / or 297A (e.g., N297A).

[0058] For example, this disclosure includes an ADC comprising an anti-EGFRvIII antibody comprising an Fc domain comprising one or more pairs or groups of mutations selected from the following group: 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 N43 4S), 257I and 311I (e.g., P257I and Q311I), 257I and 434H (e.g., P257I and N434H), 376V and 434H (e.g., D376V and N434H), 307A, 380A and 434A (e.g., T307A, E380A and N434A), and 433K and 434F (e.g., H433K and N434F). It is assumed that all possible combinations of the aforementioned Fc domain mutations and other mutations within the antibody variable domains disclosed herein are within the scope of this disclosure.

[0059] This disclosure also relates to the chimeric heavy chain constant (C H ADC containing an anti-EGFRvIII antibody having a region, and a chimeric C H The region is C of two or more immunoglobulin isotypes. H It includes segments derived from the region. For example, antibodies useful herein are derived from human IgG1, human IgG2, or human IgG4 molecules. H In combination with some or all of the three domains, C derived from human IgG1, human IgG2, or human IgG4 molecules. H Chimera C containing part or all of 2 domains H It may include a region. According to a particular embodiment, an antibody useful herein is a chimeric C having a chimeric hinge region. HThe region includes. For example, the chimeric hinge may include an "upper hinge" amino acid sequence (amino acid residues at EU numbering positions 216-227) derived from the human IgG1 hinge region, human IgG2 hinge region, or human IgG4 hinge region, combined with a "lower hinge" sequence (amino acid residues at EU numbering positions 228-236) derived from the human IgG1 hinge region, human IgG2 hinge region, or human IgG4 hinge region. According to certain embodiments, the chimeric hinge region includes amino acid residues derived from the human IgG1 upper hinge or human IgG4 upper hinge and amino acid residues derived from the human IgG2 lower hinge. Chimeric C as described herein H Antibodies containing the region exhibit, in certain embodiments, modified Fc effector functionality without negatively affecting the therapeutic or pharmacokinetic properties of the antibody. (See, for example, U.S. Provisional Application No. 61 / 759,578 filed February 1, 2013, the disclosure of which is incorporated herein by reference in its entirety.)

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

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

[0062] Tecilin has the following structure: TIFF0007923780000001.tif36152

[0063] Tecilin is also known as SG3249.

[0064] A compound having the following structure is provided herein, In formula TIFF0007923780000002.tif35155, Ab comprises an anti-EGFRvIII antibody or its antigen-binding fragment, and -S- is a sulfide bond at a cysteine ​​residue of the antibody or its antigen-binding fragment. In certain embodiments, Ab comprises three heavy-chain CDRs in the HCVR amino acid sequence containing SEQ ID NO: 2, and three light-chain CDRs in the LCVR amino acid sequence of SEQ ID NO: 10. In certain 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 certain embodiments, Ab includes an HCVR amino acid sequence having at least 95%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 2, and an LCVR amino acid sequence having at least 95%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 10. In certain embodiments, Ab includes the HCVR amino acid sequence of SEQ ID NO: 2 and / or the LCVR amino acid sequence of SEQ ID NO: 10.

[0065] Compounds having the following structure are also provided herein, In formula TIFF0007923780000003.tif35155, Ab comprises an anti-EGFRvIII antibody or its antigen-binding fragment, and -S- is a sulfide bond at a cysteine ​​residue of the antibody or its antigen-binding fragment. In certain embodiments, Ab is a complete antibody. In certain embodiments, Ab comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO: 18. In certain embodiments, Ab comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO: 20. In certain embodiments, Ab comprises a heavy chain and a light chain, the light chain comprising the amino acid sequence of SEQ ID NO: 22. In certain embodiments, Ab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 18 and a light chain comprising the amino acid sequence of SEQ ID NO: 22. In certain embodiments, Ab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 20 and a light chain comprising the amino acid sequence of SEQ ID NO: 22.

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

[0067] The synthesis of Tecilin can be carried out, for example, using the procedure described in Tiberghien et al. (ACS Medicinal Chemistry Letters 2016, 7(11):983-987). Tecilin contains the pyrrolobenzodiazepine warhead / payload component SG3199, which has the following structure. TIFF0007923780000004.tif16128

[0068] Epitope mapping and related technologies The antibody-bound epitopes described herein may consist of a single continuous sequence of three or more amino acids (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more) of the EGFRvIII protein. Alternatively, the epitope may consist of multiple discontinuous amino acids (or amino acid sequences) of EGFRvIII. In some embodiments, the epitope is located on or near the ligand-binding domain of EGFRvIII. In other embodiments, the epitope is located outside the ligand-binding domain of EGFRvIII, for example, on the EGFRvIII surface where the antibody binding to such an epitope does not interfere with ligand binding to EGFRvIII.

[0069] According to one particular embodiment, the antibodies and antigen-binding fragments useful herein include anti-EGFRvIII antibodies that specifically bind to EGFRvIII (but not to EGFR), and the antibody recognizes an EGFRvIII conjugated peptide (e.g., SEQ ID NO: 23). Such antibodies may be referred to herein as “conjugated peptide binders,” “EGFRvIII peptide-binding antibodies,” etc. According to another embodiment, the anti-EGFRvIII antibodies useful herein specifically bind to EGFRvIII (but not to EGFR), and the antibody does not recognize an EGFRvIII conjugated peptide (e.g., does not recognize the conjugated 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 “stereostructural binders,” “EGFRvIII stereostructural epitope binders,” etc.

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

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

[0072] This disclosure further includes an ADC comprising an anti-EGFRvIII antibody that binds to the same epitope as any of the specific exemplary antibodies described herein (e.g., antibodies comprising any of the amino acid sequences listed in Table 1 of this specification). Similarly, this disclosure also includes an ADC comprising an anti-EGFRvIII antibody that competes with any of the specific exemplary antibodies described herein (e.g., antibodies comprising any of the amino acid sequences listed in Table 1 of this specification) for binding to EGFRvIII.

[0073] By using standard methods known in the art and illustrated herein, it is readily possible to determine whether an antibody binds to the same epitope as the reference anti-EGFRvIII antibody or whether it competes for binding with the reference anti-EGFRvIII antibody. For example, to determine whether a test antibody binds to the same epitope as the reference anti-EGFRvIII antibody of this disclosure, the reference antibody is conjugated to the EGFRvIII protein. The ability of the test antibody to bind to the EGFRvIII molecule is then evaluated. If the test antibody is able to bind to EGFRvIII after saturation binding with the reference anti-EGFRvIII antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-EGFRvIII antibody. On the other hand, if the test antibody is unable to bind to the EGFRvIII molecule after saturation binding with the reference anti-EGFRvIII antibody, the test antibody may bind to the same epitope as the epitope conjugated by the reference anti-EGFRvIII antibody of this disclosure. Subsequently, additional standard experiments (e.g., peptide mutation and binding analysis) can be performed to confirm whether the observed lack of binding of the test antibody is actually due to binding to the same epitope as the reference antibody, or whether steric blockage (or another phenomenon) is the cause of the observed lack of binding. This type of experiment can be performed using ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art. According to certain embodiments of this disclosure, when measured in a competitive binding assay, if a surplus of, for example, 1x, 5x, 10x, 20x, or 100x of one antibody inhibits the binding of the other antibody by at least 50%, but preferably 75%, 90%, or even 99%, then the two antibodies bind to the same (or overlapping) epitopes (see, e.g., Junghans et al., Cancer Res. 1990:50:1495-1502). Alternatively, if essentially all amino acid mutations in an antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other antibody, then the two antibodies are considered to bind to the same epitope.If a subset of amino acid mutations that reduces or eliminates the binding of one antibody also reduces or eliminates the binding of the other antibody, then the two antibodies are considered to have a "duplicate epitope."

[0074] To determine whether an antibody competes for (or cross-competes for) binding with a reference anti-EGFRvIII antibody, the binding method described above is performed in two ways: In the first approach, the reference antibody binds to the EGFRvIII molecule under saturated conditions, and then the binding of the test antibody to the EGFRvIII molecule is evaluated. In the second approach, the test antibody binds to the EGFRvIII molecule under saturated conditions, and then the binding of the reference antibody to the EGFRvIII molecule is evaluated. If, in both approaches, only the first (saturated) antibody is able to bind to the EGFRvIII molecule, it is concluded that the test antibody and the reference antibody compete for binding to EGFRvIII. As will be understood by those skilled in the art, an antibody competing for binding to a reference antibody may not necessarily bind to the same epitope as the reference antibody, but may sterically block the binding of the reference antibody by binding to an overlapping or adjacent epitope.

[0075] Biological characteristics of anti-EGFRvIII ADCs The present invention comprises an anti-EGFRvIII-tesirin ADC that specifically binds to EGFRvIII. In some embodiments, the ADC comprises an anti-EGFRvIII antibody or its antigen-binding fragment and does not bind to (i) the conjugation peptide of SEQ ID NO: 23 or (ii) the peptide of SEQ ID NO: 24. In some embodiments, the ADC has an equilibrium dissociation constant (K) for human EGFRvIII monomers of approximately 500 nM, as measured by a surface plasmon resonance assay at 37°C. D The ADC comprises an anti-EGFRvIII antibody or its antigen-binding fragment exhibiting ) ). In some embodiments, the ADC has an equilibrium dissociation constant (K) for human EGFRvIII dimers of about 10 nM or less, as measured by a surface plasmon resonance assay at 37°C. DThe ADC comprises an anti-EGFRvIII antibody or its antigen-binding fragment that exhibits the following characteristics. In some embodiments, the ADC comprises an anti-EGFRvIII antibody or its antigen-binding fragment that does not bind to the EGFR dimer at a level detectable by surface plasmon resonance assay.

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

[0077] Preparation of human antibodies The anti-EGFRvIII antibodies or antigen-binding fragments useful herein may be fully human antibodies. Methods for producing monoclonal antibodies, including fully human monoclonal antibodies, are known in the art. Any such known method can be used in the context of this disclosure to produce human antibodies that specifically bind to human EGFRvIII.

[0078] For example, using VELOCIMMUNE™ technology or any other similar known method for generating fully human monoclonal antibodies, a high-affinity chimeric antibody against EGFRvIII, possessing a human variable region and a mouse constant region, is initially isolated. As in the experimental sections below, the antibody is characterized and selected for desirable features, including affinity, ligand blocking activity, selectivity, and epitope. If necessary, the mouse constant region is replaced with a desired human constant region, e.g., wild-type or modified IgG1 or IgG4, to generate a fully human anti-EGFRvIII antibody. While the selected constant region may vary depending on the specific application, the high-affinity antigen-binding and target-specific features reside in the variable region. In a particular example, the fully human anti-EGFRvIII antibody is isolated directly from antigen-positive B cells.

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

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

[0081] Two antigen-binding proteins or antibodies are considered bioequivalent or pharmaceutically equivalent if, under similar experimental conditions, they are administered in the same molar dose, either as a single or multiple doses, and their absorption rates and ranges do not show significant differences. Some antibodies may be considered equivalent or pharmaceutically equivalent if their absorption ranges are equivalent but their absorption rates are not. However, if this difference in absorption rate is intentional, reflected in the labeling, and is not considered medically important to the specific drug being tested (for example, not essential for achieving effective drug concentrations in the body over long-term use), then those antibodies may be considered bioequivalent.

[0082] In one embodiment, the two antigen-binding proteins are bioequivalent if there are no clinically significant differences in their safety, purity, and potency.

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

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

[0085] Bioequivalence can be demonstrated by in vivo and in vitro methods. Methods for measuring bioequivalence include, for example, (a) in vivo tests in humans or other mammals in which the concentration of the antibody or its metabolites in blood, plasma, serum or other biological fluids is measured as a function of time; (b) in vitro tests that correlate with and reasonably predict human in vivo bioavailability data; (c) in vivo tests in humans or other mammals in which the appropriate acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) appropriately controlled clinical trials that demonstrate the safety, efficacy, bioavailability, or bioequivalence of the antibody.

[0086] Bioequivalent variants of anti-EGFRvIII antibodies useful in the present invention may be constructed, for example, by making various substitutions of residues or sequences, or by deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity may be deleted or substituted with other amino acids to prevent the formation of unnecessary or inaccurate intramolecular disulfide crosslinks during regeneration. In other circumstances, bioequivalent antibodies may include anti-EGFRvIII antibody variants that include amino acid changes that alter the glycosylation characteristics of the antibody, such as mutations that eliminate or remove glycosylation.

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

[0088] Therapeutic preparations and administration This disclosure provides pharmaceutical compositions comprising anti-EGFRvIII antibody-tecillin conjugates, i.e., anti-EGFRvIII antibody-tecillin ADCs. The pharmaceutical compositions of this disclosure are formulated with suitable carriers, excipients, and other agents that provide improvements in transport, delivery, and tolerability. Numerous suitable formulations can be found in the prescription collection known to all pharmacists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, vesicle-containing lipids (cationic or anionic) (e.g., LIPOFECTIN®, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowaxes (polyethylene glycol 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 52:238-311.

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

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

[0091] The pharmaceutical compositions of this disclosure may be provided in a container. The pharmaceutical compositions of this disclosure may be provided in an injection device. The pharmaceutical compositions may be delivered subcutaneously using a standard needle and syringe, or intravenously. Furthermore, with respect to subcutaneous delivery, pen-type delivery devices are readily applicable to the delivery of the pharmaceutical compositions of this disclosure. Such pen-type delivery devices may be reusable or disposable. Reusable pen-type delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition inside the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen-type delivery device can then be reused. Disposable pen-type delivery devices do not have replaceable cartridges. Rather, disposable pen-type delivery devices are sold pre-filled with the pharmaceutical composition held in a reservoir inside the device. Once the pharmaceutical composition in the reservoir is empty, the entire device is discarded.

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

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

[0094] Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injection, intravenous infusion, etc. These injectable preparations may be prepared by known methods. For example, injectable preparations may be prepared by dissolving, suspending, or emulsifying the antibodies or salts thereof described above in a sterile aqueous or oily medium conventionally used for injection. Examples of aqueous media for injection include physiological saline, isotonic solutions containing glucose, and other adjuvants, which may be used in combination with suitable solubilizers such as alcohol (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. Examples of oily media include sesame oil and soybean oil, which may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The injection solutions thus prepared are preferably filled into suitable ampoules.

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

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

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

[0098] Therapeutic use of anti-EGFRvIII antibody conjugates This disclosure includes a method for administering to a subject in need of such treatment a therapeutic composition comprising an antibody-drug conjugate containing an anti-EGFRvIII antibody conjugated to tesirin (e.g., an anti-EGFRvIII antibody or ADC containing either an HCVR / LCVR or CDR sequence as listed in Table 1 of this specification). The therapeutic composition may include either an anti-EGFRvIII antibody conjugated to tesirin or its antigen-binding fragment, and a pharmaceutically acceptable carrier or diluent.

[0099] The ADCs of this disclosure are useful in treating, preventing, and / or improving any disease or disorder that can be treated by blocking the interaction between EGFRvIII and EGFR ligands, or by blocking the expression or activity, or overexpression, of EGFRvIII, or by otherwise inhibiting the activity and / or signaling of EGFRvIII, and / or by promoting receptor internalization and / or reducing the number of cell surface receptors. For example, the ADCs of this disclosure are useful in treating tumors that express EGFRvIII and / or respond to ligand-mediated signaling. The ADCs of this disclosure may also be used to treat primary and / or metastatic tumors occurring in the brain and meninges, oropharynx, lungs and bronchial trees, gastrointestinal tract, male and female reproductive organs, muscles, bones, skin and appendages, connective tissue, spleen, immune system, hematopoietic cells and bone marrow, liver and urinary tract, and special sensory organs such as the eye. 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, malignant glioma, osteosarcoma, colorectal cancer, gastric cancer (e.g., gastric cancer with MET amplification), malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, breast cancer (ductal or intraductal), or melanoma.

[0100] In the context of the therapeutic methods described herein, the anti-EGFRvIII antibody-tecilin conjugate 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).

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

[0102] Combination therapies and formulations This disclosure includes compositions and therapeutic formulations comprising any of the anti-EGFRvIII antibody-tecilin conjugates described herein in combination with one or more additional therapeutic active ingredients, as well as methods of treatment comprising administering such combination to a subject in need.

[0103] The anti-EGFRvIII antibody-tecilin conjugates useful herein may be co-formulated with and / or administered in combination with one or more additional therapeutic active ingredients 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], or small molecule inhibitors of EGFR [e.g., gefitinib or erlotinib]), antagonists of other EGFR family members such as Her2 / ErbB2, ErbB3 or ErbB4 (e.g., anti-ErbB2 [e.g., trastuzumab or T-DM1 {KADCYLA®}]), anti-ErbB3 or anti-ErbB4 antibodies, or Small molecule inhibitors of ErbB2, ErbB3, or ErbB4 activity), cMET antagonists (e.g., anti-cMET antibodies), IGF1R antagonists (e.g., anti-IGF1R antibodies), B-raf inhibitors (e.g., vemurafenib, sorafenib, GDC-0879, PLX-4720), PDGFR-α inhibitors (e.g., anti-PDGFR-α antibodies), PDGFR-β inhibitors (e.g., anti-PDGFR-β antibodies or small molecule kinase inhibitors, e.g., imatinib mesylate or sunitinib malate), PDGF ligand inhibitors (e.g., anti-PDGF-A, -B, -C, or -D antibodies, aptamers, siRNA, etc.), VEGF antagonists (e.g., VEGF-Trap such as aflibercept, e.g., US7,087,See 411 (also referred to herein as “VEGF inhibitory fusion proteins”), anti-VEGF antibodies (e.g., bevacizumab), small molecule kinase inhibitors of the VEGF receptor (e.g., sunitinib, sorafenib, or pazopanib), DLL4 antagonists (e.g., anti-DLL4 antibodies disclosed in US2009 / 0142354 such as REGN421), Ang2 antagonists (e.g., anti-Ang2 antibodies disclosed in US2011 / 0027286 such as H1H685P), FOLH1 antagonists (e.g., anti-FOLH1 antibodies), STEAP1 or STEAP2 antagonists (e.g., anti-STEAP1 antibodies or anti-STEAP2 antibodies), TMPRSS2 antagonists (e.g. Examples include anti-TMPRSS2 antibodies, MSLN antagonists (e.g., anti-MSLN antibodies), CA9 antagonists (e.g., anti-CA9 antibodies), uroplakin antagonists (e.g., anti-uroplakin [e.g., anti-UPK3A] antibodies), MUC16 antagonists (e.g., anti-MUC16 antibodies), Tn antigen antagonists (e.g., anti-Tn antibodies), CLEC12A antagonists (e.g., anti-CLEC12A antibodies), TNFRSF17 antagonists (e.g., anti-TNFRSF17 antibodies), LGR5 antagonists (e.g., anti-LGR5 antibodies), monovalent CD20 antagonists (e.g., monovalent anti-CD20 antibodies such as rituximab), PD-1 antibodies, PD-L1 antibodies, CD3 antibodies, CTLA-4 antibodies, etc. Other agents that may be beneficially administered in combination with the anti-EGFRvIII antibody-tecilin conjugate of this disclosure include, for example, tamoxifen, aromatase inhibitors, and cytokine inhibitors, such as 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, and IL-18, or their respective receptors.

[0104] This disclosure includes compositions and therapeutic formulations comprising any of the anti-EGFRvIII antibodies described herein in combination with one or more chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents, e.g., thiotepa and cyclophosphamide (Cytoxan®); alkyl sulfonates, e.g., busulfan, improsulfan and pigosulfan; aziridines, e.g., benzodopa, carbocone, meturedopa and uredopa; altoretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomellamine (trimethylolomellamine). Ethyleneimines and methylamelamines (including melamine); Nitrogen mustards, e.g., chlorambucil, chlornafadin, chlorophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, novembichin, fenesterine, prednimustine, trophosphamide, uracil mustard; Nitrosoureas, e.g., carmustine, chlorozotosine, fotemustine, lomustine, nimustine, ranimustine; Anti Biomolecules, for example, acrasinomycin, actinomycin, autramycin, azaserin, bleomycin, kactinomycin, carabicin, carminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, mykov Enolates, nogaramycin, olibomycin, peplomycin, potfiromycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zolubicin; antimetabolites, e.g., methotrexate and 5-fluorouracil (5-FU); folate analogs, e.g., denopterin, methotrexate, pteropterin, trimethrexate;Purine analogs, e.g., fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, phloxuridine; androgens, e.g., carsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; anti-adrenal agents, e.g., aminoglutethimide, mitotane, trilostane; folic acid replenishers, e.g., folinic acid; acegraton; aldophosphamide glycoside glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisanthren; edatraxate; defofamine; demecoltin; diaziquan; elfornithine; eriptinium acetate; etoglucide; gallium nitrate; hydroxyurea; lentinan; ronidamin; mitogluazone; mitoxantrone; mopidamole; nitracrine; pentostatin; fenamet; pirarubicin; pod Phyric acid; 2-ethylhydrazide; procarbazine; PSK (trademark); razoxane; schizophyllan; spirogermanium; tenuazonic acid; triadiquan; 2,2',2”-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitractol; pipobromane; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, e.g., paclitaxel (Taxol (trademark), Bristol-Myers Squibb Oncology (Princeton, NJ) and docetaxel (Taxotere®, Aventis Antony France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, e.g., cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone;Examples include teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicin; capecitabine; and any pharmaceutically acceptable salts, acids, or derivatives of any of the above. This definition also includes antihormone agents that act to control or inhibit the action of hormones on tumors, such as antiestrogens including tamoxifen, raloxifene, aromatase inhibitor 4(5)-imidazole, 4-hydroxytamoxifen, trioxyfen, keoxifene, LY117018, onapristone, and toremifene (Fareston); antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and any pharmaceutically acceptable salts, acids, or derivatives of any of the above.

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

[0106] Additional therapeutic active ingredients, such as any of the drugs listed above or their derivatives, may be administered immediately before, simultaneously with, or immediately after the administration of the anti-EGFRvIII antibody-tecilin conjugate of the Disclosure (for the purposes of the Disclosure, such an administration regimen is considered to be an administration of the anti-EGFRvIII antibody-tecilin conjugate "in combination with" the additional therapeutic active ingredient). The Disclosure includes pharmaceutical compositions in which the anti-EGFRvIII antibody-tecilin conjugate of the Disclosure is co-formulated with one or more of the additional therapeutic active ingredients described elsewhere in this Spec.

[0107] Administration regimen According to certain embodiments of the Disclosure, multiple doses of an anti-EGFRvIII antibody-tecillin conjugate (or a pharmaceutical composition comprising any combination of the anti-EGFRvIII antibody-tecillin conjugate and any additional therapeutic activators referred to herein) may be administered to a subject over a specified period of time. Methods according to these embodiments of the Disclosure include sequential administration of multiple doses of the anti-EGFRvIII antibody-tecillin conjugate of the Disclosure to a subject. As used herein, “sequential administration” means that each dose of the anti-EGFRvIII antibody is administered to the subject at different times, for example, on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The Disclosure includes methods that include sequentially administering to a patient a single initial dose of the anti-EGFRvIII antibody-tecillin conjugate, followed by one or more secondary doses of the anti-EGFRvIII antibody-tecillin conjugate, and optionally followed by one or more tertiary doses of the anti-EGFRvIII antibody-tecillin conjugate.

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

[0109] In the specific exemplary applications of this disclosure, each secondary and / or tertiary dose is administered 1 to 26 weeks after the immediately preceding dose (e.g., 1 week, 1.5 weeks, 2 weeks, 2.5 weeks, 3 weeks, 3.5 weeks, 4 weeks, 4.5 weeks, 5 weeks, 5.5 weeks, 6 weeks, 6.5 weeks, 7 weeks, 7.5 weeks, 8 weeks, 8.5 weeks, 9 weeks, 9.5 weeks, 10 weeks, 10.5 weeks, 11 weeks, 11.5 weeks, 12 weeks, 12.5 weeks, 13 weeks It is administered at intervals of 13.5 weeks, 14 weeks, 14.5 weeks, 15 weeks, 15.5 weeks, 16 weeks, 16.5 weeks, 17 weeks, 17.5 weeks, 18 weeks, 18.5 weeks, 19 weeks, 19.5 weeks, 20 weeks, 20.5 weeks, 21 weeks, 21.5 weeks, 22 weeks, 22.5 weeks, 23 weeks, 23.5 weeks, 24 weeks, 24.5 weeks, 25 weeks, 25.5 weeks, 26 weeks, 26.5 weeks, or later. As used herein, the phrase "immediately preceding dose" means the dose of anti-EGFRvIII antibody-tecilin conjugate administered to the patient immediately before the next dose in a sequence of multiple doses, without any intervening doses.

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

[0111] In embodiments including multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1 to 2 weeks or 1 to 2 months after the previous dose. Similarly, in embodiments including multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2 to 12 weeks after the previous dose. In certain embodiments of this disclosure, the frequency at which secondary and / or tertiary doses are administered to the patient may vary throughout the course of the treatment regimen. The frequency of administration may be adjusted by the physician during the course of treatment in accordance with the individual patient's needs after clinical examinations.

[0112] This disclosure includes a dosing regimen in which 2 to 6 loading doses are administered to a patient at a first frequency (e.g., once a week, once every two weeks, once every three weeks, once a month, once every two months, etc.), followed by two or more maintenance doses administered to the patient at a less frequent basis. For example, according to this embodiment of the disclosure, if the loading dose is administered at a frequency of once a month, the maintenance dose may be administered to the patient at a frequency of once every six weeks, once every two months, once every three months, etc. [Examples]

[0113] The following examples are provided to give a complete disclosure and explanation of how the methods and compositions of this disclosure are prepared and used, and are not intended to limit the scope of what the inventors consider to be their invention. Although efforts have been made to ensure accuracy to the figures used, some experimental errors and deviations should be taken into account. Unless otherwise indicated, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are atmospheric pressure or near atmospheric pressure.

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

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

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

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

[0118] (Table 1) Sequence identifiers of the variable region amino acid sequences of REGN1076 and REGN3124 TIFF0007923780000005.tif17133

[0119] (Table 2) Sequence identifiers of the complete heavy chain and light chain amino acid sequences of REGN1076 and REGN3124 TIFF0007923780000006.tif22147

[0120] (Table 3) Sequence identifiers of variable region nucleic acid sequences of REGN1076 and REGN3124 TIFF0007923780000007.tif17133

[0121] As will be understood by those skilled in the art, an antibody having a particular Fc isotype can be converted to an antibody having a different Fc isotype (for example, an antibody having mouse IgG1 Fc can be converted to an antibody having human IgG4), but in any case the variable domain (including the CDR) indicated by the numerical identifier shown in Table 1 remains the same, and the binding properties are expected to be identical or substantially similar regardless of the nature of the Fc domain.

[0122] The antibody was found to rapidly intercalate into EGFRvIII-positive tumor cells. Certain additional biological properties of exemplary anti-EGFRvIII antibodies produced according to the method of this embodiment are described in detail in the examples below.

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

[0124] Controls 1932 and 3892 are isotype control antibodies. Control 1932 does not have an Fc modification, while control 3892 has an N297Q modification.

[0125] Example 2. Conjugation and Characterization Analysis of Tecilin-Antibody Antibodies REGN1076 and REGN3124, along with 10 mg / mL each of isotype control antibodies, control 1932 (no Fc modification) and control 3892 (with N297Q modification), were treated with 1 mM dithiothreitol at 37°C for 30 minutes. After gel filtration (G-25, pH 4.5 sodium acetate), maleimide linker payload tesirin (also known as SG3249, synthesized as disclosed in Tiberghein et al., 2016, ACS Medicinal Chemistry Letters 7(11):983-987) (1.2 equivalents / SH group) in DMSO (10 mg / mL) was added to the reductive antibody, and the mixture was adjusted to pH 7.0 with 1 M HEPES (pH 7.4). The conjugate was purified by size exclusion chromatography and filtered sterile. Protein concentrations were determined by UV spectroscopy, and payload-to-antibody ratios were determined by mass spectrometry. Size exclusion HPLC confirmed that all conjugates used were monomers exceeding 95%, and LC-MS confirmed that less than 0.5% of the linker payload was non-conjugate. The payload-to-antibody ratios are shown in Table 4.

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

[0127] For this assay, 50 μg of conjugate was diluted with Milli-Q water to a final concentration of 1 mg / mL. 10 μL of PNGase F solution [PNGase F solution was prepared by adding 150 μL of PNGase F stock (New England Biolabs, catalog number P0704L) and 850 μL of Milli-Q water and mixing well] was added to the diluted conjugate solution and incubated overnight at 37°C. 2.4 μL of 0.5 M TCEP was added to the sample so that the resulting substance had a final TCEP concentration of 20 mM, and this was incubated at 50°C for 30 minutes. 10 μL of each sample was injected onto an LC-MS (Waters Synat G2-Si) and eluted over 25 minutes with a gradient mobile phase of 0.1 mL / min (20-40%) (mobile phase A: 0.1% v / v FA in H2O, mobile phase B: 0.1% v / v FA in acetonitrile). LC separation was performed using a Waters Acquity BEH C18 column (1.0 × 50 mM, 1.7 μM).

[0128] The mass spectrometry spectra were deconvoluted. The identified light and heavy chain peaks represent the light chain (L) with linker-payload values ​​of 0 and 1, and the heavy chain (H) with linker-payload values ​​of 0, 1, 2, and 3. From the various intensity values, the drug-to-antibody ratio (DAR) was calculated for the homodimeric antibody conjugates using Equation 1 below. The DARs for each conjugate are provided in Table 4. Equation 1: TIFF0007923780000008.tif17128

[0129] (Table 4) Yield and payload vs. antibody ratio TIFF0007923780000009.tif29128

[0130] Example 3. Biacore binding rate of EGFRvIII monoclonal antibody Equilibrium dissociation constant (K) for EGFRvIII binding of anti-EGFRvIII antibodies to PDB conjugates DThe values ​​were determined using a real-time surface plasmon resonance biosensor assay on a Biacore 2000 or 3000 instrument. The Biacore sensor surface was derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody (GE Healthcare, #BR-1008-39) to capture the anti-EGFRvIII antibody drug conjugate and parental unmodified antibody expressed together with the human constant region. Biacore binding studies were performed in 0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, and 0.05% v / v Surfactant P20 (HBS-EP running buffer). Human EGFRvIII extracellular domains (hEGFRvIII-MMH, SEQ ID NO: 29 (ranging from 600 nM to 22.2 nM)) expressed at different concentrations (3-fold dilution) with a C-terminal myc-myc-hexahistidine tag prepared in HBS-EP running buffer were injected at a flow rate of 50 μL / min onto anti-EGFRvIII antibody drug conjugates or antibody capture surfaces. The association of hEGFRvIII-MMH to each of the captured antibody drug conjugates and monoclonal antibodies was monitored for 4 minutes. Subsequently, hEGFRvIII-MMH dissociation was monitored in HBS-EP running buffer for 6-8 minutes. Anti-human Fc surfaces were regenerated using injection of 20 mM H3PO4. All binding kinetics experiments were performed at 25°C. Kinetic association (k) was determined by fitting real-time sensorgrams to a 1:1 binding model using Scrubber 2.0c curve fitting software. a ) and dissociation (k d The rate constant was determined. All sensorgrams were double-referenced by subtracting the buffer-injected sensorgram signal from the corresponding analyte sensorgram, thereby removing artifacts caused by antibody dissociation from the capture surface. Binding-dissociation equilibrium constant (K D ) and dissociation half-life (t 1 / 2 ) to the following: It was calculated from the velocity constant, as shown in TIFF0007923780000010.tif5128.

[0131] The binding rate parameters of the anti-EGFRvIII antibody drug conjugate and the binding of hEGFRvIII-MMH to the antibody at 25°C are shown in Table 5. As shown, the parental antibody and its corresponding antibody drug conjugate showed similar binding rates to hEGFRvIII-MMH under the tested conditions. D The value was shown.

[0132] (Table 5) Biacore rate of human EGFRvIII-MMH binding to anti-EGFRvIII conjugate and unmodified parental antibody TIFF0007923780000011.tif48137

[0133] Example 4. Cytotoxic activity of anti-EGFRvIII antibody-Tesirin ADC To determine the relative cytotoxicity of the anti-EGFRvIII antibody drug conjugate of the present invention, a cytotoxicity assay was performed on a cell line expressing human EGFRvIII. To develop the cell line, U251 cells (Sigma, #9063001) expressing human EGFRvIII (hEGFRvIII; amino acids 1-380 of accession number NP_005219.2, i.e., SEQ ID NO: 25), referred herein as U251MG / hEGFRvIII, were generated using Lipofectamine LTX containing Plus Reagent. The U251 cell line was maintained in full growth medium (MEM R salt + 10% FBS + 1% L-glutamine / penicillin / streptomycin + 1% non-essential amino acids + sodium pyruvate).

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

[0135] As summarized in Table 6, the anti-EGFRvIII antibody-drug conjugates REGN3124 tesirine and REGN1076 tesirine, a glycosylated version of REGN3124, reduce cell viability, and the IC 50The values were 33 pM for REGN3124 tesirine and 84 pM for REGN1076 tesirine in U251MG / hEGFRvIII cells. REGN3124 tesirine and REGN1076 tesirine kill parental U251MG cells, with IC 50 values were 2.6 nM for REGN3124 tesirine and 4.9 nM for REGN1076 tesirine. Control 3892-tesirine, a similarly conjugated isotype control antibody, reduces cell viability, with IC 50 values were 3.9 nM in U251MG / hEGFRvIII cells and 1.8 nM in U251MG parental cells. The free payload of tesirine (SG3199) has an IC of 10 pM 50 value to kill U251MG / hEGFRvIII cells, with an IC of 2 pM 50 value to kill U251MG parental cells.

[0136] REGN1076 conjugated to a comparative control MMAF payload (REGN1076-MMAF) was also tested for cytotoxicity. Like the other tested anti-EGFRvIII ADCs, REGN1076-MMAF has an IC of 47 pM 50 value to kill U251MG / hEGFRvIII cells. The anti-EGFRvIII ADC REGN1076-MMAF has an IC of 52 nM in parental U251MG cells 50 value to show weak cytotoxicity. Unconjugated, similarly conjugated isotype control antibody against MMAF (control 1932-MMAF) has an IC exceeding 100 nM 50 , showing weak cytotoxicity in all tested cell lines.

[0137] (Table 6) Cell viability in U251 / hEGFRvIII and the parental cell line TIFF0007923780000012.tif77142

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

[0139] REGN3124-Tesirine and REGN1076-Tesirine have ICs of 43 pM and 82 pM, respectively. 50 The values ​​killed U251MG / hEGFRvIII cells from co-cultures, and the killing was similar to that observed in U251MG / hEGFRvIII monocultures. REGN3124-Tesirin and REGN1076-Tesirin also showed IC25 levels of 28 pM and 59 pM, respectively. 50The values ​​indicate that these ADCs killed U251MG parent cells from co-cultures, suggesting bystander-killing activity. The unbound ADC control 3892-tecilin showed IC50 values ​​of 4.0 nM and 2.4 nM, respectively. 50 The values ​​killed U251MG / hEGFRvIII and U251MG parent cells.

[0140] REGN1076 conjugated in a control MMAF payload (REGN1076-MMAF) was also tested for bystander activity. REGN1076-MMAF showed 15 pM IC50 activity. 50 The values ​​showed potent cytotoxicity against U251MG / hEGFRvIII cells from co-cultures. In contrast to the tesyrin conjugate, REGN1076-MMAF showed a 28 nM IC50. 50 In the co-culture assay, the U251 parent cells showed weak cytotoxicity. Unbound ADCs conjugated to MMAF (control 1932-MMAF) showed IC50 levels of less than 100 nM in the co-culture assay. 50 The values ​​indicated weak cytotoxicity.

[0141] Example 5. Hydrogen / deuterium (H / D) exchange-based epitope mapping of anti-EGFRvIII antibody on human epidermal growth factor receptor variant (hEGFRvIII). Hydrogen-deuterium exchange mass spectrometry (HDX-MS) was performed to determine the amino acid residues of epidermal growth factor receptor variant 3 (hEGFRvIII ECD(L25-A380).mmH (SEQ ID NO: 29), amino acid sequence in Appendix) that interacts with REGN3124. A summary 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.

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

[0143] Labeled solutions were prepared at pD7.0 in PBS buffer in D2O (equivalent to 10 mM phosphate buffer, 140 mM NaCl, and 3 mM KCl, pH 7.4 at 25°C). For deuterium labeling, 10 μL of EGFRvIII (EGFRvIII extracellular domain with myc histidine tag (L25-A380), SEQ ID NO: 29, 66 μM) or EGFRvIII (Ag-Ab complex) premixed with REGN3124 in a 1:0.6 molar ratio was incubated with 90 μL of D2O-labeled solution at 20°C at various time points (e.g., 0 seconds for non-deuterated controls, and 5 minutes, 20 minutes, and 80 minutes for deuterium-labeled samples). The experiment was performed in pairs for each time point. The deuteration reaction was quenched by adding 100 μL of pre-cooled quench buffer (0.5 M TCEP-HCl, 8 M urea, and 1% formic acid) to 100 μL of the sample. The mixed sample was incubated at 20°C for 5 minutes. The quenched sample was then injected into a Waters HDX Manager for online pepsin / protease XIII digestion. The digested peptides were captured on a 1.0 mm × 50 mm C8 column (NovaBioassays) and separated by gradient separation of 10% to 32% B (mobile phase A: 0.5% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile) for 13 minutes. The separated peptides were analyzed by Q Exactive HF mass spectrometry in LC-MS / MS mode or LC-MS mode.

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

[0145] For a given peptide, the centroid mass (intensity-weighted average mass) of the spectrum is first calculated for the non-deuterated (0 sec) control. The mean centroid mass of the non-deuterated control of the antigen and Ag-Ab complex is considered the mass with 0% deuterium incorporation (mass of 0% D). For each deuterated sample, absolute D incorporation is defined as the mass difference between the centroid mass of the deuterated sample and the mass of 0% D. The percentage of deuterium incorporation (%D) is determined by comparing the centroid mass to the masses of 0 and 100% D (the maximum D incorporation mass shift defined as 80% of the mass difference between N-2 deuterium atoms and N-2 hydrogen atoms, where N is equal to the number of non-proline amino acids in the peptide). TIFF0007923780000013.tif34156

[0146] For each peptide, absolute D uptake and %D values ​​were calculated individually for two copies at each HDX time point. For each HDX time point, overlapping absolute D uptake and %D values ​​were averaged for the antigen and Ag-Ab complex. The average %D values ​​at the 5-minute and 20-minute HDX time points are then presented as a single %D value for the antigen or Ag-Ab complex, defined as "antigen %D" or "Ag-Ab %D". The difference between antigen %D and Ag-Ab %D is defined as delta %D (Δ%), representing the overall change in deuterium uptake comparing the antigen and Ag-Ab complex for a given peptide.

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

[0148] (Table 7) hEGFRvIII peptides with significant protection upon formation of the hEGFRvIII-REGN3124 complex compared to hEGFRvIII alone TIFF0007923780000014.tif61151

[0149] EGFRvlll ECD(L25-A380).mmH(the mmH tag is underlined) TIFF0007923780000015.tif42156

[0150] Example 6. Anti-EGFRvIII Antibody-Tesirine ADCs Demonstrate Significant Antitumor Efficacy Against EGFRvIII-Transfected Glioblastoma Multiforme Cell Line Xenografts The antitumor efficacy of REGN1076-tesirine and REGN3124-tesirine ADCs was first evaluated in a glioblastoma cell line xenograft model transfected to express EGFRvIII, due to loss of endogenous target expression after in vitro culture. The first model evaluated was U251 / EGFRvIII, and tumors were 10×10 6 cells mixed 1:1 with Matrigel were established by subcutaneous implantation on the right flank of male SCID mice. Tumors were grown to approximately 130 mm approximately 30 days after implantation, before the initiation of treatment. 3 The efficacy of ADC was also evaluated in U87 / EGFRvIII, and tumors were 3×10 6The cells were implanted by subcutaneous transplantation onto the right flank of male SCID mice. The U87 / EGFvIII tumor was approximately 190 mm before treatment and approximately 25 days after transplantation. 3 The mice were allowed to grow to this stage. Mice were randomized into groups of 7-8 and treated with a single dose of either a test ADC or a control ADC. Tumor growth was monitored for 60-70 days after treatment.

[0151] Experimental results: Initial studies in U251 / EGFRvIII xenograft-bearing mice evaluated the activity of REGN1076-Tesirin and REGN3124-Tesirin anti-EGFRvIII ADCs after single-dose administration designed to deliver 2.5 or 5 ug / kg PBD payloads (Table 8). Growth of xenografts treated with control-Tesirin or control-N297Q-Tesirin ADC was not significantly delayed compared to vehicle-controlled tumors. However, throughout the course of the study, significant delay in tumor growth was observed in tumors treated with 2.5 ug / kg payload doses of REGN1076-Tesirin or REGN3124-Tesirin ADC. Higher ADC doses delivering a 5 ug / kg PBD payload had a greater antitumor effect compared to control treatment. REGN3124-Tesirin ADC resulted in a more sustained antitumor effect compared to REGN1076-Tesirin at equivalent dose levels. Overall, all anti-EGFRvIII treatment groups survived until the completion of the study, approximately 60 days after administration. No weight-related treatments were observed, and all groups showed an increase of approximately 10–15% in body weight throughout the study.

[0152] The activity of REGN1076-Tesirin ADC and REGN3124-Tesirin ADC was also evaluated in a U87 / EGFRvIII tumor xenograft model (Table 9). Here, single doses of REGN1076-Tesirin and REGN3124-Tesirin ADC were compared at doses delivering a 2.5 ug / kg PBD payload. This model showed very rapid growth, and animals treated with vehicle controls were euthanized 10 days after administration because the tumors reached the study endpoint. Control-Tesirin or control-N297Q-Tesirin ADC mediated some degree of delay in tumor growth, but all tumors grew, and animals were euthanized 24 days after administration because the tumors reached the study endpoint. Both REGN1076-Tesirin and REGN3124-Tesirin delivered with a 2.5 ug / kg PBD payload mediated significant and sustained regression of tumor xenografts. All animals treated with anti-EGFRvIII survived until the completion of the study at 70 days post-administration. A single tumor in the REGN1076-Tesirin group showed regrowth towards the end of the study. All tumors treated with REGN3124-Tesirin remained suppressed. No weight-related treatments were observed, and all groups showed an increase of approximately 5% in body weight throughout the course of the study.

[0153] (Table 8) Anti-EGFRvIII-tesillin PBD ADC mediated regression of U251 / EGFRvIII xenografts compared to controls (36 days post-treatment). TIFF0007923780000016.tif81154

[0154] (Table 9) Anti-EGFRvIII-tesillin conjugate mediated regression of U87 / EGFRvIII xenografts compared to controls (10 days post-treatment). TIFF0007923780000017.tif63154

[0155] Example 7. The anti-EGFRvIII antibody-Tesirin ADC showed significant antitumor efficacy against xenografts derived from EGFRvIII-positive glioblastoma multiforme patients, which were orthotopically positioned. To evaluate the efficacy of anti-EGFRvIII ADCs against orthotopic GBM tumors in the brain, xenograft (PDX) tumors derived from intracranial GBM6 (high and homogeneous EGFRvIII expression) or GBM59 (moderate and heterogeneous EGFRvIII expression) patients were subjected to a 3 × 10⁻¹⁴ study. 5 Individual PDX cells were immobilized by injection. Intracranial injections were performed at a depth of 3 mm, 1 mm anterior and 2 mm lateral to the anterior apex. All orthotopic GBM PDX studies were conducted by Translational Drug Development Inc. After immobilizing orthotopic GBM6 PDX for 14 ± 1 day and GBM59 PDX for 25 ± 1 day, mice were randomized into groups of 7-8 and treated with a single dose of either the test ADC or the control ADC. Mice were monitored for signs of peri-morbidity for approximately 90 days and euthanized before reaching a mortal state.

[0156] Experimental results: In an initial study (Study A) using mice carrying orthotopic GBM6 PDX tumors, vehicles-treated mice showed rapidly deteriorating clinical signs, and 7 out of 8 mice were euthanized within 30 days of treatment (Table 10). Isotype-controlled ADCs produced no clinical effect, and mice in this group were rapidly euthanized due to tumor-induced per-morbidity. In contrast to the short median survival of 25 and 26.5 days observed in the control group, treatment with REGN3124-Tesirin (DAR 3.4) at a payload dose of 7 ug / kg resulted in a very significant extension of survival. Since 5 out of 8 mice survived to the final observation point of 94 days after administration, a median survival for the anti-EGFRvIII-Tesirin ADC group could not be obtained.

[0157] A second study (Study B) was initiated in mice carrying orthotopically located GBM6 PDX tumors (Table 11). Again, the mediated isotype control ADC did not extend survival compared to vehicle-treated mice, with a median survival of nearly 20 days in both groups and no surviving mice. REGN3124-Tesirin (DAR 3.4) extended survival at both 3.5 and 7 ug / kg payload dose levels, with higher doses resulting in more mice (5 out of 8) surviving to study completion at 95 days post-treatment. REGN3124-Tesirin (DAR 1.9) was similarly effective against REGN3124-Tesirin (DAR 3.4), with a median survival of 77 days post-treatment and 4 out of 8 surviving to study completion. Rapid deterioration of animal body weight was observed in mice exhibiting tumor-induced peri-morbidity. In contrast, animals treated with REGN3124-Tesirin, which demonstrated long-term survival, showed a relevant 10–15% weight gain throughout the post-treatment observation period.

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

[0159] (Table 10) Anti-EGFRvIII-tesillin ADC significantly extended the survival of mice with intracranial GBM6 GBM PDX (Study A). TIFF0007923780000018.tif45154

[0160] (Table 11) Anti-EGFRvIII-tesillin ADC significantly extended the survival of mice with intracranial GBM6 GBM PDX (Study B). TIFF0007923780000019.tif63154

[0161] (Table 12) Anti-EGFRvIII-tesillin ADC significantly extended the survival of mice with intracranial GBM59 GBM PDX (Study C). TIFF0007923780000020.tif54154

[0162] Example 8. REGN1076-Tesirin and REGN3124-Tesirin ADC demonstrate significant antitumor efficacy against xenografts derived from EGFRvIII-positive glioblastoma multiforme patients. The efficacy of REGN1076-tecilin and REGN3124-tecilin ADCs was further investigated using patient-derived xenografts with endogenous EGFRvIII expression, which is characteristic of glioblastoma multiforme tumor biology. The GBM PDX study was conducted by Translational Drug Development Inc. Subcutaneous tumors of GBM6 or GBM59 PDX were established by transplanting approximately 50 mg of PDX fragments into the flanks of nude mice. Tumor volume was approximately 125 mm² at 16-18 days post-transplant. 3 Upon reaching a certain stage, mice were randomized into groups of 7-8 and treated with a single dose of either a test ADC or a control ADC delivered at a dose equal to either 3.5 or 7 ug / kg of pyrrolobenzodiazepine (PBD) payload. Tumor growth was monitored for 60 days post-treatment.

[0163] Experimental results: In GBM6 PDX tumor-bearing mice treated with the vehicle, rapid tumor growth was observed, and the tumor reached the study endpoint 18 days after treatment. Isotype control-tecilin ADC mediated only a slight delay in tumor growth, and the tumor reached the study endpoint 22 days after treatment. In contrast to vehicle and control-treated tumors, anti-EGFRvIII ADC mediated very significant and sustained tumor regression (Table 13). In the GBM6 model, payload doses of 3.5 ug / kg from REGN1076-tecilin or REGN3124-tecilin generally had comparable antitumor efficacy, with 5 out of 8 and 4 out of 8 animals being tumor-free at the completion of the study at 60 days after treatment. Treatment with REGN1076-tesirin or REGN3124-tesirin delivered with a 7ug / kg PBD payload resulted in greater and more sustained efficacy, with 7 out of 8 and 8 out of 8 animals being tumor-free at the completion of the study 60 days after treatment. No treatment-related weight loss was observed, and animal body weight increased by approximately 15% throughout the course of the study.

[0164] (Table 13) Anti-EGFRvIII-tesirin ADC mediated regression of GBM6 PDX tumors compared to controls (18 days post-treatment). TIFF0007923780000021.tif81154

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

[0166] (Table 14) Anti-EGFRvIII tesirine conjugates mediated regression of GBM59 PDX tumors compared to controls (day 19 post-treatment) TIFF0007923780000022.tif72154

[0167] Example 9. Evaluation of a split dose schedule of REGN3124 tesirine ADC in EGFRvIII-positive GBM59 PDX tumor-bearing mice To evaluate the effects of various dose schedules of REGN3124-tecilin conjugate on antitumor efficacy, a study was conducted using a subcutaneous GBM59 PDX tumor model. This study was also conducted by Translational Drug Development Inc. Subcutaneous GBM59 PDX tumors were established by transplanting approximately 50 mg of PDX fragments into the flanks of nude mice. The tumor volume was approximately 125 mm² on post-transplant day 13. 3 Upon reaching a certain stage, mice were randomized into groups of seven and treated with either the experimental ADC or a control ADC. The isotype-controlled ADC was administered in a single dose equal to a 7 ug / kg PBD payload. Animals in the low-dose group received ADC REGN3124-tecillin conjugate at 1.75 ug / kg per dose on days 0 and 4 post-treatment, resulting in a cumulative PBD dose of 3.5 ug / kg. Further groups received REGN3124-tecillin delivering a cumulative dose of 7 ug / kg. This was divided into individual doses of 3 × 2.33 ug / kg, 2 × 3.5 ug / kg, or 1 × 7 ug / kg, delivered on days 0, 4, and 8 (2.33 ug / kg), days 0 and 4 (3.5 ug / kg), or day 0 (7 ug / kg). Tumor growth was monitored for 60 days post-treatment.

[0168] Experimental results: In this study, the isotype-controlled ADC did not cause any delay in tumor growth compared to the vehicle control, and both groups were euthanized on day 19 post-treatment because the mean tumor volume reached the study endpoint (Table 15). In animals treated with 2 × 1.75 ug / kg of REGN3124-Tesilin, a significant inhibition of tumor volume was observed, and all mice survived until the completion of the study 60 days after treatment. All dose schedules resulting in a cumulative PBD payload dose of 7 ug / kg yielded further and very significant antitumor effects. In mice administered with REGN3124-Tesilin at a PBD dose of 3 × 2.33 ug / kg, 3 out of 7 mice were tumor-free at the completion of the study, and the mean tumor volume was 90 mm². 3The results were less than 5 mm. In the groups receiving REGN3124-Tesillin delivered at PBD doses of 2 × 3.5 ug / kg and 1 × 7 ug / kg, 2 out of 7 mice and 3 out of 7 mice were tumor-free at the completion of the study, and the mean tumor volume in both groups was 5 mm. 3 The weight loss was less than 10%, which demonstrates the significant and sustained efficacy of REGN3124-Tesirin in this study. No treatment-related weight loss was observed, and the animals' body weight increased by approximately 10% throughout the course of the study.

[0169] (Table 15) Anti-EGFRvIII-tesirin conjugate mediated regression of GBM6 PDX tumors compared to controls (18 days post-treatment). TIFF0007923780000023.tif72154

[0170] Example 10. Comparison of anti-EGFRvIII-tesillin ADC with anti-EGFRvIII-meitansinoid DM1 ADC To allow the tumor to take hold, 0.5 × 10 6 Individual MMT-EGFRvIII cells were subcutaneously injected into the flank of female SCID mice. The tumor volume was approximately 140 mm². 3 Upon reaching this stage (day 8), mice were randomized into groups of 7 and treated with either tesirin or a DM1 payload in test ADCs and control ADCs. The drugs were administered 3 days over 17 days. Tumor growth was monitored for 61 days post-transplant.

[0171] Next, the antitumor efficacy of each EGFRvIII ADC was evaluated over time (Figure 1). In mice treated with 1 mg / kg REGN1076-Tesirin ADC, complete tumor eradication was observed throughout the duration of the study. The control-Tesirin ADC mediated a transient antitumor effect, but all tumors eventually showed rapid progression toward the protocol endpoint in tumor volume. In contrast to the significant efficacy observed after administration of REGN1076-Tesirin, REGN1076-DM1 administered at either 1 or 15 mg / kg induced only a moderate delay in tumor growth, and all tumors progressed rapidly toward the protocol endpoint. The control-DM1 ADC had no effect compared to the vehicle control. The anti-EGFRvIII results at the 1 mg / kg dose level indicate greater potency of the anti-EGFRvIII-Tesirin conjugate compared to the anti-EGFRvIII-Meitansinoid DM1 conjugate. Neither treatment induced severe weight loss in this study.

[0172] Example 11. Evaluation of anti-EGFRvIII-tesirin conjugate and COMP-MMAF ADC in an EGFRvIII-positive tumor model. The activity of REGN3124-Tesillin ADC was evaluated simultaneously with the activity of a control ADC, COMP-MMAF (monomethyl auristatin F, an auristatin-based ADC prepared according to the procedure described in Phillips et al., 2016, Mol Cancer Ther. 15(4):661-669; see also Doronina et al., 2006, Bioconjugate Chem. 17:114-124) in U251 / EGFRvIII tumor xenograft models and intracranial orthotopic GBM59 PDX models. For the initial xenograft study, U251 / EGFRvIII tumors were 10 × 10⁶ 6 The cells were mixed with Matrigel in a 1:1 ratio and subcutaneously transplanted onto the right flank of male SCID mice to establish the tumor. The tumor size was approximately 175 mm before treatment and approximately 30 days after transplantation. 3The mice were allowed to grow to this stage. Mice were randomized into groups of eight and treated with either a single dose of the experimental ADC or a control ADC. Tumor growth was monitored for 71 days after treatment.

[0173] To evaluate the efficacy of REGN3124-tecilin and COMP-MMAF ADC against orthotopic GBM tumors in the brain, intracranial GBM59 PDX tumors were treated with 3 × 10⁻¹⁶ treatment. 5 Individual PDX cells were immobilized by injection. Intracranial injections were performed at a depth of 3 mm, 1 mm anterior and 2 mm lateral to the anterior apex. All orthotopic GBM PDX studies were conducted by Translational Drug Development Inc. After immobilization of orthotopic GBM59 PDX for 25 days, mice were randomized into groups of 8 and treated with a single dose of either the test ADC or the control ADC. Mice were monitored for signs of peri-morbidity for 94 days and euthanized before reaching a mortal state.

[0174] Experimental results: Studies in U251 / EGFRvIII xenograft-bearing mice evaluated the activity of REGN3124-Tesirin, designed to deliver a 7 ug / kg PBD payload, as well as the activity of COMP-MMAF ADCs at ADC doses of 1, 2.5, and 5 mg / kg (Table 16). Isotype controls were included for all dose levels in this study, but these control agents showed only moderate antitumor effects. REGN3124-Tesirin demonstrated clear antitumor activity in this study, as a single dose of 0.53 mg / kg ADC (7 ug / kg PBD payload dose) was able to induce sustained regression of the xenograft. Sustained regression was not observed in tumors treated with 1 mg / kg COMP-MMAF. While activity against tumors was observed using COMP-MMAF at 2.5 mg / kg, 5 mg / kg of COMP-MMAF was required to achieve sustained activity similar to that achieved with REGN3124-Tesillin treatment at the completion of the study on day 71 post-administration. All animals in this study showed a 10% increase in body weight over the post-treatment observation period.

[0175] (Table 16) Regression of U251 / EGFRvIII xenografts mediated by anti-EGFRvIII-tesirin conjugate and COMP-MMAF conjugate compared to controls (36 days post-treatment). TIFF0007923780000024.tif73154

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

[0177] (Table 17) Activity of REGN3124-tecillin conjugate and COMP-MMAF conjugate in mice with orthotopically located GBM59 GBM PDX tumors TIFF0007923780000025.tif72154

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

[0179] (Table 18) Sequence identifiers and corresponding nucleic acid and amino acid sequences TIFF0007923780000026.tif43162TIFF0007923780000027.tif239162TIFF00079237800 00028.tif240162TIFF0007923780000029.tif242162TIFF0007923780000030.tif170162

[0180] This disclosure is not limited to the scope of the specific embodiments described herein. In fact, various modifications of this disclosure will be apparent to those skilled in the art from the above description and accompanying drawings, in addition to those described herein. Such modifications are intended to fall within the scope of the appended claims.

Claims

1. An antibody-drug conjugate (ADC) comprising an antibody that specifically binds to EGFRvIII, wherein the antibody is a human immunoglobulin molecule comprising two heavy chains and two light chains interconnected by disulfide bonds, each heavy chain comprising a heavy chain variable region (HCVR), and each light chain comprising a light chain variable region (LCVR), The HCVR includes three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) within the HCVR that contain the amino acid sequence of SEQ ID NO: 2, The LCVR includes three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) within the LCVR containing the amino acid sequence of SEQ ID NO:

10. The antibody is aglycosylated and contains the N297Q mutation. The aforementioned antibody is conjugated with tesirin. The aforementioned antibody-drug conjugate (ADC).

2. Anti-EGFRvIII antibody, (i) Also in the conjugated peptide of SEQ ID NO: 23 (ii) Also in the peptide of Sequence ID No. 24 The ADC according to claim 1, which does not bond.

3. When the antibody is measured by a surface plasmon resonance assay at 37°C, the equilibrium dissociation constant (K) for human EGFRvIII monomers is approximately 500 nM. Ｄ The ADC according to claim 1, which shows ).

4. When the antibody is measured by a surface plasmon resonance assay at 37°C, the equilibrium dissociation constant (K) for the human EGFRvIII dimer is approximately 10 nM or less. Ｄ The ADC according to claim 1, which shows ).

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

6. The aforementioned antibody HCDR1 containing the amino acid sequence of SEQ ID NO: 4, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, and HCDR3 containing the amino acid sequence of SEQ ID NO: 8 HCVR including, LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence of SEQ ID NO: 14, and LCDR3 containing the amino acid sequence of SEQ ID NO: 16 LCVR including The ADC according to claim 1, including the following:

7. The aforementioned antibody HCVR containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 2, LCVR containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 10 The ADC according to claim 1, including the following:

8. The aforementioned antibody HCVR containing an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 2, LCVR containing an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 10 The ADC according to claim 1, including the following:

9. The aforementioned antibody HCVR containing an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 2, LCVR containing an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 10 and The ADC according to claim 1, including the following:

10. The aforementioned antibody HCVR containing the amino acid sequence of SEQ ID NO: 2, LCVR containing the amino acid sequence of Sequence ID No. 10 and The ADC according to claim 1, including the following:

11. The ADC according to claim 1, wherein the antibody is a complete antibody.

12. The ADC according to claim 1, wherein the antibody comprises a heavy chain and a light chain, and each heavy chain comprises the amino acid sequence of SEQ ID NO:

20.

13. The ADC according to claim 1, wherein the antibody comprises a heavy chain and a light chain, and each light chain comprises the amino acid sequence of SEQ ID NO:

22.

14. The ADC according to claim 1, wherein the antibody comprises a heavy chain and a light chain, each heavy chain comprising the amino acid sequence of SEQ ID NO: 20, and each light chain comprising the amino acid sequence of SEQ ID NO:

22.

15. The ADC according to claim 1, wherein the drug-to-antibody ratio (DAR) is approximately 1 to approximately 4.

16. The ADC according to claim 1, wherein the antibody is aglycosylated at N297.

17. The ADC according to claim 1, wherein the antibody contains the N297Q mutation determined by EU index numbering in hIgG1Fc.

18. The ADC according to claim 1, wherein the antibody interacts with at least one residue in the amino acid sequence of SEQ ID NO:

26.

19. The following features: (a) Showing reduced in vivo survival rate in EGFRvIII-expressing cells, (b) Demonstrating in vivo bystander cytotoxicity against non-EGFRvIII-expressing cells co-cultured with EGFRvIII-expressing cells, (c) In mice with intracranial glioblastoma expressing EGFRvIII, prolonged survival was observed. (d) Exhibits antitumor effects in mice with EGFRvIII-expressing tumors in the absence of treatment-related weight loss. (e) In mice with patient-derived glioblastoma multiforme, tumor regression is observed. (f) Compared to a comparative control antibody conjugated to MMAF, it exhibits greater tumor-killing activity at lower doses, and (g) In tumor-bearing mice, it exhibits greater antitumor efficacy than anti-EGFRvIII-maytansinoid ADC. The ADC according to claim 1, having one or more of the following.

20. A complex comprising the ADC according to claim 1, wherein the antibody is bound to EGFRvIII.

21. A container or injection device comprising the ADC described in claim 1.

22. A pharmaceutical composition comprising the ADC described in claim 1, and a pharmaceutically acceptable carrier or diluent.

23. The pharmaceutical composition according to claim 22, further comprising one or more additional therapeutic agents selected from the group consisting of chemotherapeutic agents, anti-inflammatory agents, and analgesics.

24. A composition comprising an ADC according to any one of claims 1 to 19 or a pharmaceutical composition according to claim 22 for use in a method of treating cancer in a subject in need thereof, wherein the subject is suffering from an EGFRvIII-expressing tumor.

25. A composition comprising an ADC according to any one of claims 1 to 19 or a pharmaceutical composition according to claim 22, for use in a method for treating cancer or tumors, reducing tumor growth, and / or inducing tumor regression in a subject where such treatment is necessary.

26. The composition according to claim 24, wherein the cancer or tumor is selected from the group consisting of glioblastoma, ductal carcinoma or intraductal carcinoma, non-small cell lung cancer, ovarian cancer, prostate cancer, and squamous cell carcinoma of the head and neck.

27. The composition according to claim 24, wherein the method further comprises administering one or more additional therapeutic agents selected from the group consisting of chemotherapeutic agents, anti-inflammatory agents, and analgesics.

28. The composition according to claim 24, wherein the method further comprises administering a second ADC comprising an antibody and a cytotoxin, wherein the antibody of the second ADC specifically binds to EGFRvIII and also binds to the conjugation peptide of SEQ ID NO: 23 and / or the peptide of SEQ ID NO:

24.

29. A composition for use in a method for administering the ADC described in any one of claims 1 to 19 to a subject body, wherein the method comprises injecting the ADC into the subject body.

30. The composition according to claim 29, wherein the ADC is injected subcutaneously, intravenously, or intramuscularly into the body of the subject.

31. A method for producing an ADC according to any one of claims 1 to 19, comprising culturing a host cell containing an immunoglobulin comprising the HCVR of the ADC and a polynucleotide encoding the immunoglobulin comprising the LCVR of the ADC in a culture medium under conditions favorable for the expression of the polynucleotide.

32. The method according to claim 31, further comprising conjugating tesirin to one or more of the immunoglobulins.

33. The method according to claim 32, wherein the conjugation is carried out by reducing the immunoglobulin chain in the presence of a reducing agent and incubating the reduced immunoglobulin chain with tesirin.

34. The method according to claim 33, wherein the reducing agent is dithiothreitol.

35. The method according to claim 31, further comprising recovering the immunoglobulin from the culture medium.

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