Epidermal growth factor receptor variant III antibody

Abstract

Anti-EGFRvIII antibodies and binding fragments thereof are provided herein. The anti-EGFRvIII antibodies of the present disclosure are useful for treating cancer, for example, by antibody-dependent cellular cytotoxicity (ADCC). Methods of producing and using anti-EGFRvIII antibodies for treating cancer, as well as polynucleotides encoding the same, are also provided herein.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application Nos. 63 / 415,051, filed October 11, 2022, and 63 / 515,366, filed July 25, 2023, the entire contents of which are incorporated herein by reference.

[0002] This document relates to materials and methods for treating cancer, and in particular to the use of anti-EGFRvIII antibodies to reduce or eliminate cells expressing truncated EGFRvIII and treat cancer.

[0003] INCORPORATION-BY-REFERENCE OF MATERIAL FILED ON A COMPACT DISC This application contains a Sequence Listing that has been submitted electronically in .XML format and is incorporated herein by reference in its entirety. Said .XML copy, created on October 10, 2023, is named "IBIO2033WO.xml" and is 166,302 bytes in size. The Sequence Listing contained in this .XML file is a part of the present specification and is hereby incorporated by reference in its entirety. [Background technology]

[0004] Without limiting the scope of the present invention, its background is described with respect to anti-EGFRvIII-expressing cancers.

[0005] The epidermal growth factor receptor (EGFR1 / ErbB1 / HER1) is a member of the tyrosine receptor family and is activated by EGF ligand. Overexpression of EGFR1 is commonly observed in many types of cancer. EGFR1 has been found to be mutated in certain tumors, with the most common mutation being EGFR variant III (EGFRvIII). EGFRvIII contains a unique in-frame deletion of 267 amino acids in exons 2 to 7 within the ECD of EGFR, rendering it unable to bind EGF ligand. EGFRvIII expression confers resistance to conventional EGFR1-targeted therapies.

[0006] Although the frequency of EGFRvIII expression in tumors varies based on tumor type, EGFRvIII expression is specific to tumor cells only. Therefore, EGFRvIII is a desirable therapeutic target due to its specific expression in tumor cells. EGFRvIII is responsible for 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 colorectal cancers. Summary of the Invention [Problem to be solved by the invention]

[0007] What is needed are novel antibodies that specifically bind to EGFRvIII without binding to wild-type EGFR. Novel antibodies that enhance antibody-dependent cellular cytotoxicity are also needed. [Means for solving the problem]

[0008] As exemplified and broadly described herein, aspects of the disclosure include an anti-epidermal growth factor receptor version III (EGFRvIII) antibody, or antigen-binding domain thereof, wherein the antibody or antigen-binding domain comprises a heavy chain variable domain (VH) complementarity-determining region (CDR) 1, a VH CDR2, and a VH CDR3, each comprising the amino acid sequence of any one of SEQ ID NOs: 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. or 126, 127, 128. In one embodiment, the antibody comprises a light chain variable domain (VL) CDR1, VL CDR2, and VL CDR3, each comprising the amino acid sequence of any one of SEQ ID NOs: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, or 121. In another embodiment, the antibody comprises a VL comprising the amino acid sequence of any one of SEQ ID NOs: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, or 122. In another embodiment, the antibody is a monoclonal antibody. In another embodiment, the antibody is a full-length antibody. In another embodiment, the antibody is an antibody fragment. In another embodiment, the antibody is fused to the Fc domain of any one of human IgG1, human IgG2, human IgG3, and human IgG4. In another embodiment, the antibody heavy chain comprises an amino acid sequence having at least 80%, 85%, 90%, 95, 96, 97, 98, 99%, or 100% sequence identity to SEQ ID NOs: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, or 121, or an antibody comprising same.In another embodiment, the antibody light chain 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. In another embodiment, the antibody comprises heavy chain variable domains and light chain variable domains of SEQ ID NOs: 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, respectively. In another embodiment, the antibody heavy chain is encoded by a nucleic acid and the antibody light chain is encoded by a nucleic acid having at least 80%, 85%, 90%, 95, 96, 97, 98, 99%, or 100% sequence identity to SEQ ID NOs: 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. In another embodiment, the antibody or binding domain is defucosylated. In another embodiment, the antibody or binding domain is produced in a bacterial, fungal, mammalian, insect, or plant cell. As exemplified and broadly described herein, aspects of the present disclosure relate to a method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody described herein. In one embodiment, the disease is cancer. In another embodiment, 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 colorectal cancer, and the cancer expresses EGFRvIII. In another embodiment, cancer cells of the cancer are killed by antibody-dependent cellular cytotoxicity (ADCC). In another embodiment, the subject is a human. In another embodiment, the antibody or binding fragment thereof does not bind to EGFR1.

[0009] As exemplified and broadly described herein, embodiments of the present disclosure relate to polynucleotides comprising a nucleic acid sequence having at least 80%, 85%, 90%, 95, 96, 97, 98, 99%, or 100% sequence identity to SEQ ID NOs: 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. In another embodiment, the subject is a human. In another embodiment, the antibody or binding fragment thereof does not bind to EGFR1.

[0010] As exemplified and broadly described herein, aspects of the disclosure relate to vectors comprising the polynucleotides described herein. As exemplified and broadly described herein, aspects of the disclosure relate to host cells comprising the vectors described herein. As exemplified and broadly described herein, aspects of the disclosure relate to methods of making anti-EGFRvIII antibodies, the method comprising expressing in a cell a nucleic acid encoding the antibody described herein. In another aspect, the subject is a human. In another aspect, the antibody or binding fragment thereof does not bind to EGFR1. [Brief explanation of the drawings]

[0011] For a more complete understanding of the features and advantages of the present invention, reference is now made to the following detailed description of the invention taken in conjunction with the accompanying drawings. [Figures 1A-1C] Figure 1A compares the binding of various antibodies to EGFRvIII and EGFR1 by surface plasmon resonance (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). [Figures 2A-2D] FIG. 1 compares binding to human EGFRvIII and EGFR1 by a hIgG1 isotype control antibody, cetuximab, and chimeric and humanized antibodies of the invention by ELISA. [Figures 3A-3F] Figure 3 compares binding by chimeric and humanized anti-EGFRvIII antibodies of the present invention. The listed antibodies specifically bind to human EGFRvIII but not wild-type human EGFR1. FACS analysis shows anti-EGFRvIII antibodies specifically binding to F98 rat glioblastoma (Figure 3A), U87MG human glioblastoma (Figure 3C), and FaDu human head and neck cancer cells (Figure 3E), which overexpress human EGFRvIII. No binding was detected to F98 cells overexpressing wild-type human EGFR1 (Figure 3B) or wild-type U87MG (Figure 3D) and FaDu (Figure 3F) cells. Plotted values ​​are median fluorescence intensity. EC50 values ​​are the mean of n=3 experiments. n / a, no activity. [Figures 4A-4F] Figure 4 shows chimeric and humanized anti-EGFRvIII antibodies of the present invention exhibiting potent ADCC activity against F98 (Figure 4A), U87MG (Figure 4C), and FaDu cells (Figure 4E) overexpressing human EGFRvIII, but not against F98 (Figure 4B), wild-type U87MG (Figure 4D), and FaDu (Figure 4F) cells expressing human EGFR1. The ratio of dead cells to total cells was used to determine the percentage of cell lysis. EC50 values ​​are the average of n=1 to 5 experiments. n / a, no activity. [Figures 5A-5E]Figure 5A shows the study design for the in vivo efficacy of the anti-EGFRvIII antibody SD-233883-afuc against FaDu EGFRvIII tumor cells in a nude mouse model. Figure 5B (FaDu-EGFRvIII tumor volume after first drug treatment) and Figure 5C (% change in FaDu-EGFRvIII tumor volume after first drug treatment) show that SD-233883-afuc significantly inhibited FaDu-EGFRvIII tumor growth throughout the observation time window compared with the hIgG1 negative control (t-test, P<0.05). Figure 5D shows that at the study endpoint (day 22), FaDu-EGFRvIII tumor weight was significantly reduced after cetuximab or SD-233883-afuc treatment compared with the hIgG1 negative control (t-test, P<0.05). Figure 5E shows mouse body weight after the first drug treatment. DETAILED DESCRIPTION OF THE INVENTION

[0012] While the making and use of various embodiments of the invention are described in detail below, it should be understood that the invention provides many applicable inventive concepts that can be embodied in a variety of specific contexts. The specific embodiments described herein are merely illustrative of specific ways to make and use the invention and do not limit the scope of the invention.

[0013] To facilitate understanding of the present invention, several terms are defined below. Terms defined herein have meanings as commonly understood by one of ordinary skill in the art in the areas relevant to the present invention. Terms such as "a," "an," and "the" are not intended to refer to a singular entity only, but include a general class of which a particular example may be used for illustration. While the terms herein are used to describe particular embodiments of the present invention, their usage does not limit the present invention except as outlined in the claims.

[0014] Unless expressly indicated, in any method described or disclosed herein that includes two or more acts, the order of the acts is not necessarily limited to the order in which the acts of the method are recited, although it should be understood that the present disclosure encompasses example embodiments in which the order of the acts is very limited.

[0015] The epidermal growth factor receptor (EGFR1 / ErbB1 / HER1) is a member of the tyrosine receptor family and is activated by epidermal growth factor (EGF) ligands. Overexpression of EGFR1 is commonly observed in many types of cancer. EGFR1 has also been found to be mutated in certain tumors, with the most common mutation being EGFR variant III (EGFRvIII). EGFRvIII has a unique in-frame deletion of 267 amino acids in exons 2 to 7 in the EGFR ECD, rendering it unable to bind to EGF ligands. While the frequency of EGFRvIII expression in tumors varies based on tumor type, EGFRvIII expression is specific to tumor cells. In EGFRvIII-expressing cancers, EGFRvIII is a desirable therapeutic target due to its specific expression in tumor cells. Importantly, EGFRvIII expression confers resistance to conventional EGFR1-targeted therapies.

[0016] The present invention is a novel antibody that is specific for EGFRvIII and does not bind to native or wild-type EGFR1. The defucosylated antibody enhances ADCC responses and provides a better safety profile because it does not bind to wild-type or native EGFR1. The anti-EGFRvIII-specific ADCC approach allows for the selective destruction of EGFRvIII-expressing tumor cells and minimizes off-target effects.

[0017] As used herein, the term "mesoscale molecules (MEMs)" refers to modified peptides and polypeptides of about 1 kDa to about 10 kDa. Throughout this specification, the term "MEMs-nanoparticles" includes MEMs conjugated to nanoparticles (e.g., ferritin nanoparticles).

[0018] As used herein, a "subject" can be a mammalian or avian subject. Mammalian subjects include humans, non-human primates, rodents (e.g., rats, mice), lagomorphs (e.g., rabbits), ungulates (e.g., cattle, sheep, pigs, horses, goats, etc.), etc. 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 (e.g., cat, dog).

[0019] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0020] antibody As used herein, the term "antibody" refers to an intact antibody or a binding fragment thereof that specifically binds to a target antigen, in this case EGRFvIII. Binding fragments are produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Binding fragments include Fab, Fab', F(ab')2, Fv, and single-chain variable fragment (scFv) antibodies. An antibody substantially inhibits receptor attachment to a counterreceptor if excess antibody reduces the amount of receptor bound to the counterreceptor by at least about 20%, 40%, 60%, or 80%, more usually by more 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 region-containing antibodies, such as bivalent scFv Fc fusion antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, scFv), so long as they exhibit the desired biological activity. Antibodies (Ab) and immunoglobulins (Ig) are glycoproteins with the same structural characteristics. The present invention includes monoclonal antibodies (and binding fragments thereof) that are fully recombinant, i.e., have complementary determining regions (CDRs) genetically inserted into a human antibody framework, often referred to as veneered antibodies. Thus, in certain aspects, monoclonal antibodies are fully synthetic antibodies. In certain embodiments, monoclonal antibodies (and binding fragments thereof) can be produced in bacteria or eukaryotic cells, including mammalian, yeast, and plant cells.

[0021] As used herein, the term "antibody fragment" refers to a portion of a full-length antibody, generally the antigen-binding or variable region, including Fab, Fab', F(ab')2, Fv, and scFv fragments. The antibody fragments or domains of the present disclosure retain EGFRvIII antigen-binding specificity. Papain digestion of an antibody produces two identical antigen-binding fragments, called Fab fragments, each containing one antigen-binding site and a residual "Fc" fragment, so named because of its ability to readily crystallize. Pepsin treatment produces an F(ab')2 fragment containing two antigen-binding fragments capable of cross-linking antigen and another residual fragment (termed pFc'). As used herein, "functional fragments" with respect to antibodies refer to Fv, F(ab), and F(ab')2 fragments.

[0022] As used herein, an "Fv" fragment is the minimum antibody fragment containing a complete antigen-recognition and binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association (VH-VL dimer). In this configuration, the three CDRs from each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) has the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site.

[0023] Fab fragments, also designated F(ab), also contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. As used herein, Fab'-SH is the designation for Fab' in which the cysteine ​​residues of the constant domains bear free thiol groups. F(ab') fragments are produced by cleavage of the disulfide bond at the hinge cysteines of the F(ab')2 pepsin digestion product. Additional chemical couplings of antibody fragments are known to those skilled in the art.

[0024] Natural antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by at least one covalent disulfide bond, although the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by a constant domain. Each light chain has a variable domain (VL) at one end and a constant domain at its other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains (Clothia et al., J. Mol. Biol. 186, 651-66, 1985); Novotny and Haber, Proc. Natl. Acad. Sci. USA 82 4592-4596 (1985), relevant portions of which are incorporated herein by reference.

[0025] 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 is produced. Contaminant components of its production environment are materials that would interfere with diagnostic or therapeutic uses of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain embodiments, the antibody will be purified measurably by at least three different methods: 1) greater than 50% antibody by weight as determined by the Lowry method, such as greater than 75%, or greater than 85%, or greater than 95%, or greater than 99% by weight; 2) sufficient to obtain at least 10 residues of N-terminal or internal amino acid sequence, such as at least 15 residues of sequence, by use of a spinning cup sequenator; or 3) homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. An isolated antibody will be free of at least one component of the antibody's natural environment and thus includes the antibody in situ within recombinant cells. Ordinarily, however, an isolated antibody will be prepared by at least one purification step.

[0026] As used herein, the term "antibody mutant" or "antibody variant" refers to an amino acid sequence variant of an antibody in which one or more amino acid residues have been modified. Such mutants necessarily have less than 100% sequence identity or similarity with an amino acid sequence having at least 75%, 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 with the amino acid sequence of either the heavy or light chain variable domain of the antibody.

[0027] As used herein, the term "variable" in the context of antibody variable domains refers to the fact that certain portions of the variable domains differ significantly in sequence among antibodies and are used in the binding and specificity of each particular antibody for its specific antigen. However, the variation is not uniformly distributed throughout the variable domains of antibodies. It is concentrated in three segments, called complementarity-determining regions (CDRs), also known as hypervariable regions, found in both the light and heavy chain variable domains. There are at least two techniques for determining CDRs: (1) methods based on cross-species sequence variation [i.e., Kabat et al., Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. 1987)]; and (2) methods based on crystallographic studies of antigen-antibody complexes (Chothia, et al. (1989), Nature 342:877), or both Chothia and Kabat. The more highly conserved portions of variable domains are called framework regions (FRs). The variable domains of naturally occurring heavy and light chains each contain four FR regions that are primarily arranged in a β-sheet configuration, connected by three CDRs that form loops that connect, and in some cases form part of, the β-sheet structure. The CDRs within each chain are held together in close proximity by the FR regions and, with the CDRs of the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al.). The constant domains are not directly involved in binding the antibody to its cognate antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

[0028] The light chains of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly specific types, called kappa and lambda, based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the heavy chain constant domain, "immunoglobulins" can be assigned to different classes. There are at least five 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 configurations of the different classes of immunoglobulins are well known.

[0029] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which generally contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in the presently disclosed and claimed inventions can be made by the hybridoma method first described by Kohler and Milstein, Nature 256, 495 (1975), relevant portions of which are incorporated herein by reference.

[0030] All monoclonal antibodies used in accordance with the presently disclosed and claimed inventions will be either (1) the result of a deliberate immunization protocol, as described in more detail below; or (2) the result of an immune response that naturally leads to the production of antibodies during the course of disease or cancer.

[0031] Use of the monoclonal antibodies of the presently disclosed and claimed invention may require the administration of 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, administration of such antibodies to a human patient typically provokes an immune response, which is directed against the antibody itself. Such a response limits the duration and effectiveness of such therapy. To address this problem, the monoclonal antibodies of the presently disclosed and claimed invention can be "humanized," i.e., modified so that their antigenic portions are removed and analogous portions of human antibodies are replaced, thus preserving the antibody's affinity for EGFRvIII. This modification may involve only a few amino acids or may involve the entire framework region of the antibody, leaving only the complementarity-determining regions of the antibody intact. Several methods for humanizing antibodies are known to those skilled in the art and are disclosed in U.S. Pat. No. 6,180,370, issued to Queen et al. on January 30, 2001; U.S. Pat. No. 6,054,927, issued to Brickell on April 25, 2000; U.S. Pat. No. 5,869,619, issued to Studnicka on February 9, 1999; U.S. Pat. No. 5,861,155, issued to Lin on January 19, 1999; U.S. Pat. No. 5,712,120, issued to Rodriquez et al. on January 27, 1998; and U.S. Pat. No. 4,816,567, issued to Cabilly et al. on March 28, 1989, the relevant portions of which are incorporated herein by reference.

[0032] Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fab, Fab', F(ab')2, Fv, scFv, or other antigen-binding subsequences of antibodies) composed primarily of human immunoglobulin sequences, containing minimal sequences derived from non-human immunoglobulins. Humanization can be performed by substituting non-human (i.e., rodent, chicken) CDRs or CDR sequences for the corresponding sequences of a human antibody according to the method of Winter and coworkers (Jones et al., 1986; Riechmann et al., 1988; Verhoeyen et al., 1988); see, e.g., U.S. Patent No. 5,225,539. In some cases, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues from the donor antibody. Humanized antibodies can also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody will comprise substantially all, at least one, and usually two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin variable domain and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. Optimally, the humanized antibody will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0033] A fully human antibody essentially refers to an antibody molecule in which the entire sequences of both the light and heavy chains, including the CDRs, arise 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 trioma technology; human B-cell hybridoma technology [see Kozbor, et al., Hybridoma, 2:7 (1983)] and EBV hybridoma technology for producing human monoclonal antibodies [see Cole, et al., PNAS 82:859 (1985)], or by the techniques taught herein. Human monoclonal antibodies may be utilized in the practice of the presently disclosed and claimed invention, and may be produced by using human hybridomas [see Cote, et al., PNAS 80:2026 (1983)] or by transforming human B cells in vitro with Epstein-Barr virus (see Cole et al., 1985), relevant portions of which are incorporated herein by reference.

[0034] Additionally, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This technique is described, for example, but not by way of limitation, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016, as well as 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), relevant portions of which are incorporated herein by reference.

[0035] A method for producing a desired antibody, such as a human antibody, is disclosed in U.S. Patent No. 5,916,771, issued June 29, 1999 to Hori et al., which is incorporated herein by reference. The method comprises introducing into one mammalian host cell in culture an expression vector containing a nucleotide sequence encoding the heavy chain and into another mammalian host cell an expression vector containing a nucleotide sequence encoding the light chain, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing the heavy and light chains.

[0036] As used herein, the term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.

[0037] As used herein, the term "disorder" refers to any condition that would benefit from treatment with a polypeptide, including chronic and acute disorders or diseases, including infectious or pathological conditions that predispose a mammal to the disorder in question.

[0038] Antibodies or antibody fragments can be produced with altered sequences or glycosylation states to confer desired levels of activity in antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP) or antibody-dependent complement deposition (ADCD) function as measured by bead-based or cell-based assays or in vivo studies in animal models.

[0039] Alternatively, or in addition, it may be useful to combine an amino acid modification with one or more additional amino acid modifications that alter the complement component Clq binding and / or complement-dependent cytotoxicity (CDC) function of the Fc region of an IL-23p19-binding molecule. Binding polypeptides of particular interest may be those that bind Clq and exhibit complement-dependent cytotoxicity. Polypeptides with pre-existing Clq-binding activity, and optionally, the additional ability to mediate CDC, may be modified to enhance one or both of these activities. Amino acid modifications that alter Clq and / or modify its complement-dependent cytotoxicity function are described, for example, in WO / 0042072, incorporated herein by reference.

[0040] The Fc region of an antibody can be engineered to alter effector function, for example, by modifying Clq binding and / or FcγR binding, thereby altering complement-dependent cytotoxicity (CDC) and / or antibody-dependent cell-mediated cytotoxicity (ADCC) activity. These "effector functions" are involved in activating or attenuating 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; and down-regulation of cell surface receptors (e.g., B cell receptors; BCRs). Such effector functions may require the Fc region to be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays (e.g., Fc binding assays, ADCC assays, CDC assays, etc.).

[0041] For example, a variant Fc region of an antibody can be generated that has improved Clq binding and improved FcγRIII binding (e.g., both improved ADCC activity and improved CDC activity). Alternatively, if reduced or eliminated effector function is desired, the variant Fc region can be engineered to have reduced CDC activity and / or reduced ADCC activity. In other embodiments, only one of these activities may be increased, optionally with a decrease in the other activity (e.g., generating an Fc region variant that has improved ADCC activity but reduced CDC activity, and vice versa).

[0042] Single-chain variable fragments (scFvs) are fusions of the variable regions of immunoglobulin heavy and light chains, linked together by a short (usually serine or glycine) linker. These chimeric molecules retain the specificity of the original immunoglobulin despite the removal of the constant regions and the introduction of a linker peptide. This modification usually leaves specificity unaltered. These molecules were historically created to facilitate phage display, where it is very convenient to express the antigen-binding domain as a single peptide. Alternatively, scFvs can be generated directly from subcloned heavy and light chains derived from hybridomas or B cells. Single-chain variable fragments lack the constant Fc region found in intact antibody molecules and therefore lack the common binding sites (e.g., protein A / G) used to purify antibodies. These fragments can often be purified / immobilized using protein L, as protein L interacts with the variable region of the kappa light chain.

[0043] Flexible linkers are generally composed of amino acid residues that promote helices and turns, such as alanine, serine, and glycine. However, other residues may function as well. Using phage display, tailored linkers for single-chain antibodies (scFv) can be rapidly selected from protein linker libraries. A random linker library was constructed in which heavy and light chain variable domain genes were linked by segments encoding 18-amino acid polypeptides of various compositions. The scFv repertoire (approximately 5 × 10 6 The antibody fragments (different members of the species) were displayed on filamentous phage and subjected to affinity selection with haptens. The population of selected variants exhibited significantly increased binding activity while retaining considerable sequence diversity. Sequence analysis revealed a conserved proline in the linker two residues after the VH C-terminus and abundant arginine and proline at other positions as the only common features of the selected chains. In certain embodiments, the antibody fragments are further modified to increase their serum half-life by modifying the Fc region or using mutations to various constant regions, as known to those skilled in the art.

[0044] In certain embodiments, the antibodies of the invention are formulated for administration to humans. For example, the antibodies of the invention can be included in a pharmaceutical composition formulated for administration intranasally, intrapulmonary, intrabronchial, intravenous, oral, intraadipose, intraarterial, intra-articular, intracranial, intradermal, intralesional, intramuscular, intrapericardial, intraperitoneal, intrapleural, intravesical, topical, mucosal, parenteral, enteral, subcutaneous, sublingual, local, buccal, transdermal, by inhalation, by injection, in a cream, in a lipid composition, by catheter, by gastric lavage, by continuous infusion, by injection, by local delivery, or by regional perfusion, and the composition is a serum, drop, gel, ointment, spray, container, or atomizer.

[0045] As used herein, the term "antigen" refers to a molecule containing one or more epitopes (either linear, conformational, or both) that stimulate the host's immune system to elicit a humoral and / or cellular antigen-specific response. The antigen of the present invention is EGFRvIII, including the same MEM. The term is used interchangeably with the term "immunogen." Typically, a B-cell epitope will contain at least about 5 amino acids, but may be as small as 3-4 amino acids. A T-cell epitope, such as a CTL epitope, will contain at least about 7-9 amino acids, and a helper T-cell epitope will contain at least about 12-20 amino acids. Typically, an epitope will contain about 7-15 amino acids, such as 9, 10, 12, or 15 amino acids. The term encompasses polypeptides containing modifications, such as deletions, additions, and substitutions (generally conservative substitutions in nature), relative to the native sequence, so long as the protein retains its ability to elicit an immune response as defined herein. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the antigens.

[0046] As used herein, the term "epitope" refers to a particular amino acid sequence or molecule (e.g., carbohydrate, small molecule, lipid, etc.) that, when present in the appropriate form, provides a reactive site for an antibody (e.g., a B cell epitope) or, in the case of a peptide, a reactive site for a T cell receptor (e.g., a T cell epitope).

[0047] Portions of a given polypeptide containing B-cell epitopes can be identified using a number of epitope mapping techniques known to those skilled in the art (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed., 1996, Humana Press, Totowa, NJ). For example, linear epitopes can be determined by, for example, simultaneously synthesizing a large number of peptides corresponding to portions of a protein molecule on a solid support and reacting the peptides with an antibody while they are still bound to the support. Such techniques are known to those skilled in the art and are described, for example, in U.S. Pat. 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.

[0048] As used herein, the term "substantially purified" refers to the isolation of a substance (compound, polynucleotide, protein, polypeptide, polypeptide composition) such that the substance comprises the majority percentage of the sample in which it is present. Generally, a substantially purified component in a sample comprises 50%, preferably 80%-85%, and more preferably 90-95% of the sample. Techniques for purifying desired polynucleotides and polypeptides are well known to those of skill in the art and include, for example, ion exchange chromatography, affinity chromatography, and density-based precipitation.

[0049] The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, which are within the skill of the art. Such techniques are fully explained in the literature, e.g., Remington's Pharmaceutical Sciences, 18 thEdition (Easton, Pa.: Mack Publishing Company, 1990); Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.); and Handbook of Experimental Immunology, Vols. I-IV (DM Weir and CC Blackwell, eds., 1986, Blackwell Scientific Publications); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2 nd Edition, 1989);Short Protocols in Molecular Biology, 4 th ed. (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), 2 nd ed. (Newton & Graham eds., 1997, Springer Verlag); Fundamental Virology, Second Edition (Fields & Knipe eds., 1991, Raven Press, New York), relevant portions are incorporated herein by reference.

[0050] Conservative amino acid substitutions include 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 amino acids Ala, Ser, Thr, Met, and Gly. [Example]

[0051] Example 1. EGFRvIII MEM Mesoscale molecules (MEMs) of the present disclosure are engineered to mimic the agonist epitopes identified in Table 1 and are subsequently used to screen antibodies. An advantage of this approach is that antibody discovery can be directed away from the wild-type receptor toward desired epitopes found only in EGFRvIII. Scaffold amino acids are in bold and epitope residues are underlined.

[0052] [Table 1]

[0053] In some embodiments, MEM-nanoparticles are used to immunize subjects, thereby producing antibodies specific to MEM epitopes. Monoclonal hybridomas are then created to produce epitope-specific anti-EGFRvIII antibodies. The CDRs of humanized anti-EGFRvIII are determined based on the reference antibody.

[0054] Example 2. Discovery of anti-EGFRvIII antibodies based on modified MEM-nanoparticle immunization MEMs were designed based on the epitopes identified by SEQ ID NOS: 47, 48, and 49 and then conjugated to nanoparticles to target B cell antibody production to the epitopes. The MEMs conjugated to ferritin nanoparticles were identified by Coomassie-based Western blot and found to contain approximately 20-30 MEMs per nanoparticle. The MEM-nanoparticles demonstrated nanomolar binding affinity for anti-EGFRvIII using surface plasmon resonance (SPR). BALB / c mice were then immunized over a 5-week period with alternating doses of modified MEM-nanoparticles and / or full-length anti-EGFRvIII suspended in adjuvant, with a final boost containing a combination of the two. Mouse sera were collected and demonstrated potent anti-EGFRvIII binding as measured by ELISA.

[0055] The best monoclonal hybridoma-produced antibodies exhibit potent anti-EGFRvIII binding and agonism. Hybridomas were generated from immunized mouse B cells using standard electrofusion techniques. The resulting antibodies and several others were generated from monoclonal hybridomas and demonstrated strong anti-EGFRvIII binding by ELISA. Binding was further evaluated in vitro for anti-EGFRvIII binding and competitive binding to anti-EGFRvIII using SPR.

[0056] Antibody expression and purification Antibody expression plasmids were transiently introduced into animal cell lines using the ExpiFectamine CHO Transfection Kit (Thermo Fisher Scientific, catalog number A29129) to generate transformants producing anti-CCR8 chimeric or humanized antibodies. ExpiCHO-S (Thermo Fisher Scientific, catalog number A29127) or a CHO suspension cell line with a knockout of the α1,6 fucosyltransferase (FUT8) gene (referred to as "WT CHO" and "FUT8 CHO" in other references) was used as the host cell line. After 6-12 days of growth after DNA transfection, the WT CHO or FUT8 CHO cell suspension was harvested by centrifugation at 4,000 × 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 no. GE11-0034-93). WT CHO was used to express antibodies with standard glycosylation, and FUT8 CHO was used to express defucosylated antibodies with enhanced effector function (indicated by "-afuc").

[0057] Antibody Humanization Humanization was achieved by multiple approaches. In some cases, rationally selected framework amino acids that differed between the chimera (SD-233883) and the closest human germline were converted to match the human sequence. In other cases, CDRs were directly grafted into the human germline using publicly available tools (DOI: 10.1080 / 19420862.2021.2020203). In all cases, the CDRs were left unchanged. The humanized variants were then tested, and the potency was compared to the parent chimera.

[0058] Preparation of phage display libraries A CDR variant library was prepared based on the parent antibody. The VH and VL sequences were assembled using the Golden gate assembly method and ligated into a digested phagemid vector for phage display as ScFv. The ligation was transformed into Phage-Competent™ TG1 Cells (Antibody design labs, catalog number PC001), and library quality was determined by size and VH / VL insert percentage.

[0059] Phage display screening Selection of the synthetic library phage display was performed using soluble protein antigen. Selection was performed using biotinylated huEGFRvIII with Dynabeads M-280 Streptavidin beads Magnetic Beads (Invitrogen; catalog no. 11205D) on a KingFisher Apex. Antigen was used at various concentrations. Elution was performed with TEA (triethylamine) (Sigma; catalog no. T0886), and the selection buffer was skim milk in 1x PBS. After three rounds of panning, plasmids were extracted, and the VH / VL genes were amplified for analysis by Sanger sequencing. The desired VH / VL sequences were cloned into an IgG1 expression plasmid and subsequently transformed into DH5α. Each plasmid was extracted from DH5α and subsequently transfected into CHO cells in a 24-deep well plate using the ExpiFectamine CHO Transfection Kit for expression. Purification was performed using a KingFisher Apex. The matured phage clones were then screened using SPR.

[0060] Example 3. Kinetic analysis of anti-EGFRvIII antibodies by surface plasmon resonance. Kinetic analysis of anti-EGFRvIII antibodies by surface plasmon resonance. Antibody binding to human EGFR and EGFRvIII was assessed by surface plasmon resonance (SPR) using a Carterra LSA (Carterra). An anti-human IgG capture lawn was first prepared on an HC30M chip (Carterra, Cat. No. 4279) by primary amine coupling. Briefly, the chip surface was activated with a mixture of 133 mM EDC (Thermo Fisher, Cat. No. 22980) and 33.3 mM sulfo-NHS (Thermo Fisher, Cat. No. 24525) in 100 mM MES pH 5.5 (Carterra, Cat. No. 3625) for 10 minutes, followed by coupling of goat anti-human IgG (Southern Biotech, Cat. No. 2040-01) at 50 μg / mL in 10 mM sodium acetate buffer pH 4.5 (Carterra, Cat. No. 3622) for 15 minutes. Unconjugated sites on the chip surface were blocked with 1 M ethanolamine HCl pH 8.5 (Carterra, Cat. No. 3626) for 7 minutes. For capture kinetics, the prepared anti-human IgG surface and a 96-channel printhead (96PH) were used to capture a panel of antibodies at 1–10 μg / mL in HBSTE buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20; Carterra, Cat. No. 3630) for 10 minutes. Purified recombinant antigens (human EGFR, Acro Biosystems, Cat. No. EGR-H5222; human EGFRvIII, Cat. No. EGI-H52H4) were then injected onto the antibody panel at five concentrations in a 5-fold dilution series starting at 500 nM using a single flow cell (SFC). Each injection involved a 5-minute association phase and a 15-minute dissociation phase. The surface was regenerated between antigens with 0.425% H3PO4 (Carterra, Cat. No. 3637). The running buffer for antigen injection was HBSTE supplemented with 0.5 mg / ml BSA (VWR, Cat. No. 97061-422). Binding data were double-referenced by subtracting the interspot reference response and the buffer-only blank response.The resulting sensorgrams were globally fit to a one-to-one Langmuir binding model to estimate the association rate constant (ka), dissociation rate constant (kd), and dissociation constant (KD) using Carterra Kinetics software.

[0061] Figures 1A-1C compare the binding of various antibodies to EGFRvIII and EGFR1 by surface plasmon resonance (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).

[0062] [Table 2] JPEG2025533189000004.jpg128170

[0063] Example 4. EGFRvIII and EGFR1 binding ELISA Binding ELISA ELISA plates (Biolegend, catalog no. 423501) were first coated with either 1 μg / mL human EGFRvIII (ACRO Biosciences, catalog no. EGI-H52H4) or EGFR1 protein (ACRO Biosciences, catalog no. EGR-H5222) in 50 mM carbonate buffer pH 9.5 (Teknova, catalog no. S9225) overnight at 4° C. The next day, plates were washed three times with wash buffer [1× PBS containing 0.1% Tween-20 (Teknova, catalog no. P0207)], followed by addition of blocking buffer [1× PBS, 1% BSA (Teknova, catalog no. B0101)] at room temperature for 1 hour. After blocking, the plate was washed three times with wash buffer, followed by the addition of increasing concentrations (0.004–66.66 nM) of anti-EGFRvIII or control antibody in wash buffer at room temperature for 1 hour. After incubation, the plate was washed three times with wash buffer, followed by the addition of a goat anti-human IgG-HRP secondary antibody (Biorad, catalog no. STAR126P) diluted 1:2500 in wash buffer at room temperature for 1 hour. After the secondary antibody incubation, the plate was washed six times with wash buffer, followed by the addition of TMB substrate (VWR, catalog no. 95059-154) at room temperature for 5 minutes. After the TMB substrate incubation, ELISA stop solution (Thermo Fisher, catalog no. SS04) was added to the plate in an equal volume to the TMB substrate, and absorbance was read at 450 nm. Data were plotted using GraphPad Prism 9.3.0 software, and EC 50 The values ​​were calculated by the software.

[0064] Figures 2A-2D compare binding to human EGFRvIII and EGFR1 by a hIgG1 isotype control antibody, cetuximab, and chimeric and humanized antibodies of the invention by ELISA.

[0065] Chimeric and humanized anti-EGFRvIII antibodies specifically bind to truncated human EGFRvIII but not to full-length human EGFR1. ELISA assays showed that defucosylated anti-EGFRvIII antibodies and cetuximab bind to immobilized human EGFRvIII in a concentration-dependent manner. Plotted values ​​are absorbance measured at a wavelength of 450 nm. EC 50 Values ​​are the mean of n=3 experiments. n / a, no activity.

[0066] [Table 3]

[0067] Example 5. F98 EGFRvIII and EGFR1 cell binding assay F98npEGFRvIII (ATCC, Catalog No. CRL-2949) and F98 EGFR1 (ATCC, Catalog No. CRL-2948) cells were cultured in DMEM (Corning, Catalog No. 10-013-CV) supplemented with 10% FBS (ATCC, Catalog No. 30-2020), 1x penicillin-streptomycin (Corning, Catalog No. 30-002-CI), and 0.2 mg / ml G418 (Thermo Fisher, Catalog No. 10131035). U87MG (ATCC, Catalog No. HTB-14) and U87MG-EGFRvIII (Genscript) cells were cultured in DMEM supplemented with 10% FBS, 1x penicillin-streptomycin, and 0.5 μg / mL puromycin (for U87MG-EGFRvIII cells only, Gibco, Catalog No. A11138-03). FaDu (ATCC, Catalog No. HTB-43) and FaDu-EGFRvIII (Genscript) cells were cultured in EMEM (ATCC, Catalog No. 30-2003) supplemented with 10% FBS, 1x penicillin-streptomycin, and 2 μg / mL puromycin (for FaDu-EGFRvIII cells only).

[0068] For cell binding assays, PBS (Corning, Cat. No. 21-040-CV) supplemented with 2% FBS and 2 mM EDTA was used as the assay buffer. Target cells were counted and then resuspended in their respective culture medium at a concentration of 1 × 10 5 Cells were then seeded onto a 96-well plate (VWR, catalog no. 89089-826) at 100 cells / well and incubated on ice for 3 hours. After incubation, the plate was centrifuged, the supernatant removed, and the cells were then washed once with assay buffer, followed by centrifugation and washing. After washing, the indicated antibodies were diluted in assay buffer and added to the cells at increasing concentrations (0.004-66.66 nM) on ice for 20 minutes. After incubation, the cells were washed with assay buffer and then washed. Next, rat-anti-human IgG Fc Alexa Fluor 647 (BioLegend, catalog no. 410714) was diluted 1:200 in assay buffer and added to the cells on ice for 20 minutes. After incubation, the cells were washed once more with assay buffer and then washed. DAPI (BioLegend, Cat. No. 422801) was then diluted 1:5000 in assay buffer and added to the cells. Cell binding was analyzed using a Miltenyi MACSQuant 16 flow cytometer. Flow cytometry data were analyzed using FlowJo flow cytometry analysis software. Graphs were generated and EC 50 GraphPad Prism 9.3.0 was used to calculate the values.

[0069] Figures 3A-3F compare binding by chimeric and humanized anti-EGFRvIII antibodies of the present invention. The listed antibodies specifically bind to human EGFRvIII but not wild-type human EGFR1. FACS analysis shows anti-EGFRvIII antibodies specifically binding to F98 rat glioblastoma (Figure 3A), U87MG human glioblastoma (Figure 3C), and FaDu human head and neck cancer cells (Figure 3E), which overexpress human EGFRvIII. No binding was detected to F98 cells overexpressing wild-type human EGFR1 (Figure 3B) or wild-type U87MG (Figure 3D) and FaDu (Figure 3F) cells. Plotted values ​​are median fluorescence intensity. EC 50 Values ​​are the mean of n=3 experiments. n / a, no activity.

[0070] [Table 4]

[0071] [Table 5]

[0072] [Table 6]

[0073] Example 7. In vitro ADCC assay F98npEGFRvIII (ATCC, Catalog No. CRL-2949) and F98 EGFR1 (ATCC, Catalog No. CRL-2948) cells were cultured in DMEM (Corning, Catalog No. 10-013-CV) supplemented with 10% FBS (ATCC, Catalog No. 30-2020), 1x penicillin-streptomycin (Corning, Catalog No. 30-002-CI), and 0.2 mg / ml G418 (Thermo Fisher, Catalog No. 10131035). U87MG (ATCC, Catalog No. HTB-14) and U87MG-EGFRvIII (Genscript) cells were cultured in DMEM supplemented with 10% FBS, 1x penicillin-streptomycin, and 0.5 μg / mL puromycin (for U87MG-EGFRvIII cells only, Gibco, Catalog No. A11138-03). FaDu (ATCC, Catalog No. HTB-43) and FaDu-EGFRvIII (Genscript) cells were cultured in EMEM (ATCC, Catalog No. 30-2003) supplemented with 10% FBS, 1x penicillin-streptomycin, and 2 μg / mL puromycin (for FaDu-EGFRvIII cells only).

[0074] For ADCC assessment, PBMC frozen stocks purchased from STEMCELL (Cat. No. 70025) were thawed in RPMI medium supplemented with 10% FBS, 1x penicillin-streptomycin (Corning, Cat. No. 30-002-CI), 5 ng / ml IL2 (Miltenyi Biotec, Cat. No. 130-097-743) and incubated overnight in a tissue culture incubator.

[0075] Target cells were counted and cell viability was assessed. Cells were first stained with CFSE dye (Thermo Fisher, Cat. No. C34554) for 10 minutes at room temperature and then washed once with growth medium. The washed cells were then diluted to 1 × 10 6Resuspend in growth medium at a density of 1 x 10 cells / mL and then plate 1 x 10 cells per well onto a 96-well plate (Fisher Scientific, Cat. No. 07-200-89). 4 Cells were seeded and incubated overnight in a tissue culture incubator. After cell incubation, 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 assay medium were added to the cells at increasing concentrations (0.0004-6.66 nM for FaDu and U87 cells, 0.0002-3.33 nM for F98 cells) at 37°C and 5% CO2 for 10 minutes. Then, 2 x 10 peripheral blood mononuclear cells (PBMCs) were added to the assay plates. 5 100 mg of CFSE+ was added per well of a 96-well plate. Cells and antibodies were incubated for 24 hours at 37°C in a 5% CO2 incubator. Samples were stained with the LIVE / DEAD™ Fixable Aqua Dead Cell Stain Kit (Thermo Fisher, Cat. No. L34957) and analyzed using a MACSQuant 16 flow cytometer. Live cells were gated (aqua-, CFSE+) and dead cells were gated (aqua+, CFSE+). The ratio of dead cells to total target cells was used to determine the percentage of cell lysis. Flow cytometry data were analyzed with FlowJo flow cytometry analysis software. Graphs were generated and EC 50 GraphPad Prism 9.3.0 was used to calculate the values.

[0076] Figures 4A-4F show that chimeric and humanized anti-EGFRvIII antibodies of the present invention exhibit potent ADCC activity against F98 (Figure 4A), U87MG (Figure 4C), and FaDu cells (Figure 4E) overexpressing human EGFRvIII, but not against F98 (Figure 4B), wild-type U87MG (Figure 4D), and FaDu (Figure 4F) cells expressing human EGFR1. The ratio of dead cells to total cells was used to determine the percentage of cell lysis. EC 50 Values ​​are the average of n = 1 to 5 experiments. n / a, no activity.

[0077] [Table 7]

[0078] [Table 8]

[0079] [Table 9]

[0080] In vivo efficacy assay Nine-week-old female nude mice (Charles River Laboratories, Cat. No. 088Nu / Nu) were used in this assay. 2 × 10 cells were cultured in 100 μl of a mixture of PBS and MatriGel (Corning, Cat. No. 354234) (v:v = 1:1). 6 FaDu-EGFRvIII cells were inoculated into the upper left side of each nude mouse by subcutaneous injection. Tumor growth and mouse body weight were monitored twice weekly. For each individual tumor, the longest longitudinal diameter (length) and widest transverse diameter (width) were measured using a Traceable Digital Caliper (VWR, Cat. No. 62379-531). Tumor volume (TV) was calculated using the formula TV = [length × (width)]. 2 The mean tumor volume was calculated as follows: 152-153 mm 3Once the mice reached 100 mg / day, they were randomized, with each group containing 10 mice. The hIgG1 negative control, cetuximab, and SD-233883-afuc were each prepared in a 3 mg / ml stock solution in PBS. The volume of antibody administered to each mouse was calculated using the formula: volume (μl) = mouse body weight (g) × 10 μl / g. The antibody drug was administered intravenously twice a week for a total of seven doses (biw × 7). After the first drug treatment, the percentage change in each tumor was calculated using the formula: TV change % = [(TV - TVday0) / TVday0] × 100.

[0081] Figures 5A-5E show the following: Figure 5A shows the study design for the in vivo efficacy of the anti-EGFRvIII antibody SD-233883-afuc against FaDu EGFRvIII tumor cells in a nude mouse model. Figure 5B (FaDu-EGFRvIII tumor volume after first drug treatment) and Figure 5C (% change in FaDu-EGFRvIII tumor volume after first drug treatment) show that SD-233883-afuc significantly inhibited FaDu-EGFRvIII tumor growth throughout the observation time window compared to the hIgG1 negative control (t-test, P less than 0.05). Figure 5D shows that at the study endpoint (day 22), FaDu-EGFRvIII tumor weight was significantly reduced after cetuximab or SD-233883-afuc treatment compared to the hIgG1 negative control (t-test, P less than 0.05). Figure 5E shows mouse body weight after the first drug treatment.

[0082] Antibodies that bind to truncated EGFRvIII are provided herein. These antibodies are referred to herein as anti-EGFRvIII antibodies. A number of discovery strategies were utilized to obtain exemplary antibodies of the present disclosure, which are discussed further below.

[0083] Those skilled in the art will appreciate that antibodies that exhibit little or no binding to a target antigen have a low affinity for the target antigen and a low equilibrium dissociation constant (K DThose skilled in the art will recognize that an antibody that exhibits little or no binding to a collective assembly of target antigenic epitopes may be described as having a low affinity for the collective assembly of target antigenic epitopes and a high equilibrium dissociation constant (K D ) may be described as being high.

[0084] In some embodiments, the binding affinity (K) for EGFRvIII is about 5 μM to about 5 pM, about 1 μM to about 5 pM, about 0.5 μM to about 5 pM, about 0.1 μM to about 5 pM, about 50 nM to about 5 pM, about 10 nM to about 5 pM, about 5 nM to about 5 pM, about 1 nM to about 5 pM, about 0.5 nM to about 5 pM, about 0.1 nM to about 5 pM, about 50 pM to about 5 pM, or about 10 pM to about 5 pM. D ) is provided herein.

[0085] In some embodiments, the anti-EGFRvIII antibody has a binding affinity (EC ) for EGFRvIII of 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, or about 0.5 pM to about 0.1 pM. 50 )

[0086] In some embodiments, the anti-EGFRvIII antibody has a half-maximum effective concentration (EC ) of 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, or about 0.005 nM to about 0.001 nM against EGFRvIII. 50 )

[0087] In some embodiments, the anti-EGFRvIII antibody is a full-length antibody (referring to an antibody having two heavy chains and two light chains joined together in an Fc domain, forming a "Y" configuration). In some embodiments, the Fc domain (or simply referred to as Fc) is a human Fc domain. In some embodiments, the Fc domain of a humanized antibody is derived from human IgG1, human IgG2, human IgG3, or human IgG4.

[0088] Example 8. Exemplary Anti-EGFRvIII Antibodies—CDR Sequences The sequences of exemplary anti-EGFRvIII antibodies of the present disclosure are provided herein, including the complementarity determining region (CDR) sequences and variable heavy and light domain (VH, VL) sequences that constitute the EGFRvIII antigen-binding domain of the present disclosure. The discovery of these antibodies is described in detail in the Examples section.

[0089] As referred to below, the light chain variable (VL) domain CDR1 region is referred to as CDR-L1; the VL CDR2 region is referred to as CDR-L2; the VL CDR3 region is referred to as CDR-L3; the heavy chain variable (VH) domain CDR1 region is referred to as CDR-H1; the VH CDR2 region is referred to as CDR-H2; and the VH CDR3 region is referred to as CDR-H3. Table 10 provides exemplary CDR combinations for antibodies of the disclosure.

[0090] [Table 10]

[0091] [Table 11] JPEG2025533189000014.jpg251170 JPEG2025533189000015.jpg251170JPEG2025533189000016.jpg251170 JPEG2025533189000017.jpg246170JPEG2025533189000018.jpg251170JPEG2025533189000019.jpg251170 JPEG2025533189000020.jpg246170JPEG2025533189000021.jpg251170JPEG2025533189000022.jpg251170 JPEG2025533189000023.jpg251170 JPEG2025533189000024.jpg35170

[0092] In some embodiments, provided herein is an anti-EGFRvIII antibody, wherein the antibody comprises the amino acid sequences of three VH CDRs, which are SEQ ID NOs: 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.

[0093] In some embodiments, provided herein are anti-EGFRvIII antibodies, wherein the antibodies comprise the amino acid sequences of the three VL CDRs are 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.

[0094] In some embodiments, provided herein is an anti-EGFRvIII antibody, wherein the antibody comprises an amino acid sequence of a VH comprising the amino acid sequence of any one of SEQ ID NOs: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121.

[0095] In some embodiments, provided herein is an anti-EGFRvIII antibody, wherein the antibody comprises an amino acid sequence of a VL comprising the amino acid sequence of any one of SEQ ID NOs: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, or 122.

[0096] In some embodiments, provided herein are anti-EGFRvIII antibodies, wherein the antibodies comprise paired heavy and light chain amino acid sequences of SEQ ID NOs: 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, and 121 and 122, respectively.

[0097] In some embodiments, anti-EGFRvIII antibodies are provided, wherein the antibody heavy and light chains are encoded by nucleic acids having at least 80%, 85%, 90%, 95, 96, 97, 98, 99%, or 100% sequence identity to SEQ ID NOs: 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.

[0098] Example 9. scFv-Fc anti-EGFRvIII In some embodiments, the present disclosure provides tandem scFv antibodies with multiple anti-EGFRvIII binding sites. The tandem scFv-Fc antibodies of the present disclosure are comprised of two or more scFv binding sites in tandem on each antibody arm, which may be linked by a linker or may be linked by a flexible linker. In some embodiments, the tandem scFv antibodies have a total of four or five or more scFv binding sites within a single scFv-Fc format antibody.

[0099] The VH1 and VL1 of each scFV1 may be connected by a linker, for example a flexible linker.

[0100] The VH2 and VL2 of each scFV2 ​​may be connected by a linker, for example a flexible linker.

[0101] The scFvs on each antibody arm may be connected by a linker, e.g., a flexible linker. An exemplary linker comprises the amino acid sequence: GGGSGGGGSGGGGS (SEQ ID NO: 131).

[0102] Example 10. Therapeutic anti-EGFRvIII antibodies In some embodiments, the anti-EGFRvIII antibodies provided herein are useful for treating EGFRvIII-expressing cancers, such as glioblastoma, head and neck squamous cell carcinoma, non-small cell lung cancer-squamous cell carcinoma (NSCLC-SCC), prostate cancer, breast cancer, and colorectal cancer.

[0103] Example 11. Administration of therapeutic anti-EGFRvIII antibodies In vivo administration of the therapeutic anti-EGFRvIII antibodies described herein may be performed intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, intrathecally, intraventricularly, intranasally, transmucosally, by implantation, or by inhalation. Intravenous administration may be performed by injection or infusion. In some embodiments, the anti-EGFRvIII antibodies of the present disclosure are administered intravenously. In some embodiments, the anti-EGFRvIII antibodies of the present disclosure are administered subcutaneously. Administration of the therapeutic anti-EGFRvIII antibodies can be performed with any suitable excipient, carrier, or other agent to provide suitable or improved tolerance, transfer, delivery, etc.

[0104] It is contemplated that any embodiment discussed herein can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, the compositions of the invention can be used to achieve the methods of the invention.

[0105] It will be understood that the specific embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this 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 no more than routine experimentation, many equivalents to the specific procedures described herein. Such equivalent procedures are considered to be within the scope of the invention and are covered by the claims.

[0106] All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0107] The use of the word "a" or "an," when used in conjunction with the term "comprising" in the claims and / or specification, can mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more than one." The use of the term "or" in the claims is used to mean "and / or," unless expressly stated to refer to alternatives only or that the alternatives are mutually exclusive. However, this disclosure supports definitions and "and / or" that refer to alternatives only. Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error that exists among devices, the method utilized to determine the value, or the study subject.

[0108] As used in this specification and claims, the words "comprising" (and all forms of "comprising", such as "comprise" and "comprises"), "having" (and all forms of "having", such as "have" and "has"), "including" (and all forms of "including", such as "includes" and "include"), or "containing" (and all forms of "containing", such as "contains" and "contain") are all inclusive or open-ended and do not exclude additional, unrecited elements or method steps. In any of the embodiments of the compositions and methods provided herein, "comprising" can be replaced with "consisting essentially of" or "consisting of." As used herein, the phrase "consisting essentially of" requires the specified integers or steps and those that do not have a material effect on the nature or function of the claimed invention. As used herein, the term "consisting of" is used to indicate that only the enumerated integer (e.g., feature, element, attribute, property, method / method step, or limitation) or group of integers (e.g., feature, element, attribute, property, method / method step, or limitation) is present.

[0109] 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 A, B, C, AB, AC, BC, or ABC, and, where order is important in the particular context, BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Following this example, combinations containing one or more repeats of an item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, etc., are expressly included. Those skilled in the art will understand that generally, there is no limitation on the number of items or terms in any combination unless otherwise clear from the context.

[0110] As used herein, without limitation, approximation words such as "about," "substantially," or "substantially," when so modified, are understood to be not necessarily absolute or complete, but refer to a state that would be considered close enough to a person skilled in the art to justify specifying the state as it exists. The extent to which deviations from this specification may occur will depend on how significant a change can be made and still enable a person skilled in the art to recognize that the modified feature still possesses the required properties and capabilities of the unmodified feature. Generally, however, in light of the preceding considerations, numerical values ​​herein modified by approximation words such as "about" may vary from the stated value by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12, or 15%.

[0111] Furthermore, the headings herein are provided to provide organizational guidance otherwise consistent with the suggestions of 37 CFR 1.77. These headings are not intended to limit or characterize the invention(s) set forth in any claims that may arise from this disclosure. In particular and by way of example, headings may refer to "Field of the Invention," but such claims should not be limited by the language of these headings to describe the so-called technical field. Furthermore, the description of a technology in the "Background" section should not be construed as an admission that that technology is prior art to any invention(s) in this disclosure. Neither should the "Summary of the Invention" be deemed a characterization of the invention(s) set forth in the issued claims. Furthermore, any reference to the singular "invention" in this disclosure should not be used to assert that there is only one novelty in this disclosure. Multiple inventions may be set forth pursuant to the limitations of the multiple claims issuing from this disclosure, and such claims accordingly define the present invention and any equivalents thereof protected thereby. In all instances, such claims should be considered on their own merits in light of this disclosure, but should not be bound by the headings set forth herein.

[0112] For each claim, each dependent claim may depend on both the independent claim and each preceding dependent claim, so long as the preceding claim provides appropriate antecedent for the claim term or element.

[0113] In order to assist the Patent Office, and any reader of any patent that may issue on this application, in interpreting the claims attached hereto, applicants are advised that they do not intend to invoke paragraph 6 of 35 U.S.C. § 112, paragraph (f) of 35 U.S.C. § 112, or the equivalent, as they existed on the filing date of this document, in any of the appended claims, unless the words "means" or "step" are expressly used in a particular claim.

[0114] All of the compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. 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 variations can be applied to the compositions and / or methods and to the steps or sequence of steps of the methods described herein without departing from the concept, spirit 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 concept of the invention as defined by the appended claims.

Claims

1. An anti-epidermal growth factor receptor version III (EGFRvIII) antibody, or an antigen-binding domain thereof, comprising: a. SEQ ID NOs: 3, 4, 5; 13, 14, 15; 23, 24, 25; 33, 34, 35; 43, 44, 45; ５３、５４、５５； ６３、６４、６５； ７３、７４、７５； a heavy chain variable domain (VH) complementarity determining region (CDR) 1, VH CDR2, and VH CDR3 each comprising any one of the amino acid sequences: 83, 84, 85; 93, 94, 95; 103, 104, 105; 113, 114, 115; or 123, 124, 125; and b. SEQ ID NOs: 6, 7, 8; 16, 17, 18; 26, 27, 28; 36, 37, 38; 46, 47, 48; ５６、５７、５８； ６６、６７、６８； ７６、７７、７８； a light chain variable domain (VL) CDR1, VL CDR2, and VL CDR3 each comprising any one of the amino acid sequences: 86, 87, 88; 96, 97, 98; 106, 107, 108; 116, 117, 118; or 126, 127, 128; The antibody or binding domain comprising:

2. 2. The antibody or binding domain of claim 1, wherein the antibody comprises a VH comprising the amino acid sequence of any one of SEQ ID NOs: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, or 121.

3. 2. The antibody or binding domain of claim 1, wherein the antibody comprises a VL comprising the amino acid sequence of any one of SEQ ID NOs: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, or 122.

4. An 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 of any one of claims 1 to 4, wherein the antibody is a full-length antibody.

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

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

8. 2. The antibody or binding domain of claim 1, wherein the antibody heavy chain 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 same.

9. 2. The antibody or binding domain of 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 to SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, or 122.

10. 2. The antibody or binding domain of claim 1, wherein the antibody comprises heavy chain variable domains and light chain variable domains of SEQ ID NOs: 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, respectively.

11. 2. The antibody or binding domain of claim 1, wherein the antibody heavy chain is encoded by a nucleic acid and the antibody light chain is encoded by a nucleic acid, each nucleic acid having at least 80%, 85%, 90%, 95, 96, 97, 98, 99%, or 100% sequence identity to SEQ ID NOs: 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 of claim 1 , wherein the antibody or binding domain is defucosylated.

13. 10. The antibody or binding domain of claim 1, wherein the antibody or binding domain is produced in a bacterial cell, a fungal cell, a mammalian cell, an insect cell, or a plant cell.

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

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

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

17. 16. The method of claim 15, wherein 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 colorectal cancer, and the cancer expresses EGFRvIII.

18. 17. The method of claim 16, wherein cancer cells in the cancer are killed by antibody-dependent cellular cytotoxicity (ADCC).

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

20. A polynucleotide comprising a nucleic acid sequence having at least 80%, 85%, 90%, 95, 96, 97, 98, 99%, or 100% sequence identity to SEQ ID NOs: 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. 15. A method for producing an anti-EGFRvIII antibody, the method comprising the step of expressing in a cell a nucleic acid encoding the antibody of any one of claims 1 to 14.

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