Epidermal growth factor receptor variant III antibodies

Abstract

Provided herein are anti-EGFRvIII antibodies and binding fragments thereof. The anti-EGFRvIII antibodies of the present disclosure are useful for the treatment of cancer by, for example, antibody dependent cytotoxicity (ADCC). Also provided herein are methods of making and using anti-EGFRvIII antibodies for the treatment of cancer, as well as polynucleotides encoding the antibodies.

Description

[0001] Cross - reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 415,051, filed on October 11, 2022, and U.S. Provisional Application No. 63 / 515,366, filed on July 25, 2023, the entire contents of which are incorporated herein by reference.

[0003] Technical field of the invention

[0004] This document relates to materials and methods for treating cancer, and particularly to the use of anti - EGFRvIII antibodies to reduce or eliminate cells expressing truncated EGFRvIII and for treating cancer.

[0005] Incorporation by reference of materials submitted on compact disc

[0006] This application includes a sequence listing, which has been electronically submitted in.XML format and is hereby incorporated by reference in its entirety. The.XML copy was created on October 10, 2023, named "IBIO2033WO.xml" and is 166302 bytes in size. The sequences contained in this.XML file are part of the specification and are hereby incorporated by reference in their entirety into this text. Background of the invention

[0008] Without limiting the scope of the present invention, its background is described in the context of cancers expressing anti - EGFRvIII.

[0009] Epidermal growth factor receptor (EGFR1 / ErbB1 / HER1) is a member of the tyrosine receptor family and is activated by the ligand EGF. Overexpression of EGFR1 is common in many cancer types. EGFR1 is found to be mutated in certain tumors, and the most common mutation is EGFR variant III (EGFRvIII). EGFRvIII has a unique in - frame deletion of 267 aa from exons 2 to 7 in the ECD of EGFR, resulting in the inability to bind the EGF ligand. Expression of EGFRvIII leads to resistance to conventional EGFR1 - targeted therapies.

[0010] Although the frequency of EGFRvIII expression in tumors varies according to tumor type, EGFRvIII expression is specific only to tumor cells. Thus, EGFRvIII is an ideal therapeutic target due to its specific expression in tumor cells. EGFRvIII is present in 30 - 40% of glioblastomas, 8 - 42% of head and neck squamous cell carcinomas, 3 - 16% of non - small cell lung cancer - squamous cell carcinomas (NSCLC - SCC), up to 6.5% of prostate cancers, up to 27% of breast cancers, and up to 8% of colon cancers.

[0011] What is needed are novel antibodies that specifically bind to EGFRvIII but not to wild-type EGFR. Also needed are novel antibodies that enhance antibody-dependent cellular cytotoxicity. SUMMARY OF THE INVENTION

[0013] As embodied and broadly described herein, one aspect of the current disclosure relates to anti-epidermal growth factor receptor type III (EGFRvIII) antibodies or antigen-binding domains thereof, wherein the antibody or antigen-binding domain comprises: heavy chain variable domain (VH) complementarity-determining regions (CDRs) 1, VH CDR2, and VH CDR3, each comprising the amino acid sequence of any one of SEQ ID NO: 3, 4, 5; 13, 14, 15; 23, 24, 25; 33, 34, 35; 43, 44, 45; 53, 54, 55; 63, 64, 65; 73, 74, 75; 83, 84, 85; 93, 94, 95; 103, 104, 105; 113, 114, 115; or 123, 124, 125; and light chain variable domain (VL) CDRs 1, VL CDR2, and VL CDR3, each comprising the amino acid sequence of any one of SEQ ID NO: 6, 7, 8; 16, 17, 18; 26, 27, 28; 36, 37, 38; 46, 47, 48; 56, 57, 58; 66, 67, 68; 76, 77, 78; 86, 87, 88; 96, 97, 98; 106, 107, 108; 116, 117, 118; or 126, 127, 128. In one aspect, the antibody comprises a VH that comprises the amino acid sequence of any one of SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, or 121. In another aspect, the antibody comprises a VL that comprises the amino acid sequence of any one of SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, or 122. In another aspect, the antibody is a monoclonal antibody. In another aspect, the antibody is a full-length antibody. In another aspect, the antibody is an antibody fragment. In another aspect, the antibody is fused to the Fc domain of any one of: human IgG1, human IgG2, human IgG3, and human IgG4. In another aspect, the heavy chain of the antibody comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, or 121, or an antibody that comprises the amino acid sequence. In another aspect, the light chain of the antibody comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, or 122.On the other hand, the antibody comprises a heavy chain variable domain and a light chain variable domain, which are respectively: SEQ ID NO: 1 and 2, 11 and 12, 21 and 22, 31 and 32, 41 and 42, 51 and 52, 61 and 62, 71 and 72, 81 and 82, 91 and 92, 101 and 102, 111 and 112, or 121 and 122. On the other hand, the heavy chain of the antibody is encoded by a nucleic acid, and the light chain of the antibody is encoded by a nucleic acid, and the nucleic acid has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 9 and 10, 19 and 20, 29 and 30, 39 and 40, 49 and 50, 59 and 60, 69 and 70, 79 and 80, 89 and 90, 99 and 100, 109 and 110, 119 and 120, or 129 and 130. On the other hand, the antibody or binding domain is non-fucosylated. On the other hand, the antibody or binding domain is produced in bacterial, fungal, mammalian, insect or plant cells. As specifically embodied and broadly described herein, one aspect of the present disclosure relates to a method of treating a disease in a subject in need thereof, which comprises administering to the subject a therapeutically effective amount of the antibody described herein. On the one hand, the disease is cancer. On the other hand, the disease is a cancer selected from glioblastoma, head and neck squamous cell carcinoma, non-small cell lung cancer-squamous cell carcinoma (NSCLC-SCC), prostate cancer, breast cancer and colon cancer, wherein the cancer expresses EGFRvIII. On the other hand, the cancer cells of the cancer are killed by antibody-dependent cell cytotoxicity (ADCC). On the other hand, the subject is a human. On the other hand, the antibody or its binding fragment does not bind to EGFR1.

[0014] As specifically embodied and broadly described herein, one aspect of the present disclosure relates to a polynucleotide, which comprises a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 9 and 10, 19 and 20, 29 and 30, 39 and 40, 49 and 50, 59 and 60, 69 and 70, 79 and 80, 89 and 90, 99 and 100, 109 and 110, 119 and 120, or 129 and 130, respectively. On the other hand, the subject is a human. On the other hand, the antibody or its binding fragment does not bind to EGFR1.

[0015] As embodied and broadly described herein, one aspect of the present disclosure relates to a vector comprising the polynucleotide described herein. As embodied and broadly described herein, one aspect of the present disclosure relates to a host cell comprising the vector described herein. As embodied and broadly described herein, one aspect of the present disclosure relates to a method of making an anti-EGFRvIII antibody, which comprises expressing a nucleic acid encoding the antibody described herein in a cell. In another aspect, the subject is a human. In another aspect, the antibody or its binding fragment does not bind to EGFR1.

[0016] Brief Description of the Drawings

[0017] To more fully understand the features and advantages of the present invention, reference is now made to the detailed description of the invention as well as the drawings, wherein:

[0018] Figures 1A - 1C compare the binding of various antibodies to EGFRvIII and EGFR1 by surface plasmon resonance binding (Figure 1A, SD-127612-afuc, SD-233883-afuc, cetuximab, SD-382591-afuc, hIgG1 negative control, SD-577776-afuc), (Figure 1B, SD-633416-afuc, SD-638526-afuc, SD-649072-afuc, SD-710726-afuc, SD-741396-afuc, SD-757052-afuc) and (Figure 1C, SD-787077-afuc, SD-837152-afuc, SD-844257-afuc).

[0019] Figures 2A - 2D compare the binding of an hIgG1 isotype control antibody, cetuximab, and the chimeric and humanized antibodies of the present invention to human EGFRvIII and EGFR1 by ELISA.

[0020] Figures 3A - 3F compare the binding of the chimeric and humanized anti-EGFRvIII antibodies of the present invention. The listed antibodies specifically bind to human EGFRvIII but do not bind to wild-type human EGFR1. FACS analysis shows that the anti-EGFRvIII antibody specifically binds to F98 rat glioblastoma (Figure 3A), U87MG human glioblastoma (Figure 3C) and FaDu human head and neck cancer cells (Figure 3E) overexpressing human EGFRvIII. No binding was detected to F98 cells overexpressing wild-type human EGFR1 (Figure 3B) or to wild-type U87MG (Figure 3D) and FaDu (Figure 3F) cells. The values plotted are the median fluorescence intensity. EC 50 The value is the average of n = 3 experiments. n / a, inactive.

[0021] Figures 4A to 4F show chimeric and humanized anti-EGFRvIII antibodies of the present invention, demonstrating potent ADCC activity against F98 cells (Figure 4A), U87MG cells (Figure 4C), and FaDu cells (Figure 4E) overexpressing human EGFRvIII, but no potent ADCC activity against F98 cells expressing human EGFR1 (Figure 4B), wild-type U87MG cells (Figure 4D), and FaDu cells (Figure 4F). The ratio of dead cells to total cells was used to determine the percentage of cell lysis. EC 50 Values are mean of n = 1 - 5 experiments. n / a, not active.

[0022] Figures 5A to 5E show: Figure 5A is the study design of the in vivo efficacy of anti-EGFRvIII antibody SD-233883-afuc against FaDu EGFRvIII tumor cells in a nude mouse model. Figures 5B (FaDu-EGFRvIII tumor volume after initial drug treatment) and 5C (% change in FaDu-EGFRvIII tumor volume after initial drug treatment) show that SD-233883-fuc significantly inhibited FaDu-EGFRvIII tumor growth (T test, P < 0.05) throughout the observation time window compared to the hIgG1 negative control. Figure 5D shows that at the end point of the study (day 22), the FaDu-EGFRvIII tumor weight was significantly reduced after treatment with cetuximab or SD-233883-fuc compared to the hIgG1 negative control (T test, P < 0.05). Figure 5E shows the body weight of the mice after initial drug treatment.

[0023] Detailed description

[0024] Although the formation and use of various embodiments of the present invention are discussed in detail below, it should be understood that the present invention provides many applicable inventive concepts that can be embodied in many specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways of forming and using the present invention and do not define the scope of the present invention.

[0025] To facilitate understanding of the present invention, several terms are defined below. The terms defined herein have the meanings commonly understood by those of ordinary skill in the relevant art of the present invention. Terms such as "a", "an", and "the" are not intended to refer only to a singular entity but include the broad classes that can be illustrated by their specific instances. The terms herein are used to describe specific embodiments of the present invention, but their use does not define the present invention unless outlined in the claims.

[0026] It should be understood that, unless explicitly stated otherwise, in any method described or disclosed herein, if it includes more than one act, the order of the acts is not necessarily limited to the order in which the acts of the method are recited, but the present disclosure includes exemplary embodiments in which the order of the acts is so limited.

[0027] The epidermal growth factor receptor (EGFR1 / ErbB1 / HER1) is a member of the tyrosine receptor family and is activated by the ligand epidermal growth factor (EGF). Overexpression of EGFR1 is common in many cancer types. Mutations in EGFR1 have also been found in certain tumors, and the most common mutation is EGFR variant III (EGFRvIII). EGFRvIII has a unique in-frame deletion of 267 amino acids from exons 2 to 7 in the ECD of EGFR, resulting in the inability to bind the EGF ligand. Although the frequency of EGFRvIII expression in tumors varies according to tumor type, EGFRvIII expression is specific only to tumor cells. For those cancers that express EGFRvIII, due to its specific expression in tumor cells, it is an ideal therapeutic target. Importantly, the expression of EGFRvIII leads to resistance to conventional EGFR1-targeted therapies.

[0028] The present invention is a novel antibody that is specific for EGFRvIII and does not bind to native or wild-type EGFR1. Non-fucosylated antibodies result in enhanced ADCC responses and do not bind to wild-type or native EGFR1, which provides better safety. The anti-EGFRvIII-specific ADCC method achieves selective destruction of tumor cells expressing EGFRvIII and minimizes off-target effects.

[0029] As used herein, the term "mesoscale molecule (MEM)" refers to engineered peptides or polypeptides of about 1 kDa to about 10 kDa. The term "MEM-nanoparticle" as used throughout this text includes MEM conjugated to a nanoparticle (such as a ferritin nanoparticle).

[0030] As used herein, a "subject" can be a mammalian or avian subject. Mammalian subjects include humans, non-human primates, rodents (such as rats, mice), lagomorphs (such as rabbits), ungulates (such as cows, sheep, pigs, horses, goats, etc.), and the like. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human primate, such as a cynomolgus monkey. In some embodiments, the subject is a companion animal (such as a cat, dog).

[0031] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent, or patent application is specifically and individually indicated to be incorporated by reference.

[0032] Antibody

[0033] As used herein, the term "antibody" refers to a full antibody or a binding fragment thereof that specifically binds to a target antigen, which in the present invention is EGRFvIII. The binding fragment is produced by recombinant DNA techniques or by enzymatic or chemical cleavage of a full antibody. Binding fragments include Fab, Fab’, F(ab’)2, Fv, and single-chain variable region fragment (scFv) antibodies. An antibody substantially inhibits the adhesion of a receptor to a counter-receptor when an excess of the antibody reduces the number of receptors bound to the counter-receptor by at least about 20%, 40%, 60%, or 80%, more commonly greater than about 85% (as measured in an in vitro competitive binding assay). The term "antibody" is used in the broadest sense and specifically encompasses monoclonal antibodies (including full-length antibodies, or other bivalent, Fc-containing antibodies, such as bivalent scFvFc fusion antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., Fab, Fab’, F(ab’)2, Fv, scFv), provided that they exhibit the desired biological activity. Antibodies (Ab) and immunoglobulins (Ig) are glycoproteins having the same structural characteristics. The present invention includes fully recombinant monoclonal antibodies (and their binding fragments), in other words, where the complementarity determining regions (CDRs) are spliced by genetic methods into a human antibody backbone, which is generally referred to as antibody veneering. Thus, in some aspects, monoclonal antibodies are fully synthetic antibodies. In certain embodiments, monoclonal antibodies (and their binding fragments) can be prepared in bacterial or eukaryotic cells, including mammalian, yeast, and plant cells.

[0034] As used herein, the term "antibody fragment" refers to a portion of a full antibody, generally the antigen-binding region or variable region, and includes Fab, Fab’, F(ab’)2, Fv, and scFv fragments. The antibody fragments or domains of the present disclosure retain the EGFRvIII antigen-binding specificity. Papain digestion of an antibody produces two identical antigen-binding fragments, called Fab fragments, each having one antigen-binding site, and a residual "Fc" fragment, so named because it can be easily crystallized. Pepsin treatment produces an F(ab’)2 fragment, which has two antigen-binding fragments capable of cross-linking antigens and a residual other fragment (called pFc’). As used herein, "functional fragments" related to an antibody refer to Fv, F(ab), and F(ab’)2 fragments.

[0035] As used herein, the term "Fv" fragment is the smallest antibody fragment that contains the complete antigen recognition and binding site. This region consists of a dimer formed by a non-covalent tight binding of a heavy chain variable domain and a light chain variable domain (VH -V L A dimer). In this conformation, the three CDRs of each variable domain interact with each other, and on the surface of the V H- V L dimer, an antigen-binding site is defined. The six CDRs together confer antigen-binding specificity on the antibody. However, even a single variable domain (or half of the Fv containing only three antigen-specific CDRs) has the ability to recognize and bind an antigen, although its affinity is lower than that of the entire binding site.

[0036] The Fab fragment, also known as F(ab), also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment in that several residues are added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH refers herein to Fab' in which the cysteine residue(s) of the constant domain has a free sulfhydryl group. The F(ab') fragment is produced by cleaving the disulfide bond of the hinge cysteine of the F(ab')2 pepsin digestion product. Other chemical conjugations of antibody fragments are known to those of ordinary skill in the art.

[0037] Native antibodies and immunoglobulins are usually heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to the heavy chain by at least one covalent disulfide bond. However, the number of disulfide bonds varies with the heavy chain of different immunoglobulin isotypes. Each heavy chain and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (V H ) at one end, followed by a constant domain. Each light chain has a variable domain (V L ) at one end and a constant domain at the other end. The constant domain of the light chain aligns with the first constant domain of the heavy chain, and the light chain variable domain aligns with the variable domain of the heavy chain. Specific amino acid residues are thought to form an interface between the light chain variable domain and the heavy chain variable domain (Clothia et al., J. Mol. Biol. 186, 651-66, 1985; Novotny and Haber, Proc. Natl. Acad. Sci. USA 82 4592-4596 (1985), the relevant parts of which are incorporated herein by reference).

[0038] As used herein, an "isolated" antibody is one that has been identified and separated and / or recovered from components of the environment in which it was produced. Contaminant components in its production environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and can include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain embodiments, the antibody will be purified, as measured by at least three different methods: 1) to greater than 50% by weight of antibody, such as greater than 75% by weight, or greater than 85% by weight, or greater than 95% by weight, or greater than 99% by weight, as determined by the Lowry method; 2) to a degree sufficient to obtain at least 10 residues of the N-terminal or internal amino acid sequence, such as at least 15 residues of the sequence, using a spinning cup sequencer; or 3) to homogeneity as determined by SDS-PAGE using Coomassie blue staining or preferably silver staining under reducing or non-reducing conditions. Isolated antibodies include in situ antibodies within recombinant cells since at least one component of the antibody's natural environment will not be present. However, typically, isolated antibodies will be prepared by at least one purification step.

[0039] As used herein, the terms "antibody mutant" or "antibody variant" refer to amino acid sequence variants of an antibody in which one or more amino acid residues have been modified. Such mutants will necessarily have less than 100% sequence identity or similarity to the amino acid sequence of the heavy chain variable domain or the light chain variable domain of the antibody, but have at least 75% amino acid sequence identity or similarity, such as at least 80%, or at least 85%, or at least 90%, or at least 95%, 96%, 97%, 98% or 99% amino acid sequence identity or similarity.

[0040] As used herein, in the context of antibody variable domains, the term "variable" refers to the fact that the sequences of certain portions of the variable domains vary widely between antibodies and are used for the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity determining regions (CDRs), also known as hypervariable regions, in both the light chain variable domain and the heavy chain variable domain. There are at least two techniques for defining CDRs: (1) a method based on cross-species sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. 1987)); and (2) a method based on crystallographic studies of antigen-antibody complexes (Chothia et al. (1989), Nature 342: 877), or both, i.e., Chothia plus Kabat. The more conserved portions of the variable domains are called framework (FR). The variable domains of native heavy and light chains each contain four FR regions, which predominantly adopt a β-sheet conformation and are connected by three CDRs, which form loops connecting the β-sheet structures and in some cases form part of the β-sheet. The CDRs in each chain are held tightly together by the FR regions and, together with the CDRs from the other chain, contribute to form the antigen-binding site of the antibody (see Kabat et al.). The constant domains do not directly participate in the binding of the antibody to its cognate antigen but exhibit various effector functions, such as antibody-dependent cell cytotoxicity in which the antibody participates.

[0041] The light chains of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two distinct types based on the amino acid sequence of their constant domains, namely one of those called κ and λ. "Immunoglobulins" can be assigned to different classes based on the amino acid sequence of their heavy chain constant domains. There are at least five (5) major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, several of which can be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3 and IgG4; IgA-1 and IgA-2. The subunit structures and three-dimensional conformations of the different classes of immunoglobulins are well known.

[0042] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. A monoclonal antibody is highly specific, being directed against a single antigenic site. Furthermore, each monoclonal antibody is directed against a single determinant (epitope) on an antigen, as compared to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes). In addition to their specificity, the advantage of monoclonal antibodies is that they are synthesized by hybridoma cultures and are not contaminated with other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring that the antibody be produced by any particular method. For example, monoclonal antibodies used in accordance with the presently disclosed and claimed invention may be prepared by the hybridoma method first described by Kohler and Milstein, Nature 256, 495 (1975), the relevant portions of which are incorporated herein by reference.

[0043] All monoclonal antibodies used in accordance with the presently disclosed and claimed invention will be the result of (1) a purposeful immunization protocol, as described in more detail below; or (2) the result of an immune response that naturally leads to antibody production during a disease or cancer process.

[0044] The use of the monoclonal antibodies of the presently disclosed and claimed invention may call for administering such or similar monoclonal antibodies to a subject, such as a human. However, when monoclonal antibodies are produced in non-human animals, such as rodents or chickens, administering such antibodies to a human patient will generally elicit an immune response, where the immune response is directed against the antibody itself. Such a response limits the duration and effectiveness of such therapies. To overcome such problems, the monoclonal antibodies of the presently disclosed and claimed invention can be "humanized," i.e., the antibody is engineered such that its antigenic portions are removed and thus replaced with analogous portions of human antibodies, while maintaining the antibody's affinity for EGFRvIII. Such engineering may involve only a few amino acids, or may include the entire framework regions of the antibody, leaving only the complementarity determining regions of the intact antibody. Several methods of humanizing antibodies are known in the art and are disclosed in the following patents: U.S. Patent No. 6,180,370, issued January 30, 2001 to Queen et al.; U.S. Patent No. 6,054,927, issued April 25, 2000 to Brickel; U.S. Patent No. 5,869,619, issued February 9, 1999 to Studnicka; U.S. Patent No. 5,861,155, issued January 19, 1999 to Lin; U.S. Patent No. 5,712,120, issued January 27, 1998 to Rodriquez et al.; and U.S. Patent No. 4,816,567, issued March 28, 1989 to Cabilly et al., the relevant portions of which patents are incorporated herein by reference.

[0045] A humanized form of an antibody is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (such as Fab, Fab’, F(ab’)2, Fv, scFv, or other antigen-binding sequences of an antibody), which consists predominantly of sequences of human immunoglobulins and contains a minimal sequence derived from a non-human immunoglobulin. Humanization can be performed according to the methods of Winter and his collaborators (Jones et al., 1986; Riechmann et al., 1988; Verhoeyen et al., 1988), by replacing the corresponding sequences of a human antibody with the CDR or CDR sequences of a non-human (i.e., rodent, chicken) antibody, see, for example, U.S. Patent No. 5,225,539. In some cases, the Fv framework residues of the human immunoglobulin are replaced with the corresponding non-human residues from the donor antibody. A humanized antibody may also contain residues that are neither present in the recipient antibody nor in the CDR or framework sequences that are introduced. Generally, a humanized antibody contains substantially all, at least one, and usually two variable domains, wherein all or substantially all of the CDR regions correspond to the CDR regions of a non-human immunoglobulin, and all or substantially all of the framework regions are framework regions of human immunoglobulin consensus sequences. A humanized antibody preferably also contains at least a portion of the immunoglobulin constant region (Fc), usually at least a portion of the immunoglobulin constant region (Fc) of a human immunoglobulin.

[0046] Fully human antibodies essentially refer to antibody molecules in which both the light chain and the heavy chain, including the entire sequence of the CDR, are derived from human genes. Such antibodies are referred to herein as "human antibodies" or "fully human antibodies". Human monoclonal antibodies can be prepared, for example, by the trioma technology for producing human monoclonal antibodies; human B-cell hybridoma technology (see Kozbor et al., Hybridoma, 2:7 (1983)) and EBV hybridoma technology (Cole et al., PNAS 82:859 (1985)) or as taught herein. Human monoclonal antibodies can be used in the practice of the presently disclosed and claimed inventions and can be produced by using human hybridomas (see Cote et al., PNAS 80:2026 (1983)) or by Epstein-Barr Virus in vitro transformation of human B cells (Cole et al., 1985), the relevant portions of which are incorporated herein by reference.

[0047] In addition, human antibodies can be prepared by introducing human immunoglobulin loci into transgenic animals, such as mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. After challenge, production of human antibodies is observed that is very similar in all respects to that seen in humans, including gene rearrangement, assembly, and antibody repertoire. This method is described in, for example but not limited to, U.S. Patents Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016 and Marks et al., J Biol. Chem. 267:16007, (1992); Lonberg et al., Nature, 368:856 (1994); Morrison, 1994; Fishwild et al., Nature Biotechnol. 14:845(1996); Neuberger, Nat. Biotechnol. 14:826 (1996); and Lonberg and Huszar, Int Rev Immunol. 13:65 (1995), the relevant portions of which are incorporated herein by reference.

[0048] A method for generating antibodies of interest, such as human antibodies, is disclosed in U.S. Patent No. 5,916,771, issued June 29, 1999 to Hori et al., which is incorporated herein by reference. It involves introducing an expression vector containing a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing the heavy and light chains.

[0049] As used herein, the term “treatment” refers to both therapeutic treatment and prophylactic or preventive measures. Persons in need of treatment include those already suffering from a disorder as well as those in need of prevention of a disorder.

[0050] As used herein, the term “disorder” refers to any condition that would benefit from treatment with a polypeptide. This includes chronic and acute disorders or diseases, including those infectious or pathological conditions that render a mammal susceptible to the disorder in question.

[0051] Antibodies or antibody fragments can be produced with engineered sequences or in a glycosylated state to confer a preferred level of activity in antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP), or antibody-dependent complement deposition (ADCD) functions, as measured by bead-based or cell-based assays or in vivo studies in animal models.

[0052] Alternatively or additionally, it may be useful to combine the amino acid modification with one or more other amino acid modifications that alter the complement-dependent cytotoxicity (CDC) function of the complement component Clq-binding and / or IL-23p19-binding molecule. Particularly interesting binding polypeptides may be polypeptides that bind to Clq and exhibit complement-dependent cytotoxicity. Polypeptides having pre-existing Clq-binding activity, optionally further having the ability to mediate CDC, can be modified such that one or both of these activities are enhanced. Amino acid modifications that alter Clq and / or modify its complement-dependent cytotoxicity function are described, for example, in WO / 0042072, which is incorporated herein by reference.

[0053] The Fc region of an antibody can be designed to alter effector functions, for example, by altering Clq-binding and / or FcγR-binding, thereby altering complement-dependent cytotoxicity (CDC) activity and / or antibody-dependent cell-mediated cytotoxicity (ADCC) activity. These "effector functions" are responsible for the activation or attenuation of biological activity (e.g., in a subject). Examples of effector functions include, but are not limited to: Clq-binding; CDC; Fc receptor-binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions may require the Fc region to bind to a binding domain (e.g., the antibody variable domain) and can be evaluated using various assays (e.g., Fc-binding assay, ADCC assay, CDC assay, etc.).

[0054] For example, variant Fc regions of antibodies with improved Clq-binding and improved FcγRIII-binding (e.g., having both improved ADCC activity and improved CDC activity) can be generated. Alternatively, if it is desired to reduce or eliminate effector functions, variant Fc regions with reduced CDC activity and / or reduced ADCC activity can be designed. In other embodiments, only one of these activities can be enhanced, and optionally, the other activity can also be reduced (e.g., generating a variant Fc region with improved ADCC activity but reduced CDC activity, and vice versa).

[0055] A single-chain variable fragment (scFv) is a fusion of the variable regions of the heavy and light chains of an immunoglobulin, linked together by a short (usually serine, glycine) linker. This chimeric molecule retains the specificity of the original immunoglobulin, despite removal of the constant regions and introduction of a linker peptide. This modification generally does not alter specificity. Historically, these molecules were created to facilitate phage display, in which it is convenient to express the antigen-binding domain as a single peptide. Alternatively, scFvs can be created directly from the heavy and light chains derived from subclones of hybridomas or B cells. Single-chain variable fragments lack the constant Fc region present in the intact antibody molecule and thus lack the common binding sites (e.g., protein A / G) used to purify antibodies. These fragments can generally be purified / immobilized using protein L, as protein L interacts with the variable region of the κ light chain.

[0056] Flexible linkers generally consist of amino acid residues that promote helices and turns, such as alanine, serine, and glycine. However, other residues can also function. Phage display can be used to rapidly select tailor-made linkers for single-chain antibodies (scFvs) from a library of protein linkers. A random linker library can be constructed in which the genes for the heavy-chain variable domain and the light-chain variable domain are linked by a fragment encoding a variable 18-amino acid polypeptide. The scFv library (about 5×10 6 different members) is displayed on filamentous phage and affinity selected with a hapten. The selected variant population shows a significant increase in binding activity but retains a substantial sequence diversity. Sequence analysis reveals conserved prolines in the linker, two residues after the VH C-terminus, and substantial arginines and prolines at other positions as the only common feature of the selected tethers. In certain embodiments, as known in the art, antibody fragments are further modified by using a modified Fc region or mutations to various constant regions to increase their serum half-life.

[0057] In certain embodiments, the antibodies of the invention are formulated for administration to a human. For example, the antibodies of the invention can be included in a pharmaceutical composition formulated for administration by: intranasal, intrapulmonary, intratracheal, intravenous, oral, intra-adipose, intra-arterial, intra-articular, intracranial, intradermal, intralesional, intramuscular, intra-pericardial, intra-peritoneal, intra-pleural, intravesical, topical, mucosal, parenteral, enteral, subcutaneous, sublingual, topical, trans-buccal, transdermal, by inhalation, by injection, as a cream, as a lipid composition, by catheter, by lavage, by continuous infusion, by infusion, by local delivery, or by local perfusion, and wherein the composition is a serum, a drop, a gel, an ointment, a spray, a depot, or an aerosol.

[0058] As used herein, the term "antigen" refers to a molecule containing one or more epitopes (linear, conformational, or both) that stimulates the host immune system to produce a humoral and / or cell antigen-specific response. The antigen of the present invention is EGFRvIII, which also includes the MEM of EGFRvIII. This term may be used interchangeably with the term "immunogen". Generally, B cell epitopes include at least about 5 amino acids, but can be as small as 3 - 4 amino acids. T cell epitopes, such as CTL epitopes, include at least about 7 - 9 amino acids, and helper T cell epitopes include at least about 12 - 20 amino acids. Generally, epitopes include about 7 to 15 amino acids, such as 9, 10, 12, or 15 amino acids. This term includes polypeptides that include modifications compared to the native sequence, such as deletions, additions, and substitutions (usually conservative in nature), so long as the protein retains the ability to elicit an immune response as defined herein. These modifications can be intentional, such as by site-directed mutagenesis, or accidental, such as by mutations in the host that produces the antigen.

[0059] As used herein, the term "epitope" refers to a specific amino acid sequence or molecule (such as, carbohydrates, small molecules, lipids, etc.) that, when present in the appropriate conformation, provides a site for antibody reaction (e.g., B cell epitope) or, in the case of a peptide, involves the T cell receptor (e.g., T cell epitope).

[0060] Any number of epitope mapping techniques known in the art can be used to identify the portion of a given polypeptide that includes a B cell epitope (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Volume 66, edited by Glenn E. Morris, 1996, Humana Press, Totowa, N.J.). For example, linear epitopes can be determined, for example, by simultaneously synthesizing a large number of peptides (the peptides corresponding to portions of the protein molecule) on a solid support and reacting the peptides with an antibody while the peptides remain attached to the solid support. Such techniques are known in the art and are described in U.S. Patent No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad Sci. USA 81:3998 - 4002; Geysen et al. (1986) Molec. Immunol. 23:709 - 715.

[0061] As used herein, the term "substantially purified" refers to a separated substance (compound, polynucleotide, protein, polypeptide, polypeptide composition) such that the substance comprises the major part of the sample in which it is present. Typically, in a sample, a substantially purified component comprises 50% of the sample, preferably 80%-85%, more preferably 90-95%. Techniques for purifying polynucleotides and polypeptides of interest are well known in the art and include, for example, ion-exchange chromatography, affinity chromatography, and density-based sedimentation.

[0062] Unless otherwise indicated, the practice of the present invention employs conventional methods of chemistry, biochemistry, molecular biology, immunology, and pharmacology within the skill of the art. Such techniques are well explained in the literature. See, for example, Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack Publishing Company, 1990); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); and Handbook of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell eds., 1986, Blackwell Scientific Publications); Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Short Protocols in Molecular Biology, 4th Edition (Ausubel et al. eds., 1999, John Wiley & Sons); Molecular Biology Techniques: An Intensive Laboratory Course, (Ream et al. eds., 1998, Academic Press); PCR (Introduction to Biotechniques Series), 2nd Edition (Newton & Graham eds., 1997, Springer Verlag); Fundamental Virology, 2nd Edition (Fields & Knipe eds., 1991, Raven Press, New York), the relevant portions of which are incorporated herein by reference.

[0063] Conservative amino acid substitutions involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu, and Ile; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gln, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.

[0064] Example 1. EGFRvIII MEM

[0065] The mesoscale molecules (MEMs) of the present disclosure are made to mimic the agonist epitopes identified in Table 1 and are subsequently used to screen for antibodies. The advantage of this method is the ability to shift antibody discovery from the wild-type receptor towards the desired epitopes found only in EGFRvIII. The backbone amino acids are shown in bold and the epitope residues are shown in Underline display.

[0066] Table 1. MEM Sequences for Immunization

[0067]

[0068] In some embodiments, MEM nanoparticles are used to immunize a subject to generate antibodies specific for the MEM epitope. Monoclonal hybridomas are then created to produce epitope-specific anti-EGFRvIII antibodies. Humanized anti-EGFRvIII CDRs are detected based on reference antibodies.

[0069] Example 2. Anti-EGFRvIII Antibody Discovery Based on Immunization with Engineered MEM Nanoparticles

[0070] MEMs are designed based on the epitopes confirmed by SEQ ID NO: 47, 48, and 49 and are then conjugated to nanoparticles to direct the production of B cell antibodies against the epitopes. MEM conjugated to ferritin nanoparticles was identified by Coomassie blue-based western blotting and was found to contain approximately 20 - 30 MEMs per nanoparticle. Using surface plasmon resonance (SPR), the MEM nanoparticles showed nanomolar binding affinity for anti-EGFRvIII. Then, BALB / c mice were immunized with alternating doses of engineered MEM nanoparticles and / or full-length anti-EGFRvIII suspended in adjuvant over a 5-week period, with the final boost consisting of a combination of both. Mouse sera were collected and produced strong anti-EGFRvIII binding as measured by ELISA.

[0071] Antibodies produced by top monoclonal hybridomas showed strong anti-EGFRvIII binding and agonistic effects. Hybridomas were created from immunized murine B cells using standard electrocell fusion methods. The resulting antibodies, along with several others, were produced by monoclonal hybridomas and demonstrated strong anti-EGFRvIII binding via ELISA. Binding was further evaluated in vitro using SPR, namely anti-EGFRvIII binding and competitive binding to anti-EGFRvIII.

[0072] Antibody expression and purification. Antibody expression plasmids were transiently transfected into animal cell lines using the ExpiFectamine CHO transfection kit (Thermo Fisher, catalog number A29129) to obtain transfectants that produced anti-CCR8 chimeric or humanized antibodies. For the host cell lines, ExpiCHO-S (Thermo Fisher, catalog number A29127) or a suspension CHO cell line with the α1,6-fucosyltransferase (FUT8) gene knocked out (referred to as "WT CHO" and "FUT8CHO" in other references) was used. After 6 - 12 days of growth following DNA transfection, the cell suspensions of WT CHO or FUT8 CHO were harvested by centrifugation at 4000×g for 20 minutes and then filtered using a 0.2 µm disposable PES filter unit (Fisher Scientific, catalog number FB12566504). Antibodies were recovered from the filtrate using protein A purification (HiTrap MabSelect SuRe; Cytiva, catalog number GE11-0034-93). WT CHO was used to express antibodies with standard glycosylation, and FUT8 CHO was used to express afucosylated antibodies with enhanced effector function (denoted as "-afuc").

[0073] Antibody humanization. Humanization was achieved by multiple methods. In some cases, framework amino acids that differed between a rationally selected chimera (SD-233883) and the closest human germline were converted to match the human sequence. In other cases, the CDRs were directly transplanted onto the human germline using publicly available tools (DOI: 10.1080 / 19420862.2021.2020203). In all cases, the CDRs remained unchanged. Then, the humanized variants were tested and their potencies compared to the parental chimera.

[0074] Preparation of phage display library. A CDR variant library was prepared based on the parental antibody. The VH and VL sequences were assembled using Golden Gate assembly and ligated to the digested phagemid vector for phage display as ScFv. The ligation was transformed into Phage-Competent™ TG1 cells (Antibody design labs, catalog number PC001), and the library quality was determined by size and VH / VL insert percentage.

[0075] Phage display screening. Synthetic library phage display selection was performed using soluble protein antigen. Selection was performed using Dynabeads M-280 streptavidin beads (Invitrogen; catalog number 11205D) on a KingFisher Apex with biotinylated huEGFRvIII. A series of antigen concentrations were used. Elution was performed with TEA (triethylamine) (Sigma; catalog number T0886), and the selection buffer was skim milk in 1×PBS. After three rounds of panning, plasmids were extracted, VH / VL genes were amplified, and analyzed by Sanger sequencing. The VH / VL sequences of interest were cloned into an IgG1 expression plasmid and then transformed into DH5α. Each plasmid was extracted from DH5α and then transfected into CHO cells in a 24-well deep plate using the ExpiFectamine CHO transfection kit for expression. Purification was performed using a KingFisher Apex. Then, phage-matured clones were screened using SPR.

[0076] Example 3. Kinetic analysis of anti-EGFRvIII antibodies by surface plasmon resonance

[0077] Kinetic analysis of anti-EGFRvIII antibodies by surface plasmon resonance. Binding of antibodies to human EGFR and EGFRvIII was evaluated by surface plasmon resonance (SPR) using a Carterra LSA (Carterra, Inc.). First, an anti-human IgG capture layer was prepared by primary amine coupling on an HC30M chip (Carterra, catalog number 4279). Briefly, the chip surface was activated for 10 minutes with a mixture of 133 mM EDC (Thermo Fisher, catalog number 22980) and 33.3 mM sulfo-NHS (Thermo Fisher, catalog number 24525) in 100 mM MES pH 5.5 (Carterra, catalog number 3625), after which goat anti-human IgG (Southern Biotech, catalog number 2040-01) was coupled at 50 µg / mL for 15 minutes in 10 mM sodium acetate buffer pH 4.5 (Carterra, catalog number 3622). Unconjugated sites on the chip surface were blocked with 1 M ethanolamine HCl pH 8.5 (Carterra, catalog number 3626) for 7 minutes. For capture kinetics, the prepared anti-human IgG surface and a 96-channel printhead (96PH) were used to capture the antibody set at 1-10 µg / mL for 10 minutes in HBSTE buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20; Carterra, catalog number 3630). Then, using a single flow cell (SFC), purified recombinant antigens (human EGFR, Acro Biosystems catalog number EGR-H5222; human EGFRvIII, catalog number EGI-H52H4) were injected over the antibody set at concentrations starting from 500 nM in a 5-fold dilution series of 5. Each injection employed a 5-minute binding phase and a 15-minute dissociation phase. The surface was regenerated between antigens with 0.425% H3PO4 (Carterra, catalog number 3637). The running buffer for antigen injection was HBSTE supplemented with 0.5 mg / mL BSA (VWR, catalog number 97061-422). The binding data were double-reference processed by subtracting the inter-point reference response and the blank response of buffer only. Using Carterra Kinetics software, the resulting sensorgrams were globally fit to a 1:1 Langmuir binding model to estimate the association rate constant (ka), dissociation rate constant (kd), and dissociation constant (KD).

[0078] Figures 1A to 1C compare the binding of various antibodies to EGFRvIII and EGFR1 by surface plasmon resonance binding (Figure 1A, SD-127612-afuc, SD-233883-afuc, cetuximab, SD-382591-afuc, hIgG1 negative control, SD-577776-afuc), (Figure 1B, SD-633416-afuc, SD-638526-afuc, SD-649072-afuc, SD-710726-afuc, SD-741396-afuc, SD-757052-afuc), and (Figure 1C, SD-787077-afuc, SD-837152-afuc, SD-844257-afuc).

[0079] Table 2. Chimeric and humanized anti-EGFRvIII antibodies specifically bind to human EGFRvIII but not to full-length human EGFR1. Cetuximab recognizes both EGFR1 and EGFRvIII, while the human IgG1 negative control antibody binds to neither. Kinetic parameters were measured by SPR using Carterra LSA and fitting the resulting sensorgrams to a 1:1 Langmuir binding model. NB: Not bound.

[0080]

[0081] Example 4. EGFRvIII and EGFR1 Binding ELISA

[0082] In combination with ELISA. First, ELISA plates (Biolegend, catalog number 423501) were coated with human EGFRvIII (ACRO Biosciences, catalog number EGI-H52H4) or EGFR1 protein (ACRO Biosciences, catalog number EGR-H5222) at 1 µg / mL in 50 mM pH 9.5 carbonate buffer (Teknova, catalog number S9225) and kept overnight at 4°C. The next day, the plates were washed three times with wash buffer [1×PBS containing 0.1% Tween-20 (Teknova, catalog number P0207)], followed by the addition of blocking buffer [1×PBS, 1% BSA (Teknova, catalog number B0101)] and kept for 1 hour at room temperature. After blocking, the plates were washed three times with wash buffer, and then increasing concentrations (0.004 – 66.66 nM) of anti-EGFRvIII or control antibody in wash buffer were added and kept for 1 hour at room temperature. After incubation, the plates were washed three times with wash buffer, and then a goat anti-human IgG-HRP secondary antibody (Biorad, catalog number STAR126P) diluted 1:2500 in wash buffer was added and kept for 1 hour at room temperature. After incubation with the secondary antibody, the plates were washed six times with wash buffer, and then TMB substrate (VWR, catalog number 95059-154) was added and kept for 5 minutes at room temperature. After incubation with the TMB substrate, ELISA stop solution (Thermo Fisher, catalog number SS04) was added to the plates in an equal volume to the TMB substrate, and the absorbance was read at 450 nm. The data was plotted using GraphPad Prism 9.3.0 software, and the EC 50 values were calculated by this software.

[0083] Figures 2A to 2D compared the binding of hIgG1 isotype control antibody, cetuximab, and the chimeric and humanized antibodies of the present invention to human EGFRvIII and EGFR1 by ELISA.

[0084] The chimeric and humanized anti-EGFRvIII antibodies specifically bind to truncated human EGFRvIII but not to full-length human EGFR1. ELISA assays showed that the afucosylated anti-EGFRvIII antibody and cetuximab bind to immobilized human EGFRvIII in a concentration-dependent manner. The values plotted were the absorbance measured at 450 nm wavelength. The EC 50 values were the mean of n = 3 experiments. n / a, inactive.

[0085] Table 3. The chimeric and humanized anti-EGFRvIII antibodies specifically bind to truncated human EGFRvIII but not to full-length human EGFR1.

[0086]

[0087] Example 5. F98 EGFRvIII and EGFR1 Cell Binding Assay

[0088] Culture F98npEGFRvIII cells (ATCC, catalog number CRL-2949) and F98 EGFR1 cells (ATCC, catalog number CRL-2988) in DMEM (Corning, catalog number 10-013-CV) supplemented with 10% FBS (ATCC, catalog number 30-2020), 1× penicillin-streptomycin (Corning, catalog number 30-002-CI), and 0.2 mg / ml G418 (Thermo Fisher, catalog number 10131035). Culture U87MG cells (ATCC, catalog number HTB-14) and U87MG-EGFRvIII cells (Genscript) in DMEM supplemented with 10% FBS, 1× penicillin-streptomycin, and 0.5 μg / mL puromycin (for U87MG-EGFRvIII cells only, Gibco, catalog number A11138-03). Culture FaDu cells (ATCC, catalog number HTB-43) and FaDu-EGFRvIII cells (Genscript) in EMEM (ATCC, catalog number 30-2003) supplemented with 10% FBS, 1× penicillin-streptomycin, and 2 μg / mL puromycin (for FaDu-EGFRvIII cells only).

[0089] For the cell binding assay, PBS (Corning, catalog number 21-040-CV) supplemented with 2% FBS and 2 mM EDTA was used as the assay buffer. Count the target cells, then resuspend them in their respective media, and then at 1×10 5Cells were seeded at 50 cells / well into a 96-well plate (VWR, catalog number 89089-826) and incubated on ice for 3 hours. After incubation, the plate was centrifuged and the supernatant was removed. Then the cells were washed once with detection buffer, centrifuged again, and the wash buffer was removed. After removing the wash buffer, the indicated antibodies were diluted in detection buffer and added to the cells at increasing concentrations (0.004 – 66.66 nM) and kept on ice for 20 minutes. After incubation, the cells were washed with detection buffer and the wash buffer was removed. Next, rat anti-human IgG Fc Alexa Fluor 647 (BioLegend, catalog number 410714) diluted 1:200 in detection buffer was added to the cells and kept on ice for 20 minutes. After incubation, the cells were washed once more with detection buffer and the wash buffer was removed. Next, DAPI (BioLegend, catalog number 422801) diluted 1:5000 in detection buffer was added to the cells. Cell binding was analyzed on a Miltenyi MACSQuant 16 flow cytometer. Flow cytometer data were analyzed using FlowJo flow cytometry analysis software. GraphPad Prism 9.3.0 was used for graph plotting and calculation of EC

[0090] Values. Figures 3A to 3F compare the binding of the chimeric and humanized anti-EGFRvIII antibodies of the present invention. The listed antibodies specifically bind to human EGFRvIII but not to wild-type human EGFR1. FACS analysis shows that the anti-EGFRvIII antibodies specifically bind to F98 rat glioblastoma (Figure 3A), U87MG human glioblastoma (Figure 3C), and FaDu head and neck cancer cells (Figure 3E) overexpressing human EGFRvIII. No binding was detected to cells overexpressing wild-type human EGFR1 - F98 cells (Figure 3B) or to wild-type U87MG (Figure 3D) and FaDu (Figure 3F) cells. The values plotted are the median fluorescence intensity. EC 50 Values are the mean of n = 3 experiments. n / a, inactive.

[0091] Table 4. Chimeric and humanized anti-EGFRvIII antibodies specifically bind to human EGFRvⅢ but not to human EGFR1 overexpressed in the F98 rat glioblastoma cell line

[0092]

[0093] Table 5. Chimeric and humanized anti-EGFRvIII antibodies specifically bind to human EGFRvIII overexpressed in FaDu head and neck cancer cells but not to wild-type FaDu cells.

[0094]

[0095] Table 6. Chimeric and humanized anti-EGFRvIII antibodies specifically bind to human EGFRvIII overexpressed in U87MG human glioblastoma cells, but do not bind to wild-type U87MG cells.

[0096]

[0097] Example 7. In vitro ADCC assay

[0098] F98npEGFRvIII cells (ATCC, catalog number CRL-2949) and F98 EGFR1 cells (ATCC, catalog number CRL-2988) were cultured in DMEM (Corning, catalog number 10-013-CV) supplemented with 10% FBS (ATCC, catalog number 30-2020), 1× penicillin-streptomycin (Corning, catalog number 30-002-CI), and 0.2 mg / ml G418 (Thermo Fisher, catalog number 10131035). U87MG cells (ATCC, catalog number HTB-14) and U87MG-EGFRvIII cells (Genscript) were cultured in DMEM supplemented with 10% FBS, 1× penicillin-streptomycin, and 0.5 µg / mL puromycin (only for U87MG-EGFRvIII cells, Gibco, catalog number A11138-03). FaDu cells (ATCC, catalog number HTB-43) and FaDu-EGFRvIII cells (Genscript) were cultured in EMEM (ATCC, catalog number 30-2003) supplemented with 10% FBS, 1× penicillin-streptomycin, and 2 µg / mL puromycin (only for FaDu-EGFRvIII cells).

[0099] For ADCC assessment, a frozen stock solution of PBMCs purchased from STEMCELL (catalog number 70025) was thawed in RPMI medium containing 10% FBS, 1× penicillin-streptomycin (Corning, catalog number 30-002-CI), 5 ng / ml IL2 (Miltenyi Biotec, catalog number 130-097-743), and incubated overnight in a tissue culture incubator.

[0100] Target cells were counted and cell viability was assessed. First, the cells were stained with CFSE dye (ThermoFisher, catalog number C34554) for 10 minutes at room temperature and then washed once with growth medium. The washed cells were resuspended in growth medium to a density of 1×10 6 cells / mL, and then 1×10 4Cells were seeded onto 96-well plates (Fisher Scientific, catalog number 07-200-89) and incubated overnight in a tissue culture incubator. After incubating the cells, the growth medium in the assay plates was replaced with assay medium consisting of RPMI 1640 medium supplemented with 10% FBS, 1% penicillin-streptomycin, and 5 ng / mL IL2 (Miltenyi Biotec, catalog number 130-097-743). Control or anti-EGFRvIII antibodies in the assay medium were added to the cells at increasing concentrations (0.0004 to 6.66 nM for FaDu and U87 cells, 0.0002 to 3.33 nM for F98 cells) and kept at 37°C, 5% CO2 for 10 minutes. Subsequently, 2×10 5 peripheral blood mononuclear cells (PBMCs) were added to each well of the 96-well plates. The cells and antibodies were incubated in a 37°C, 5% CO2 incubator for 24 hours. Samples were stained with the LIVE / DEAD™ Fixable Aqua Dead Cell Stain Kit (Thermo Fisher, catalog number L34957) and analyzed using a MACSQuant 16 flow cytometer. Gates were set for live cells (aqua-, CFSE+) and dead cells (aqua+, CFSE+). The ratio of dead cells to total target cells was used to determine the percentage of cell lysis. Flow cytometer data were analyzed using FlowJo flow cytometry analysis software. GraphPad Prism 9.3.0 was used for graph plotting and calculation of the EC 50 values.

[0101] Figures 4A to 4F show the chimeric and humanized anti-EGFRvIII antibodies of the present invention, demonstrating potent ADCC activity against F98 cells (Figure 4A), U87MG cells (Figure 4C), and FaDu cells (Figure 4E) overexpressing human EGFRvIII, but no activity against F98 cells expressing human EGFR1 (Figure 4B), wild-type U87MG cells (Figure 4D), and FaDu cells (Figure 4F). The ratio of dead cells to total cells was used to determine the percentage of cell lysis. The EC 50 values are the mean of n = 1 - 5 experiments. n / a, no activity.

[0102] Table 7. Chimeric and humanized anti-EGFRvIII antibodies show potent ADCC activity against F98.

[0103]

[0104] Table 8. Chimeric and humanized anti-EGFRvIII antibodies exhibit potent ADCC activity against overexpressed human EGFRvIII in U87MG human glioblastoma cells, but no activity against wild-type U87MG cells.

[0105]

[0106] Table 9. Chimeric and humanized anti-EGFRvIII antibodies exhibit potent ADCC activity against overexpressed human EGFRvIII in FaDu human head and neck cancer cells, but no activity against wild-type FaDu cells.

[0107]

[0108] In vivo potency assay. Nine-week-old female nude mice (Charles River Laboratories, catalog number 088Nu / Nu) were used in this assay. 2×10 6 FaDu-EGFRvIII cells in a mixture of 100 μl PBS and Matrigel (Corning, catalog number 354234) (v:v = 1:1) were inoculated subcutaneously into the upper left flank of each nude mouse. Tumor growth and mouse body weight were monitored twice a week. For each individual tumor, the longest longitudinal diameter as length and the widest transverse diameter as width were measured using a Traceable digital caliper (VWR, catalog number 62379-531). Then, the tumor volume (TV) was calculated by the formula TV = [length × (width)2] / 2. When the average tumor volume reached 152-153 mm 3 , the mice were randomly grouped with 10 mice in each group. Each of hIgG1 negative control, cetuximab, and SD-233883-afuc was prepared in PBS as a 3 mg / ml stock solution. For each mouse, the volume of the administered antibody was calculated by the formula volume (μl) = mouse body weight (g) × 10 μl / g. The antibody drugs were administered via the intravenous route twice a week for a total of 7 times (biw×7). After the first drug treatment, the percentage change of each tumor was calculated by the formula: % change in TV = [(TV – TV at day 0) / TV at day 0] × 100.

[0109] Figures 5A to 5E show: Figure 5A is a study design of the in vivo efficacy of anti-EGFRvIII antibody SD-233883-afuc against FaDu EGFRvIII tumor cells in a nude mouse model. Figure 5B (FaDu-EGFRvIII tumor volume after initial drug treatment) and Figure 5C (% change in FaDu-EGFRvIII tumor volume after initial drug treatment) show that compared with the hIgG1 negative control, SD-233883-fuc significantly inhibited the growth of FaDu-EGFRvIII tumors throughout the observation time window (T-test, P<0.05). Figure 5D shows that at the end point of the study (day 22), compared with the hIgG1 negative control, after treatment with cetuximab or SD-233883-fuc, the weight of FaDu-EGFRvIII tumors was significantly reduced (T-test, P<0.05). Figure 5E shows the body weight of the mice after initial drug treatment.

[0110] The present disclosure provides antibodies that bind to truncated EGFRvIII. These antibodies are referred to herein as anti-EGFRvIII antibodies. A variety of discovery strategies have been employed to obtain the exemplary antibodies of the present disclosure, which are further discussed below.

[0111] Those skilled in the art will recognize that an antibody that exhibits little or no binding to a target antigen can be described as having low affinity for the target antigen and a high equilibrium dissociation constant (K D ). Those skilled in the art will also recognize that an antibody that exhibits little or no binding to an epitope ensemble of a target antigen can be described as having low avidity for the epitope ensemble of the target antigen and a high equilibrium dissociation constant (K D ).

[0112] In some embodiments, the present disclosure provides anti-EGFRvIII antibodies that have a binding affinity (K D ) for EGFRvIII of from about 5 μM to about 5 pM, from about 1 μM to about 5 pM, from about 0.5 μM to about 5 pM, from about 0.1 μM to about 5 pM, from about 50 nM to about 5 pM, from about 10 nM to about 5 pM, from about 5 nM to about 5 pM, from about 1 nM to about 5 pM, from about 0.5 nM to about 5 pM, from about 0.1 nM to about 5 pM, from about 50 pM to about 5 pM, from about 10 pM to about 5 pM.

[0113] In some embodiments, the anti-EGFRvIII antibody has a binding avidity (EC 50)(is) from about 500 nM to about 0.1 pM, about 100 nM to about 0.1 pM, about 50 nM to about 0.1 pM, about 10 nM to about 0.1 pM, about 5 nM to about 0.1 pM, about 1 nM to about 0.1 pM, about 0.5 nM to about 0.1 pM, about 0.1 nM to about 0.1 pM, about 50 pM to about 0.1 pM, about 10 pM to about 0.1 pM, about 5 pM to about 0.1 pM, about 1 pM to about 0.1 pM, about 0.5 pM to about 0.1 pM.

[0114] In some embodiments, the half-maximal effective concentration (EC 50 )(is) from about 500 nM to about 0.001 nM, about 100 nM to about 0.001 nM, about 50 nM to about 0.001 nM, about 10 nM to about 0.001 nM, about 5 nM to about 0.001 nM, about 1 nM to about 0.001 nM, about 0.5 nM to about 0.001 nM, about 0.1 nM to about 0.001 nM, about 0.05 nM to about 0.001 nM, about 0.01 nM to about 0.001 nM, about 0.005 nM to about 0.001 nM.

[0115] In some embodiments, the anti-EGFRvIII antibody is a full-length antibody (an antibody in which two heavy chains and two light chains are linked to an Fc domain, in a "Y" shape). In some embodiments, the Fc domain (or simply Fc) is a human Fc domain. In some embodiments, the Fc domain of the humanized antibody is from human IgG1, human IgG2, human IgG3, or human IgG4.

[0116] Example 8. Exemplary Anti-EGFRvIII Antibodies - CDR Sequences

[0117] Sequences of exemplary anti-EGFRvIII antibodies of the present disclosure are provided herein. Complementary determining region (CDR) sequences and variable heavy and light chain domain sequences (VH, VL) that constitute the EGFRvIII antigen-binding domain of the present disclosure are included. The discovery of these antibodies is described in detail in the Examples section.

[0118] As referred to below, the CDR1 region of the light chain variable (VL) domain is designated CDR-L1; the VL CDR2 region is designated CDR-L2; the VL CDR3 region is designated CDR-L3; the CDR1 region of the heavy chain variable (VH) domain is designated CDR-H1; the VH CDR2 region is designated CDR-H2; and the VH CDR3 region is designated CDR-H3. Table 10 provides exemplary CDR combinations of the antibodies of the present disclosure.

[0119] Table 10. Anti-EGFRvIII Name and Type

[0120]

[0121] Table 11. Full-Length Humanized and Chimeric Anti-EGFRvIII Antibodies

[0122] Name SEQ ID NO Sequence SD-233883_VH 1 EVQLQQFGAELVKPGASVKLSCKASGYTFTSYDINWVRQRPEQGLEWIGWIFPGDGTSKYNEKFKGKATLSTDKSSSTAYMQLSRLTIEDSAVYFCARRLGSHWGQGSTLTVSS SD-233883_VL 2 DIVLTQSPASLAVSLGQRATISCKASQSVDSDGDSYMNWYQQKPGQPPKLLIYGASNLESGIPARFSGSGSGTDFSLNIHPVEEEDAATYYCQQSHEYPFTFGGGTKLEIK SD-233883_hCDR1 3 GYTFTSYD SD-233883_hCDR2 4 IFPGDGTS SD-233883_hCDR3 5 ARRLGSH SD-233883_lCDR1 6 QSVDSDGDSY SD-233883_lCDR2 7 GAS SD-233883_lCDR3 8 QQSHEYPFT SD-233883_VH_ DNA 9 GAAGTGCAACTCCAGCAATTCGGTGCCGAGCTTGTGAAGCCTGGGGCCTCAGTGAAGCTGAGCTGCAAGGCCTCCGGATATACCTTCACCTCCTACGATATCAACTGGGTCCGGCAGAGGCCGGAACAGGGCCTGGAGTGGATCGGTTGGATCTTCCCCGGCGACGGGACCTCGAAGTACAACGAAAAGTTCAAGGGAAAAGCAACGCTGTCCACCGACAAGTCCTCATCCACTGCGTACATGCAGCTGTCCCGCCTGACTATTGAGGACTCCGCTGTGTACTTTTGTGCCCGGAGACTCGGAAGCCACTGGGGACAGGGCAGCACCTTGACTGTGTCGTCG SD-233883_VL_ DNA 10 GATATTGTGCTGACTCAAAGCCCTGCGTCCCTGGCTGTCTCCCTGGGACAGCGCGCCACCATTTCATGCAAAGCCTCCCAGTCCGTGGACAGCGACGGGGACAGCTATATGAACTGGTACCAGCAGAAGCCCGGGCAGCCTCCGAAGCTGCTTATCTACGGTGCCTCCAACTTGGAGTCGGGAATCCCCGCACGGTTCTCCGGATCGGGCTCCGGAACTGACTTCTCGCTCAACATCCACCCAGTGGAAGAAGAAGATGCCGCCACCTACTACTGTCAGCAATCACATGAGTACCCGTTTACCTTCGGTGGCGGCACCAAGCTCGAGATCAAG SD-633416_VH 11 EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQAPGQGLEWMGWIFPGDGTSKYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARRLGSHWGQGTTVTVSS SD-633416_VL 12 DIQMTQSPSSLSASVGDRVTITCKASQSVDSDGDSYMNWYQQKPGKAPKLLIYGASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHEYPFTFGQGTKLEIK SD-633416_hCDR1 13 GYTFTSYD SD-633416_hCDR2 14 IFPGDGTS SD-633416_hCDR3 15 ARRLGSH SD-633416_lCDR1 16 QSVDSDGDSY SD-633416_lCDR2 17 GAS SD-633416_lCDR3 18 QQSHEYPFT SD-633416_VH_ DNA 19 GAAGTGCAACTGGTGCAAAGCGGTGCCGAAGTCAAGAAGCCCGGAGCCTCAGTGAAAGTGTCCTGCAAGGCTTCGGGCTACACCTTCACCTCCTACGACATTAACTGGGTCAGACAGGCACCTGGACAGGGCCTGGAGTGGATGGGCTGGATCTTCCCGGGCGACGGAACTTCGAAATACGCCCAGAAGTTTCAGGGTCGCGTGACTATGACTCGGGATACTTCCACCTCCACCGTGTACATGGAACTCAGCTCCCTTCGGTCCGAGGACACCGCCGTCTACTATTGTGCGAGGAGACTGGGGTCACACTGGGGACAGGGGACGACCGTGACCGTGTCGAGC SD-633416_VL_ DNA 20 GATATTCAGATGACGCAGAGCCCCTCGTCCCTCTCCGCTTCCGTGGGAGATCGCGTCACCATTACTTGCAAAGCCAGCCAGTCCGTGGACTCGGACGGAGACTCCTACATGAACTGGTACCAGCAGAAGCCAGGAAAGGCCCCGAAGCTGCTTATCTACGGGGCCTCCAACTTGGAATCGGGAGTGCCTTCACGGTTCTCTGGTTCCGGCTCCGGCACTGACTTTACCCTGACCATCAGCAGCCTGCAGCCGGAGGACTTCGCGACTTACTACTGCCAACAGTCACACGAATATCCCTTCACCTTCGGCCAAGGGACCAAGCTGGAGATCAAG SD-382591_VH 21 EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQAPGQGLEWMGWIFPGDGTSKYAQKFQGRVTMTTDTSTSTAYMELSSLRSEDTAVYYCARRLGSHWGQGTTVTVSS SD-382591_VL 22 DIQLTQSPSSLSASVGDRVTITCKASQSVDSDGDSYMNWYQQKPGKAPKLLIYGASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHEYPFTFGQGTKLEIK SD-382591_hCDR1 23 GYTFTSYD SD-382591_hCDR2 24 IFPGDGTS SD-382591_hCDR3 25 ARRLGSH SD-382591_lCDR1 26 QSVDSDGDSY SD-382591_lCDR2 27 GAS SD-382591_lCDR3 28 QQSHEYPFT SD-382591_VH_ DNA 29 GAAGTGCAGCTGGTGCAGTCAGGCGCCGAGGTCAAGAAGCCCGGAGCAAGCGTGAAAGTGTCCTGCAAGGCCTCAGGGTACACTTTCACCTCCTATGACATCAACTGGGTCAGACAGGCTCCGGGACAAGGGCTCGAATGGATGGGTTGGATTTTCCCTGGCGACGGCACATCGAAATACGCGCAGAAGTTTCAGGGACGCGTGACCATGACCACCGACACGTCCACTTCCACTGCCTACATGGAACTGAGCTCGCTGCGGTCCGAGGATACCGCCGTGTACTACTGTGCCCGGAGGCTTGGCAGCCACTGGGGTCAAGGAACCACCGTGACTGTGTCCTCG SD-382591_VL_ DNA 30 GATATTCAGCTGACGCAGAGCCCCTCGTCCCTCTCCGCTTCCGTGGGAGATCGCGTCACCATTACTTGCAAAGCCAGCCAGTCCGTGGACTCGGACGGAGACTCCTACATGAACTGGTACCAGCAGAAGCCAGGAAAGGCCCCGAAGCTGCTTATCTACGGGGCCTCCAACTTGGAATCGGGAGTGCCTTCACGGTTCTCTGGTTCCGGCTCCGGCACTGACTTTACCCTGACCATCAGCAGCCTGCAGCCGGAGGACTTCGCGACTTACTACTGCCAACAGTCACACGAATATCCCTTCACCTTCGGCCAAGGGACCAAGCTGGAGATCAAG SD-741396_VH 31 EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQAPGQGLEWMGWIFPGDGTSKYAQKFQGRVTMTTDTSTSTAYMELSSLRSEDTAVYYCARRLGSHWGQGTTVTVSS SD-741396_VL 32 DIQLTQSPSSLSASVGDRATITCKASQSVDSDGDSYMNWYQQKPGKAPKLLIYGASNLESGIPSRFSGSGSGTDFTLTISSVQPEDFATYYCQQSHEYPFTFGQGTKLEIK SD-741396_hCDR1 33 GYTFTSYD SD-741396_hCDR2 34 IFPGDGTS SD-741396_hCDR3 35 ARRLGSH SD-741396_lCDR1 36 QSVDSDGDSY SD-741396_lCDR2 37 GAS SD-741396_lCDR3 38 QQSHEYPFT SD-741396_VH_ DNA 39 GAAGTGCAGCTGGTGCAGTCAGGCGCCGAGGTCAAGAAGCCCGGAGCAAGCGTGAAAGTGTCCTGCAAGGCCTCAGGGTACACTTTCACCTCCTATGACATCAACTGGGTCAGACAGGCTCCGGGACAAGGGCTCGAATGGATGGGTTGGATTTTCCCTGGCGACGGCACATCGAAATACGCGCAGAAGTTTCAGGGACGCGTGACCATGACCACCGACACGTCCACTTCCACTGCCTACATGGAACTGAGCTCGCTGCGGTCCGAGGATACCGCCGTGTACTACTGTGCCCGGAGGCTTGGCAGCCACTGGGGTCAAGGAACCACCGTGACTGTGTCCTCG SD-741396_VL_ DNA 40 GATATTCAGTTGACCCAGTCCCCGAGCTCACTGTCCGCTTCCGTGGGTGATCGCGCCACTATCACGTGTAAAGCGTCCCAGAGCGTCGACTCCGACGGGGACTCCTACATGAACTGGTATCAGCAGAAGCCCGGAAAGGCCCCTAAGCTCCTGATCTACGGCGCATCCAACCTGGAAAGCGGAATCCCCTCGCGGTTCTCGGGAAGCGGCTCTGGGACCGACTTCACCCTTACTATCTCATCGGTGCAACCGGAGGACTTCGCCACCTACTACTGCCAACAGTCCCACGAATACCCATTCACCTTTGGACAAGGCACTAAGCTGGAGATTAAG SD-844257_VH 41 EVQLVQSGAEVKKPGASVKLSCKASGYTFTSYDINWVRQAPGQGLEWIGWIFPGDGTSKYAQKFQGRATLTTDTSTSTAYMELSSLRSEDTAVYYCARRLGSHWGQGTTLTVSS SD-844257_VL 42 DIQLTQSPSSLSASVGDRVTITCKASQSVDSDGDSYMNWYQQKPGKAPKLLIYGASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHEYPFTFGQGTKLEIK SD-844257_hCDR1 43 GYTFTSYD SD-844257_hCDR2 44 IFPGDGTS SD-844257_hCDR3 45 ARRLGSH SD-844257_lCDR1 46 QSVDSDGDSY SD-844257_lCDR2 47 GAS SD-844257_lCDR3 48 QQSHEYPFT SD-844257_VH_ DNA 49 GAAGTGCAGCTCGTGCAGTCCGGAGCCGAAGTCAAGAAGCCGGGAGCCTCAGTGAAGCTCAGCTGCAAGGCCTCGGGCTACACCTTCACTTCCTACGACATTAACTGGGTCAGACAGGCACCTGGACAAGGCTTGGAGTGGATCGGATGGATCTTTCCCGGCGATGGGACTAGCAAATACGCCCAGAAGTTCCAGGGTCGCGCGACTCTGACCACCGACACCTCCACCTCAACCGCGTATATGGAACTGTCCTCCCTTCGGTCGGAGGACACTGCCGTGTACTACTGCGCTAGAAGGCTGGGCAGCCACTGGGGTCAAGGGACCACACTGACGGTGTCGTCC SD-844257_VL_ DNA 50 GATATTCAGCTGACGCAGAGCCCCTCGTCCCTCTCCGCTTCCGTGGGAGATCGCGTCACCATTACTTGCAAAGCCAGCCAGTCCGTGGACTCGGACGGAGACTCCTACATGAACTGGTACCAGCAGAAGCCAGGAAAGGCCCCGAAGCTGCTTATCTACGGGGCCTCCAACTTGGAATCGGGAGTGCCTTCACGGTTCTCTGGTTCCGGCTCCGGCACTGACTTTACCCTGACCATCAGCAGCCTGCAGCCGGAGGACTTCGCGACTTACTACTGCCAACAGTCACACGAATATCCCTTCACCTTCGGCCAAGGGACCAAGCTGGAGATCAAG SD-757052_VH 51 EVQLVQSGAEVKKPGASVKLSCKASGYTFTSYDINWVRQAPGQGLEWIGWIFPGDGTSKYAQKFQGRATLTTDTSTSTAYMELSSLRSEDTAVYYCARRLGSHWGQGTTLTVSS SD-757052_VL 52 DIQLTQSPSSLSASVGDRATITCKASQSVDSDGDSYMNWYQQKPGKAPKLLIYGASNLESGIPSRFSGSGSGTDFTLTISSVQPEDFATYYCQQSHEYPFTFGQGTKLEIK SD-757052_hCDR1 53 GYTFTSYD SD-757052_hCDR2 54 IFPGDGTS SD-757052_hCDR3 55 ARRLGSH SD-757052_lCDR1 56 QSVDSDGDSY SD-757052_lCDR2 57 GAS SD-757052_lCDR3 58 QQSHEYPFT SD-757052_VH_ DNA 59 GAAGTGCAGCTCGTGCAGTCCGGAGCCGAAGTCAAGAAGCCGGGAGCCTCAGTGAAGCTCAGCTGCAAGGCCTCGGGCTACACCTTCACTTCCTACGACATTAACTGGGTCAGACAGGCACCTGGACAAGGCTTGGAGTGGATCGGATGGATCTTTCCCGGCGATGGGACTAGCAAATACGCCCAGAAGTTCCAGGGTCGCGCGACTCTGACCACCGACACCTCCACCTCAACCGCGTATATGGAACTGTCCTCCCTTCGGTCGGAGGACACTGCCGTGTACTACTGCGCTAGAAGGCTGGGCAGCCACTGGGGTCAAGGGACCACACTGACGGTGTCGTCC SD-757052_VL_ DNA 60 GATATTCAGTTGACCCAGTCCCCGAGCTCACTGTCCGCTTCCGTGGGTGATCGCGCCACTATCACGTGTAAAGCGTCCCAGAGCGTCGACTCCGACGGGGACTCCTACATGAACTGGTATCAGCAGAAGCCCGGAAAGGCCCCTAAGCTCCTGATCTACGGCGCATCCAACCTGGAAAGCGGAATCCCCTCGCGGTTCTCGGGAAGCGGCTCTGGGACCGACTTCACCCTTACTATCTCATCGGTGCAACCGGAGGACTTCGCCACCTACTACTGCCAACAGTCCCACGAATACCCATTCACCTTTGGACAAGGCACTAAGCTGGAGATTAAG SD-787077_VH 61 EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQAPGQGLEWMGWIFPGDGTSKYAQKFQGRVTMTTDKSTSTAYMELSSLRSEDTAVYYCARRLGSHWGQGTTVTVSS SD-787077_VL 62 DIQLTQSPSSLSASVGDRVTITCKASQSVDSDGDSYMNWYQQKPGKAPKLLIYGASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHEYPFTFGQGTKLEIK SD-787077_hCDR1 63 GYTFTSYD SD-787077_hCDR2 64 IFPGDGTS SD-787077_hCDR3 65 ARRLGSH SD-787077_lCDR1 66 QSVDSDGDSY SD-787077_lCDR2 67 GAS SD-787077_lCDR3 68 QQSHEYPFT SD-787077_VH_ DNA 69 GAAGTGCAGCTCGTGCAATCCGGCGCCGAAGTCAAGAAGCCTGGGGCCTCAGTGAAGGTGTCCTGCAAAGCATCGGGGTACACCTTCACGAGCTACGACATCAACTGGGTCCGCCAAGCTCCGGGACAGGGTCTGGAGTGGATGGGCTGGATTTTTCCCGGCGACGGTACCAGCAAATACGCGCAGAAGTTCCAGGGCAGAGTGACCATGACCACCGACAAGTCCACTTCAACCGCCTACATGGAGCTGTCCTCCCTGCGGTCGGAGGATACTGCCGTGTATTACTGTGCCCGGAGGCTTGGAAGCCACTGGGGACAGGGAACTACTGTGACCGTGTCGTCC SD-787077_VL_ DNA 70 GATATTCAGCTGACGCAGAGCCCCTCGTCCCTCTCCGCTTCCGTGGGAGATCGCGTCACCATTACTTGCAAAGCCAGCCAGTCCGTGGACTCGGACGGAGACTCCTACATGAACTGGTACCAGCAGAAGCCAGGAAAGGCCCCGAAGCTGCTTATCTACGGGGCCTCCAACTTGGAATCGGGAGTGCCTTCACGGTTCTCTGGTTCCGGCTCCGGCACTGACTTTACCCTGACCATCAGCAGCCTGCAGCCGGAGGACTTCGCGACTTACTACTGCCAACAGTCACACGAATATCCCTTCACCTTCGGCCAAGGGACCAAGCTGGAGATCAAG SD-638526_VH 71 EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQAPGQGLEWMGWIFPGDGTSKYAQKFQGRVTMTTDKSTSTAYMELSSLRSEDTAVYYCARRLGSHWGQGTTVTVSS SD-638526_VL 72 DIQLTQSPSSLSASVGDRATITCKASQSVDSDGDSYMNWYQQKPGKAPKLLIYGASNLESGIPSRFSGSGSGTDFTLTISSVQPEDFATYYCQQSHEYPFTFGQGTKLEIK SD-638526_hCDR1 73 GYTFTSYD SD-638526_hCDR2 74 IFPGDGTS SD-638526_hCDR3 75 ARRLGSH SD-638526_lCDR1 76 QSVDSDGDSY SD-638526_lCDR2 77 GAS SD-638526_lCDR3 78 QQSHEYPFT SD-638526_VH_ DNA 79 GAAGTGCAGCTCGTGCAATCCGGCGCCGAAGTCAAGAAGCCTGGGGCCTCAGTGAAGGTGTCCTGCAAAGCATCGGGGTACACCTTCACGAGCTACGACATCAACTGGGTCCGCCAAGCTCCGGGACAGGGTCTGGAGTGGATGGGCTGGATTTTTCCCGGCGACGGTACCAGCAAATACGCGCAGAAGTTCCAGGGCAGAGTGACCATGACCACCGACAAGTCCACTTCAACCGCCTACATGGAGCTGTCCTCCCTGCGGTCGGAGGATACTGCCGTGTATTACTGTGCCCGGAGGCTTGGAAGCCACTGGGGACAGGGAACTACTGTGACCGTGTCGTCC SD-638526_VL_ DNA 80 GATATTCAGTTGACCCAGTCCCCGAGCTCACTGTCCGCTTCCGTGGGTGATCGCGCCACTATCACGTGTAAAGCGTCCCAGAGCGTCGACTCCGACGGGGACTCCTACATGAACTGGTATCAGCAGAAGCCCGGAAAGGCCCCTAAGCTCCTGATCTACGGCGCATCCAACCTGGAAAGCGGAATCCCCTCGCGGTTCTCGGGAAGCGGCTCTGGGACCGACTTCACCCTTACTATCTCATCGGTGCAACCGGAGGACTTCGCCACCTACTACTGCCAACAGTCCCACGAATACCCATTCACCTTTGGACAAGGCACTAAGCTGGAGATTAAG SD-710726_VH 81 EVQLVQSGAEVKKPGASVKLSCKASGYTFTSYDINWVRQAPGQGLEWIGWIFPGDGTSKYAQKFQGRATLTTDKSTSTAYMELSSLRSEDTAVYYCARRLGSHWGQGTTLTVSS SD-710726_VL 82 DIQLTQSPSSLSASVGDRVTITCKASQSVDSDGDSYMNWYQQKPGKAPKLLIYGASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHEYPFTFGQGTKLEIK SD-710726_hCDR1 83 GYTFTSYD SD-710726_hCDR2 84 IFPGDGTS SD-710726_hCDR3 85 ARRLGSH SD-710726_lCDR1 86 QSVDSDGDSY SD-710726_lCDR2 87 GAS SD-710726_lCDR3 88 QQSHEYPFT SD-710726_VH_ DNA 89 GAAGTGCAGTTGGTGCAGTCGGGAGCCGAAGTCAAGAAGCCTGGAGCGTCCGTGAAGCTGAGCTGCAAGGCCTCAGGATACACTTTCACTTCATATGACATCAACTGGGTCAGACAGGCACCGGGCCAAGGACTGGAGTGGATTGGCTGGATCTTTCCCGGGGATGGCACGAGCAAATACGCCCAGAAGTTCCAGGGTAGAGCGACCCTGACCACCGACAAGTCCACTTCGACCGCCTACATGGAACTCTCCTCGCTGCGCTCCGAGGACACCGCCGTGTACTACTGTGCTCGGAGGCTTGGGTCCCACTGGGGTCAAGGCACCACTCTCACCGTGTCCAGC SD-710726_VL_ DNA 90 GATATTCAGCTGACGCAGAGCCCCTCGTCCCTCTCCGCTTCCGTGGGAGATCGCGTCACCATTACTTGCAAAGCCAGCCAGTCCGTGGACTCGGACGGAGACTCCTACATGAACTGGTACCAGCAGAAGCCAGGAAAGGCCCCGAAGCTGCTTATCTACGGGGCCTCCAACTTGGAATCGGGAGTGCCTTCACGGTTCTCTGGTTCCGGCTCCGGCACTGACTTTACCCTGACCATCAGCAGCCTGCAGCCGGAGGACTTCGCGACTTACTACTGCCAACAGTCACACGAATATCCCTTCACCTTCGGCCAAGGGACCAAGCTGGAGATCAAG SD-577776_VH 91 EVQLVQSGAEVKKPGASVKLSCKASGYTFTSYDINWVRQAPGQGLEWIGWIFPGDGTSKYAQKFQGRATLTTDKSTSTAYMELSSLRSEDTAVYYCARRLGSHWGQGTTLTVSS SD-577776_VL 92 DIQLTQSPSSLSASVGDRATITCKASQSVDSDGDSYMNWYQQKPGKAPKLLIYGASNLESGIPSRFSGSGSGTDFTLTISSVQPEDFATYYCQQSHEYPFTFGQGTKLEIK SD-577776_hCDR1 93 GYTFTSYD SD-577776_hCDR2 94 IFPGDGTS SD-577776_hCDR3 95 ARRLGSH SD-577776_lCDR1 96 QSVDSDGDSY SD-577776_lCDR2 97 GAS SD-577776_lCDR3 98 QQSHEYPFT SD-577776_VH_ DNA 99 GAAGTGCAGTTGGTGCAGTCGGGAGCCGAAGTCAAGAAGCCTGGAGCGTCCGTGAAGCTGAGCTGCAAGGCCTCAGGATACACTTTCACTTCATATGACATCAACTGGGTCAGACAGGCACCGGGCCAAGGACTGGAGTGGATTGGCTGGATCTTTCCCGGGGATGGCACGAGCAAATACGCCCAGAAGTTCCAGGGTAGAGCGACCCTGACCACCGACAAGTCCACTTCGACCGCCTACATGGAACTCTCCTCGCTGCGCTCCGAGGACACCGCCGTGTACTACTGTGCTCGGAGGCTTGGGTCCCACTGGGGTCAAGGCACCACTCTCACCGTGTCCAGC SD-577776_VL_ DNA 100 GATATTCAGTTGACCCAGTCCCCGAGCTCACTGTCCGCTTCCGTGGGTGATCGCGCCACTATCACGTGTAAAGCGTCCCAGAGCGTCGACTCCGACGGGGACTCCTACATGAACTGGTATCAGCAGAAGCCCGGAAAGGCCCCTAAGCTCCTGATCTACGGCGCATCCAACCTGGAAAGCGGAATCCCCTCGCGGTTCTCGGGAAGCGGCTCTGGGACCGACTTCACCCTTACTATCTCATCGGTGCAACCGGAGGACTTCGCCACCTACTACTGCCAACAGTCCCACGAATACCCATTCACCTTTGGACAAGGCACTAAGCTGGAGATTAAG SD-127612_VH 101 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQAPGQRLEWMGWIFPGDGTSKYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARRLGSHWGQGTLVTVSS SD-127612_VL 102 DIVLTQSPDSLAVSPGERATISCKASQSVDSDGDSYMNWYQQKPGQPPKLLIYGASNLESGVPDRFSGSGSGTDFTLTISRVEAEDVAVYYCQQSHEYPFTFGGGTKLEIK SD-127612_hCDR1 103 GYTFTSYD SD-127612_hCDR2 104 IFPGDGTS SD-127612_hCDR3 105 ARRLGSH SD-127612_lCDR1 106 QSVDSDGDSY SD-127612_lCDR2 107 GAS SD-127612_lCDR3 108 QQSHEYPFT SD-127612_VH_ DNA 109 CAAGTTCAGCTCGTGCAGAGTGGAGCAGAAGTGAAAAAGCCAGGGGCTTCAGTTAAGGTAAGCTGTAAAGCCTCCGGGTATACATTCACATCATACGACATAAATTGGGTGAGGCAAGCCCCGGGTCAGCGGCTGGAGTGGATGGGGTGGATTTTCCCCGGCGATGGGACTTCCAAGTACAATGAAAAGTTCAAGGGGCGAGTGACAATCACTAGGGACACTTCCGCCAGCACGGCTTACATGGAACTCAGCTCACTCAGAAGTGAGGATACCGCGGTCTATTACTGTGCTCGCAGGCTGGGATCCCACTGGGGCCAAGGGACTCTGGTTACAGTCTCCTCC SD-127612_VL_ DNA 110 GACATTGTACTCACCCAGAGTCCAGACAGCTTGGCCGTCAGTCCAGGTGAGAGAGCCACCATTAGCTGCAAGGCATCTCAGAGCGTGGATAGTGATGGCGATAGCTACATGAACTGGTACCAGCAGAAACCAGGCCAGCCACCTAAGCTCCTCATCTATGGCGCCTCTAACCTTGAATCTGGAGTGCCCGACCGCTTTAGCGGTAGCGGCAGCGGCACAGATTTCACTTTGACAATTAGTCGCGTGGAGGCCGAAGACGTGGCAGTCTACTACTGCCAGCAGAGCCACGAGTACCCATTCACATTCGGGGGAGGGACAAAGTTAGAGATTAAG SD-837152_VH 111 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQAPGQRLEWMGWIFPGDGTSKYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARRLGSHWGQGTLVTVSS SD-837152_VL 112 DIVLTQSPASLAVSPGQRATITCKASQSVDSDGDSYMNWYQQKPGQPPKLLIYGASNLESGVPARFSGSGSGTDFTLTINPVEANDTANYYCQQSHEYPFTFGQGTKLEIK SD-837152_hCDR1 113 GYTFTSYD SD-837152_hCDR2 114 IFPGDGTS SD-837152_hCDR3 115 ARRLGSH SD-837152_lCDR1 116 QSVDSDGDSY SD-837152_lCDR2 117 GAS SD-837152_lCDR3 118 QQSHEYPFT SD-837152_VH_ DNA 119 CAAGTTCAGCTCGTGCAGAGTGGAGCAGAAGTGAAAAAGCCAGGGGCTTCAGTTAAGGTAAGCTGTAAAGCCTCCGGGTATACATTCACATCATACGACATAAATTGGGTGAGGCAAGCCCCGGGTCAGCGGCTGGAGTGGATGGGGTGGATTTTCCCCGGCGATGGGACTTCCAAGTACAATGAAAAGTTCAAGGGGCGAGTGACAATCACTAGGGACACTTCCGCCAGCACGGCTTACATGGAACTCAGCTCACTCAGAAGTGAGGATACCGCGGTCTATTACTGTGCTCGCAGGCTGGGATCCCACTGGGGCCAAGGGACTCTGGTTACAGTCTCCTCC SD-837152_VL_ DNA 120 GATATCGTTCTGACCCAGAGTCCAGCGTCCTTGGCCGTGTCTCCAGGCCAACGGGCTACAATAACATGCAAGGCCTCTCAGAGCGTGGACTCTGACGGCGATAGTTACATGAATTGGTATCAGCAGAAGCCTGGACAGCCCCCCAAACTGTTGATTTACGGGGCAAGCAACCTGGAGAGTGGCGTGCCCGCACGTTTTTCAGGCTCAGGGAGCGGTACCGACTTTACACTTACAATTAATCCCGTGGAGGCTAATGATACTGCCAACTACTACTGTCAGCAGAGCCACGAGTATCCTTTTACTTTTGGCCAGGGAACCAAACTGGAAATCAAA SD-649072_VH 121 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQAPGQRLEWMGWIFPGDGTSKYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARRLGTVWGQGTLVTVSS SD-649072_VL 122 DIVLTQSPASLAVSPGQRATITCKASQSVDSDGDSYMNWYQQKPGQPPKLLIYGASNLESGVPARFSGSGSGTDFTLTINPVEANDTANYYCQQSHEYPFTFGQGTKLEIK SD-649072_hCDR1 123 GYTFTSYD SD-649072_hCDR2 124 IFPGDGTS SD-649072_hCDR3 125 ARRLGTV SD-649072_lCDR1 126 QSVDSDGDSY SD-649072_lCDR2 127 GAS SD-649072_lCDR3 128 QQSHEYPFT SD-649072_VH_ DNA 129 CAAGTTCAGCTCGTGCAGAGTGGAGCAGAAGTGAAAAAGCCAGGGGCTTCAGTTAAGGTAAGCTGTAAAGCCTCCGGGTATACATTCACATCATACGACATAAATTGGGTGAGGCAAGCCCCGGGTCAGCGGCTGGAGTGGATGGGGTGGATTTTCCCCGGCGATGGGACTTCCAAGTACAATGAAAAGTTCAAGGGGCGAGTGACAATCACTAGGGACACTTCCGCCAGCACGGCTTACATGGAACTCAGCTCACTCAGAAGTGAGGATACCGCGGTCTATTACTGTGCTCGCAGGCTGGGAACCGTTTGGGGCCAAGGGACTCTGGTTACAGTCTCCTCC SD-649072_VL_ DNA 130 GATATCGTTCTGACCCAGAGTCCAGCGTCCTTGGCCGTGTCTCCAGGCCAACGGGCTACAATAACATGCAAGGCCTCTCAGAGCGTGGACTCTGACGGCGATAGTTACATGAATTGGTATCAGCAGAAGCCTGGACAGCCCCCCAAACTGTTGATTTACGGGGCAAGCAACCTGGAGAGTGGCGTGCCCGCACGTTTTTCAGGCTCAGGGAGCGGTACCGACTTTACACTTACAATTAATCCCGTGGAGGCTAATGATACTGCCAACTACTACTGTCAGCAGAGCCACGAGTATCCTTTTACTTTTGGCCAGGGAACCAAACTGGAAATCAAA

[0123] In some embodiments, provided herein is an anti-EGFRvIII antibody, wherein the antibody comprises the amino acid sequences of the following three VH CDRs: SEQ ID NO: 3, 4, 5; 13, 14, 15; 23, 24, 25; 33, 34, 35; 43, 44, 45; 53, 54, 55; 63, 64, 65; 73, 74, 75; 83, 84, 85; 93, 94, 95; 103, 104, 105; 113, 114, 115; 123, 124, 125, respectively.

[0124] In some embodiments, provided herein is an anti-EGFRvIII antibody, wherein the antibody comprises the amino acid sequences of the following three VL CDRs: 6, 7, 8; 16, 17, 18; 26, 27, 28; 36, 37, 38; 46, 47, 48; 56, 57, 58; 66, 67, 68; 76, 77, 78; 86, 87, 88; 96, 97, 98; 106, 107, 108; 116, 117, 118; 126, 127, 128, respectively.

[0125] In some embodiments, provided herein is an anti-EGFRvIII antibody, wherein the antibody comprises the amino acid sequence of VH, and the VH comprises the amino acid sequence of any one of the following SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121.

[0126] In some embodiments, provided herein is an anti-EGFRvIII antibody, wherein the antibody comprises the amino acid sequence of VL, and the VL comprises the amino acid sequence of any one of the following SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122.

[0127] In some embodiments, provided herein is an anti-EGFRvIII antibody, wherein the antibody comprises the amino acid sequences of a pair of heavy and light chains, namely SEQ ID NO: 1 and 2, 11 and 12, 21 and 22, 31 and 32, 41 and 42, 51 and 52, 61 and 62, 71 and 72, 81 and 82, 91 and 92, 101 and 102, 111 and 112, 121 and 122, respectively.

[0128] In some embodiments, provided herein is an anti-EGFRvIII antibody, wherein the heavy and light chains of the antibody are encoded by nucleic acids that have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 9 and 10, 19 and 20, 29 and 30, 39 and 40, 49 and 50, 59 and 60, 69 and 70, 79 and 80, 89 and 90, 99 and 100, 109 and 110, 119 and 120, 129 and 130, respectively.

[0129] Example 9. scFv-Fc anti-EGFRvIII

[0130] In some embodiments, the present disclosure provides tandem scFv antibodies having multiple anti-EGFRvIII binding sites. The tandem scFv-Fc antibodies of the present disclosure are composed of two or more scFv binding sites tandemly arranged on each antibody arm, optionally connected by a linker, optionally a flexible linker. In some embodiments, the tandem scFV antibody has a total of four or more scFv binding sites in a single scFv-Fc type antibody.

[0131] The VH1 and VL1 of each scFV1 can be connected by a linker, such as a flexible linker.

[0132] The VH2 and VL2 of each scFV2 can be connected by a linker, such as a flexible linker.

[0133] The scFvs on each antibody arm can be connected by a linker, such as a flexible linker. Exemplary linkers include the following amino acid sequence: GGGGSGGGGSGGGGS (SEQ ID NO: 131).

[0134] Example 10. Therapeutic anti-EGFRvIII antibody

[0135] In some embodiments, the anti-EGFRvIII antibodies provided herein are effective for treating cancers expressing EGFRvIII, such as glioblastoma, head and neck squamous cell carcinoma, non-small cell lung cancer - squamous cell carcinoma (NSCLC-SCC), prostate cancer, breast cancer, and colon cancer.

[0136] Example 11. Administration of a Therapeutic Anti-EGFRvIII Antibody

[0137] In vivo administration of the therapeutic anti-EGFRvIII antibodies described herein can be by intravenous, intramuscular, subcutaneous, topical, oral, transdermal, intraperitoneal, intraorbital, intrathecal, intracerebroventricular, intranasal, transmucosal, by implantation, or by inhalation. Intravenous administration can be by injection or infusion. In some embodiments, the anti-EGFRvIII antibodies of the present disclosure are administered by intravenous injection. In some embodiments, the anti-EGFRvIII antibodies of the present disclosure are administered subcutaneously. Administration of the therapeutic anti-EGFRvIII antibodies can be carried out with any suitable excipient, carrier, or other reagent that provides suitable or improved tolerance, transfer, delivery, etc.

[0138] It is contemplated that for any method, kit, reagent, or composition of the present invention, any embodiment discussed in this specification can be implemented, and vice versa. In addition, the compositions of the present invention can be used to implement the methods of the present invention.

[0139] It should be understood that the specific embodiments described herein are shown by way of illustration and not as a limitation of the invention. The main features of the invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize or be able to ascertain using only routine experimentation many equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of the invention and are covered by the claims.

[0140] All publications and patent applications mentioned in this specification represent the level of skill of those of ordinary skill in the art to which this invention pertains. All publications and patent applications are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0141] When used in the claims and / or the specification in conjunction with the term “comprising,” the use of the word “a” or “an” can mean “one,” but it also is in the sense of “one or more,” “at least one,” and “one or more than one.” The term “or” as used in the claims is used to mean “and / or” unless explicitly indicated to refer only to alternatives or the alternatives are mutually exclusive, although the present disclosure supports definitions that refer only to alternatives as well as “and / or.” Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, method used to determine the value, or the variation that exists among the study subjects.

[0142] As used in this specification and the claims (if any), the word "comprising" (and any form of "comprising", such as "comprise" and "comprises"), "having" (and any form of "having", such as "have" and "has"), "including" (and any form of "including", such as "includes" and "include") or "containing" (and any form of "containing", such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. In any embodiments of the compositions and methods provided herein, "comprising" may be replaced with "consisting essentially of" or "consisting of". As used herein, the phrase "consisting essentially of" requires the specified integer(s) or step(s) and those that do not materially affect the characteristics or functions of the claimed invention. As used herein, the term "consisting" is used to indicate the presence of only the listed integers (e.g., features, elements, characteristics, properties, method / process steps or limitations) or groups of integers (e.g., features, elements, characteristics, properties, method / process steps or limitations).

[0143] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the items listed before the term. For example, "A, B, C or combinations thereof" is intended to include at least one of the following: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also includes BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, combinations that contain one or more repetitions of an item or term are expressly included, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, etc. Those skilled in the art will understand that there is usually no limit to the number of items or terms in any combination, unless it is apparent from the context.

[0144] As used herein, approximate terms such as, but not limited to, "about," "substantial," or "substantially" are meant to be understood as not necessarily being absolute or perfect when so modified, but will be considered close enough by one of ordinary skill in the art to ensure that the circumstance being specified is present. The degree to which a description may vary will depend on how much change can be made and still be recognized by one of ordinary skill in the art as having the characteristics and capabilities of the unmodified feature that is desired. In general, but subject to the foregoing discussion, a numerical value modified by an approximate term such as "about" in this disclosure may vary from the stated value by at least ±1%, 2%, 3%, 4%, 5%, 6%, 7%, 10%, 12%, or 15%.

[0145] In addition, the section headings provided herein are for compliance with the suggestions under 37 CFR 1.77 or otherwise to provide organizational cues. These headings should not limit or characterize the (multiple) inventions set forth in any claims that may issue from this disclosure. Specifically, for example, although the heading is called "Field of the Invention," such claims should not be limited by the language describing the so-called field of technology under that heading. Moreover, the technical descriptions in the "Background of the Invention" section should not be construed as admitting that the technology is prior art to any (multiple) inventions in this disclosure. Nor should the "Summary" be considered as characterizing the (multiple) inventions set forth in the issued claims. Additionally, any singular reference to "the invention" in this disclosure is not to be used to argue that there is only a single novelty point in this disclosure. Multiple inventions may be set forth in the limitations of the multiple claims that may issue from this disclosure, and such claims accordingly define the (multiple) inventions so protected and their equivalents. In all cases, the scope of such claims should be considered on its own merits in light of this disclosure, but should not be limited by the headings set forth herein.

[0146] For each claim, each dependent claim may depend from an independent claim and from each of the prior dependent claims that depend from each claim, so long as the prior claim provides an appropriate prior basis for the claim item or element.

[0147] To assist the Patent Office and any readers of any patent issued based on this application in interpreting the claims appended hereto, the Applicant wishes to note that, unless the words "means for" or "step for" are expressly used in a particular claim, they do not intend for any of the appended claims to be invoked against them as a result of 35 U.S.C. § 112, paragraph 6, 35 U.S.C. § 112, paragraph (f), or equivalent provisions as they existed on the date of filing of this application.

[0148] According to the present disclosure, all of the compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that changes can be made in the compositions and / or methods described herein and in the steps or in the sequence of steps of the methods described herein, without departing from the spirit, nature, and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and nature of the invention as defined by the appended claims.

Claims

1. An anti-epidermal growth factor receptor type III (EGFRvIII) antibody or antigen-binding domain thereof, wherein the antibody or antigen-binding domain comprises: a. Heavy chain variable domain (VH) complementarity-determining regions (CDR) 1, VH CDR2, and VH CDR3, each comprising the amino acid sequence of any one of SEQ ID NO: 3, 4, 5; 13, 14, 15; 23, 24, 25; 33, 34, 35; 43, 44, 45; 53, 54, 55; 63, 64, 65; 73, 74, 75; 83, 84, 85; 93, 94, 95; 103, 104, 105; 113, 114, 115; or 123, 124, 125; and b. Light chain variable domain (VL) CDR1, VL CDR2, and VL CDR3, each comprising the amino acid sequence of any one of SEQ ID NO: 6, 7, 8; 16, 17, 18; 26, 27, 28; 36, 37, 38; 46, 47, 48; 56, 57, 58; 66, 67, 68; 76, 77, 78; 86, 87, 88; 96, 97, 98; 106, 107, 108; 116, 117, 118; or 126, 127, 128.

2. The antibody or binding domain according to claim 1, wherein the antibody comprises a VH comprising the amino acid sequence of any one of SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, or 121.

3. The antibody or binding domain according to claim 1, wherein the antibody comprises a VL comprising the amino acid sequence of any one of SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, or 122.

4. The antibody or binding domain according to any one of claims 1 to 3, wherein the antibody is a monoclonal antibody.

5. The antibody or binding domain according to any one of claims 1 to 4, wherein the antibody is a full-length antibody.

6. The antibody or binding domain according to any one of claims 1 to 4, wherein the antibody is an antibody fragment.

7. The antibody or binding domain according to claim 5, wherein the antibody is fused to the Fc domain of any one of: human IgG1, human IgG2, human IgG3, and human IgG4.

8. The antibody or binding domain according to claim 1, wherein the heavy chain of the antibody comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, or 121, or an antibody comprising the amino acid sequence.

9. The antibody or binding domain according to claim 1, wherein the antibody light chain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112 or 122.

10. The antibody or binding domain according to claim 1, wherein the antibody comprises a heavy chain variable domain and a light chain variable domain, and the heavy chain variable domain and the light chain variable domain are respectively: SEQ ID NO: 1 and 2, 11 and 12, 21 and 22, 31 and 32, 41 and 42, 51 and 52, 61 and 62, 71 and 72, 81 and 82, 91 and 92, 101 and 102, 111 and 112, or 121 and 122.

11. The antibody or binding domain according to claim 1, wherein the antibody heavy chain is encoded by a nucleic acid, and the antibody light chain is encoded by a nucleic acid, and the nucleic acids have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 9 and 10, 19 and 20, 29 and 30, 39 and 40, 49 and 50, 59 and 60, 69 and 70, 79 and 80, 89 and 90, 99 and 100, 109 and 110, 119 and 120, or 129 and 130.

12. The antibody or binding domain according to claim 1, wherein the antibody or binding domain is non-fucosylated.

13. The antibody or binding domain according to claim 1, wherein the antibody or binding domain is produced in bacterial, fungal, mammalian, insect or plant cells.

14. The antibody or binding domain according to claim 1, wherein the antibody or its binding fragment does not bind to EGFR1.

15. A method for treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the antibody according to any one of claims 1 to 14.

16. The method according to claim 15, wherein the disease is cancer.

17. The method according to claim 15, wherein the disease is cancer selected from glioblastoma, head and neck squamous cell carcinoma, non-small cell lung cancer - squamous cell carcinoma (NSCLC - SCC), prostate cancer, breast cancer and colon cancer, and wherein the cancer expresses EGFRvIII.

18. The method according to claim 16, wherein the cancer cells of the cancer are killed by antibody-dependent cell cytotoxicity (ADCC).

19. The method according to any one of claims 15 to 18, wherein the subject is a human.

20. A polynucleotide comprising nucleic acid sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 9 and 10, 19 and 20, 29 and 30, 39 and 40, 49 and 50, 59 and 60, 69 and 70, 79 and 80, 89 and 90, 99 and 100, 109 and 110, 119 and 120, or 129 and 130, respectively.

21. A vector comprising the polynucleotide of claim 20.

22. A host cell comprising the vector of claim 21.

23. A method for producing an anti-EGFRvIII antibody, comprising expressing in a cell a nucleic acid encoding the antibody of claim, the antibody of any one of claims 1 to 14.

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