Humanized 40H3 antibody

Abstract

Antibodies that specifically bind to epidermal growth factor receptor variant III (EGFRvIII) and / or gene-amplified EGFR (e.g., humanized monoclonal antibodies and antigen-binding fragments), as well as conjugates and chimeric antigen receptors (CARs) containing such antibodies, are disclosed herein. Nucleic acid molecules encoding the antibodies, conjugates, or CARs disclosed herein, and host cells expressing the nucleic acid molecules are also provided. Methods for treating or detecting tumors, such as tumors expressing EGFRvIII and / or gene-amplified EGFR, using the disclosed compositions are further disclosed.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims prior art under U.S. Provisional Application No. 63 / 525,493, filed on 7 July 2023, which is incorporated in its entirety by reference. Field of Invention

[0002] This application relates to the field of cancer biology, specifically to humanized monoclonal antibodies and antigen-binding fragments (e.g., EGFRvIII and / or gene-amplified EGFR) that specifically bind to epidermal growth factor receptor variants / variants expressed by tumor cells.

[0003] Government support description This invention was developed with government support under project number Z01#:Z1A BC 008757 by the National Institutes of Health and the National Cancer Institute. The U.S. Government has certain rights to this invention.

[0004] See sequence list The sequence listing was submitted as an XML file named "Sequence.xml" (1,386,226 bytes) created on July 8, 2024, and is incorporated herein by reference. [Background technology]

[0005] background EGFR is frequently involved in the oncogenic progression of human cancer. Various changes in expression, including gene amplification and activating mutations, contribute to carcinogenesis. The human form of this large receptor has a 621-amino acid external domain (ECD), a 23-amino acid single-pass transmembrane domain (TM), and a 542-amino acid enzymatically active intracellular domain (ICD). EGFR is a member of the receptor tyrosine kinase family and was the first receptor shown to be positively associated with human cancer. Ligand binding results in receptor dimerization and activation of the kinase domain, which signals to one of several pathways that can promote the proliferation, survival, and expansion of mammalian cells. Activating mutations can occur in either the ECD or the ICD. Gene amplification and deletion can also activate EGFR. Exemplary deletions include the loss of exons 2-7, which produces EGFR variant III (EGFRvIII), and the loss of exon 19 generates a constitutively active enzyme variant. Gene amplification of EGFR, expression of EGFRvIII, or loss of EGFR exon 19 have only been reported in cancer cells.

[0006] Antibodies generally bind strongly and specifically to their target antigens, thereby becoming useful therapeutic agents for treating a wide range of diseases characterized by altered protein expression, such as EGFRvIII or gene-amplified EGFR expression. However, in many therapeutic applications, the efficacy and safety of non-human antibodies are compromised because non-human immunoglobulin (Ig) molecules themselves are immunogenic and can therefore induce an anti-Ig immune response, severely impairing repeated doses. Therefore, modifying antibodies to reduce or eliminate immunogenicity may be beneficial for therapeutic use. Humanized antibodies capable of binding to EGFRvIII or gene-amplified EGFR, rather than wild-type EGFR, are still needed to exhibit cancer cell specificity. [Overview of the Initiative] [Means for solving the problem]

[0007] Summary of Disclosure Disclosed are isolated humanized monoclonal antibodies and their antigen-binding fragments, the monoclonal antibodies specifically binding to EGFRvIII and / or gene-amplified EGFR. In some embodiments, the monoclonal antibody or antigen-binding fragment is a) Sequence ID 5 and Sequence ID 8 (A10, VH3 + VL3), respectively; b) Sequence ID 3 and Sequence ID 6 (A2, VH1 + VL1), respectively; c) Sequence ID 3 and Sequence ID 7 (A3, VH1+VL2), respectively; d) Sequence ID 3 and Sequence ID 8 (A4, VH1 + VL3), respectively; e) Sequence ID 4 and Sequence ID 6 (A5, VH2 + VL1), respectively; f) Sequence ID 4 and Sequence ID 7 (A6, VH2 + VL2), respectively; g) Sequence ID 4 and Sequence ID 8 (A7, VH2 + VL3), respectively; h) Sequence ID 5 and Sequence ID 6 (A8, VH3 + VL1), respectively; i) Sequence ID 5 and Sequence ID 7 (A9, VH3 + VL2), respectively; j) Sequence ID 1 and Sequence ID 9 (B1, 40H3 VH+VL-EG), respectively; k) Sequence ID 1 and Sequence ID 10 (B2, 40H3 VH+VL-DA), respectively; l) Sequence ID 4 and Sequence ID 11 (B3, VH2 + VL1 - DA), respectively; m) Sequence ID 4 and Sequence ID 12 (B4, VH2 + VL2 - DA), respectively; n) Sequence ID 5 and Sequence ID 11 (B5, VH3, and VL1-DA), respectively; o) Sequence ID 31 and Sequence ID 32 (C10), respectively; p) Sequence ID 5 and Sequence ID 14 (D2), respectively; or q) Sequence ID 5 and Sequence ID 24 (D3), respectively. The heavy chain variable region (V H ) and light chain variable region (V L ) includes.

[0008] Conjugates of these antibodies and antigen-binding fragments are also disclosed. Chimeric antigen receptors (CARs) containing the disclosed antibodies or antigen-binding fragments, and cells expressing such chimeric antigen receptors (e.g., CAR T cells) (e.g., immune cells) are further disclosed.

[0009] Any of the antibodies disclosed herein H and / or V L Nucleic acid molecules encoding these nucleic acids, vectors containing these nucleic acids, and host cells transformed with these nucleic acids and / or vectors are also disclosed.

[0010] In further embodiments, the use of monoclonal antibodies or antigen-binding fragments disclosed herein for inhibiting tumors expressing EGFRvIII or gene-amplified EGFR in a subject is disclosed. In some embodiments, a subject having a tumor or cancer expressing EGFRvIII or gene-amplified EGFR is selected for treatment. In other embodiments, the use of monoclonal antibodies or antigen-binding fragments disclosed herein for detecting EGFRvIII or gene-amplified EGFR is disclosed.

[0011] The above and other features will become clearer from the following detailed description, which will proceed with reference to the attached drawings. [Brief explanation of the drawing]

[0012] [Figure 1A] Antibody binding to rat glioma cells expressing human EGFRvIII. A10 showed the highest MFI compared to other candidates, including a chimeric antibody (mouse VH and VL derived from 40H3 antibody + human IgG1CH / human IgκCL). Cetuximab is used as a universal positive control that binds to both WT and cancer-expressing EGFR (EGFRvIII). [Figure 1B]Antibody binding to rat glioma cells expressing human EGFRvIII. A10 showed the highest MFI compared to other candidates, including a chimeric antibody (mouse VH and VL derived from 40H3 antibody + human IgG1CH / human IgκCL). Cetuximab is used as a universal positive control that binds to both WT and cancer-expressing EGFR (EGFRvIII).

[0013] [Figure 2A] Antibody binding to human tumor cells expressing amplified EGFR, specifically MDA-MB-468 (Figure 2A) and A431 (Figure 2B) cells. Antibody A10 showed the highest MFI. [Figure 2B] Antibody binding to human tumor cells expressing amplified EGFR, specifically MDA-MB-468 (Figure 2A) and A431 (Figure 2B) cells. Antibody A10 showed the highest MFI.

[0014] [Figure 3A] Antibody binding to cells expressing WT EGFR, specifically WI-38 (Figure 3A) or U87MG (Figure 3B) cells. The A10 antibody does not bind to cells expressing WT EGFR. However, evidence of surface WT EGFR is confirmed via cetuximab binding. [Figure 3B] Antibody binding to cells expressing WT EGFR, specifically WI-38 (Figure 3A) or U87MG (Figure 3B) cells. The A10 antibody does not bind to cells expressing WT EGFR. However, evidence of surface WT EGFR is confirmed via cetuximab binding.

[0015] [Figure 4A] A table showing antibody characteristics including combinations of variable regions, dissociation constant (KD), cell binding, and EC50. [Figure 4B] A table showing antibody characteristics including combinations of variable regions, dissociation constant (KD), cell binding, and EC50.

[0016] [Figure 5A]Comparative antibody binding data for 40H3, A10, and cetuximab. Figure 5A shows that 40H3 and A10 strongly bind to cells expressing amplified EGFR (MDA-MB-468) but weakly bind to cells with EGFR-WT (WI-38). In contrast, cetuximab is shown to strongly bind to EGFR-WT cells. Figure 5B shows that both A10 and cetuximab strongly bind to cells with EGFRvIII. [Figure 5B] Comparative antibody binding data for 40H3, A10, and cetuximab. Figure 5A shows that 40H3 and A10 strongly bind to cells expressing amplified EGFR (MDA-MB-468) but weakly bind to cells with EGFR-WT (WI-38). In contrast, cetuximab is shown to strongly bind to EGFR-WT cells. Figure 5B shows that both A10 and cetuximab strongly bind to cells with EGFRvIII.

[0017] [Figure 6A] Crystal structure of the EGFR loop-A10 Fab. Figures 6B and 6C are enlarged views of Figure 6A to show more detail of the EGFR loop-A10 interaction. [Figure 6B] Crystal structure of the EGFR loop-A10 Fab. Figures 6B and 6C are enlarged views of Figure 6A to show more detail of the EGFR loop-A10 interaction. [Figure 6C] Crystal structure of the EGFR loop-A10 Fab. Figures 6B and 6C are enlarged views of Figure 6A to show more detail of the EGFR loop-A10 interaction.

[0018] [Figure 7] A summary of information about the cell lines shown (see column 1).

[0019] [Figure 8A] Vector maps of pMH289-A10-1 (Figure 8A, Sequence ID 51) and pMH289-A10-2 (Figure 8B, Sequence ID 52). [Figure 8B] Vector maps of pMH289-A10-1 (Figure 8A, Sequence ID 51) and pMH289-A10-2 (Figure 8B, Sequence ID 52).

[0020] [Figure 9] Cell viability of A431-luc cells 24 or 48 hours after the indicated treatment. "CAR-1" and "CAR-2" are A10 scFv-CAR-T cells (see Figure 8A and Figure 8B, respectively). The control is transduced activated T cells lacking the chimeric antigen receptor.

[0021] [Figure 10] Cell viability of MDA-MB-468 cells after treatment with two A10 antibody-drug conjugates (ADCs): A10 conjugated to deruxtecan (A10-Dxd) or A10 conjugated to monomethyl auristatin E (A10-MMAE). [Modes for carrying out the invention]

[0022] array The enumerated nucleic acid and amino acid sequences are shown using standard abbreviations for nucleotide bases and amino acids, as defined in 37C.FR1.822. Only one strand of each nucleic acid sequence is shown, but complementary strands are understood to be included by any reference to the presented strand. In heavy and light chain variable domain sequences, the CDR sequence (as determined by the Kabat, IMGT, or Chothia numbering scheme) is underlined. In SEQ ID NOs. 3–32, the bolded framework residues are, specifically, SEQ ID NOs. 1 (V H (Array) or Sequence ID 2 (V L These are humanized residues compared to the sequence.

[0023] Sequence ID 1 is the amino acid sequence of the 40H3 heavy chain variable domain. [ka]

[0024] Sequence ID 2 is the amino acid sequence of the 40H3 light chain variable domain. [ka]

[0025] Sequence ID 3 is the amino acid sequence of the VH1 heavy chain variable domain. [ka]

[0026] Sequence ID 4 is the amino acid sequence of the VH2 heavy chain variable domain. [ka]

[0027] Sequence ID 5 is the amino acid sequence of the VH3 heavy chain variable domain (which is also the D2 and D3 heavy chain variable domain). [ka]

[0028] Sequence ID 6 is the amino acid sequence of the VL1 light chain variable domain. [ka]

[0029] Sequence ID 7 is the amino acid sequence of the VL2 light chain variable domain. [ka]

[0030] Sequence ID 8 is the amino acid sequence of the VL3 light chain variable domain (which is also the D1 light chain variable domain). [ka]

[0031] Sequence ID 9 is the amino acid sequence of the VL-EG light chain variable domain. [ka]

[0032] Sequence ID 10 is the amino acid sequence of the VL-DA light chain variable domain. [ka]

[0033] Sequence ID 11 is the amino acid sequence of the VL1-DA light chain variable domain. [ka]

[0034] Sequence ID 12 is the amino acid sequence of the VL2-DA light chain variable domain. [ka]

[0035] Sequence ID 13 is the amino acid sequence of the C1 heavy chain variable domain (which is also the D1 heavy chain variable domain). [ka]

[0036] Sequence ID 14 is the amino acid sequence of the C1 light chain variable domain (which is also the D2 light chain variable domain). [ka]

[0037] Sequence ID 15 is the amino acid sequence of the C2 heavy chain variable domain. [ka]

[0038] Sequence ID 16 is the amino acid sequence of the C2 light chain variable domain. [ka]

[0039] Sequence ID 17 is the amino acid sequence of the C3 heavy chain variable domain. [ka]

[0040] Sequence ID 18 is the amino acid sequence of the C3 light chain variable domain. [ka]

[0041] Sequence ID 19 is the amino acid sequence of the C4 heavy chain variable domain. [ka]

[0042] Sequence ID 20 is the amino acid sequence of the C4 light chain variable domain. [ka]

[0043] Sequence ID 21 is the amino acid sequence of the C5 heavy chain variable domain. [ka]

[0044] Sequence ID 22 is the amino acid sequence of the C5 light chain variable domain. [ka]

[0045] Sequence ID 23 is the amino acid sequence of the C6 heavy chain variable domain. [ka]

[0046] Sequence ID 24 is the amino acid sequence of the C6 light chain variable domain (which is also the D3 light chain variable domain). [ka]

[0047] Sequence ID 25 is the amino acid sequence of the C7 heavy chain variable domain. [ka]

[0048] Sequence ID 26 is the amino acid sequence of the C7 light chain variable domain. [ka]

[0049] Sequence ID 27 is the amino acid sequence of the C8 heavy chain variable domain. [ka]

[0050] Sequence ID 28 is the amino acid sequence of the C8 light chain variable domain. [ka]

[0051] Sequence ID 29 is the amino acid sequence of the C9 heavy chain variable domain. [ka]

[0052] Sequence ID 30 is the amino acid sequence of the C9 light chain variable domain. [ka]

[0053] Sequence ID 31 is the amino acid sequence of the C10 heavy chain variable domain. [ka]

[0054] Sequence ID 32 is the amino acid sequence of the C10 light chain variable domain. [ka]

[0055] Sequence IDs 33-42 are the amino acid sequences of components of the chimeric antigen receptor.

[0056] Sequence ID 43 is the amino acid sequence of A10 VH-VL scFv. [ka]

[0057] Sequence ID 44 is an exemplary nucleic acid sequence that encodes A10 VH-VL scFv. [ka]

[0058] Sequence ID 45 is the amino acid sequence of A10 VL-VH scFv. [ka]

[0059] Sequence ID 46 is an exemplary nucleic acid sequence encoding A10 VL-VH scFv. [ka]

[0060] Sequence ID 47 is the amino acid sequence of A10 Fab (heavy chain). The sequence terminates at the site where papain cleavage occurs. [ka]

[0061] Sequence ID 48 is an exemplary nucleic acid sequence that encodes A10 Fab (heavy chain). [ka]

[0062] Sequence ID 49 is the amino acid sequence of A10 Fab (light chain). [ka]

[0063] Sequence ID 50 is an exemplary nucleic acid sequence that encodes A10 Fab (light chain). [ka]

[0064] Sequence ID 51 is an exemplary sequence that encodes the pMH289-A10-1 construct. [ka] [ka] [ka] [ka]

[0065] Sequence ID 52 is an exemplary sequence that encodes the pMH289-A10-2 construct. [ka] [ka] [ka] [ka] [ka] Detailed explanation

[0066] I. Overview Disclosed herein are humanized monoclonal antibodies and their antigen-binding fragments that specifically bind to mutant / variant EGFR expressed by cancer cells (e.g., EGFRvIII or gene-amplified EGFR) but do not bind to wild-type EGFR expressed on normal cells. These humanized monoclonal antibodies not only exhibit low immunogenicity but also increased binding to targeted EGFRvIII and / or gene-amplified EGFR.

[0067] Activation of wild-type EGFR involves dimerization, which requires a monomer-to-dimer transfer accompanied by ligand binding and associated changes in receptor conformation. Several structures of the extracellular domain of EGFR have been reported in both monomeric and dimeric conformations. Analysis of these structures reveals the presence of residues not exposed in the wild-type receptor. However, hidden structures may be exposed under oncogenic conditions where the receptor is highly expressed and may not fold correctly, or where mutant forms of the receptor are expressed. One structural element not sterically available under normal conditions is the 287–302 (numbering in the mature receptor – or 301–326 in the full-length receptor) disulfide restriction loop. This loop is exposed in EGFRvIII and may be exposed when receptor expression is very high or when ECD mutations alter the wild-type structure. In some examples, the antibody or antigen-binding fragments disclosed herein may be exposed to EGFR 287-302 It binds to the loop. EGFR exposed on the cell. 287-302Methods for inhibiting or treating tumors having loops are also provided. Methods for inhibiting tumors that overexpress EGFR in a subject are also provided.

[0068] II. Terminology Unless otherwise specified, technical terms will be used according to their conventional usage. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes X, Jones & Bartlett Publishers, 2009; and Meyers et al. (eds.), The Encyclopedia of Cell Biology and Molecular Medicine, Wiley-VCH, Vol. 16, 2008; and other similar references.

[0069] Where used herein, the singular forms “a,” “an,” and “the” refer to both singular and plural unless the context clearly indicates otherwise. For example, the term “antigen” can be considered equivalent to the phrase “at least one antigen,” including one or more antigens. Where used herein, the term “comprises” means “includes.” Furthermore, unless otherwise indicated, any base size or amino acid size, and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximations and are provided for illustrative purposes only. Many methods and materials similar or equivalent to those described herein can be used, but certain suitable methods and materials are described herein. In case of any conflict, this specification, including the definitions of terms, shall prevail. Furthermore, the materials, methods, and examples are illustrative and not intended to limit the scope of the invention.

[0070] To facilitate the examination of various aspects, the following definitions of terms are provided. Approximately: Unless otherwise indicated in the context, "approximately" refers to ±5% of the base value. For example, "approximately" 100 refers to a range of 95 to 105.

[0071] Administration: Introduction of the composition to the target via a selected route. Administration may be topical or systemic. For example, if the selected route is intravenous, the composition (e.g., a composition containing a disclosed humanized monoclonal antibody or antigen-binding fragment, etc.) is administered by introducing the composition into the vein of the target. Exemplary routes of administration, but not limited to, include oral, injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, intratumoral, and intravenous), sublingual, rectal, percutaneous (e.g., topical), intranasal, vaginal, and inhalation routes. In one example, administration is systemic. In another example, administration is topical, for example, directly to a tumor.

[0072] Drug: Any substance or any combination of substances that is useful in achieving a purpose or result, for example, a substance or combination of substances that is useful in inhibiting tumor growth or metastasis in a subject. Drugs include proteins, nucleic acid molecules, compounds, small molecules, organic compounds, inorganic compounds, or other molecules of purpose. Drugs may include therapeutic agents (such as chemotherapeutic agents), diagnostic agents, or pharmaceutical agents. In some examples disclosed herein, drugs are humanized monoclonal antibodies that specifically bind to EGFRvIII and / or gene-amplified EGFR, their antigen-binding fragments, their conjugates, or chimeric antigen receptors (CARs) containing antibodies or antigen-binding fragments. Those skilled in the art will understand that a particular drug may be useful in achieving two or more results.

[0073] Amino acid substitution: Substitution of one amino acid in a polypeptide with a different amino acid, or without an amino acid (i.e., deletion). In some examples, an amino acid in a polypeptide is substituted with an amino acid from a homologous polypeptide; for example, an amino acid in a humanized monoclonal antibody or its antigen-binding fragment that specifically binds to EGFRvIII may be substituted with a corresponding amino acid from another antibody or its antigen-binding fragment that specifically binds to EGFRvIII.

[0074] Antibody: An immunoglobulin, antigen-binding fragment, or derivative thereof that specifically binds to and recognizes an analyte (antigen), for example, EGFRvIII. The term "antibody" is used herein in the broadest sense and encompasses various antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antigen-binding fragments, as long as they exhibit the desired antigen-binding activity.

[0075] Non-limiting examples of antibodies include intact immunoglobulins and their variants and antigen-binding fragments that retain binding affinity for an antigen. Examples of antibody fragments include Fv, Fab, dsFv, Fab’, Fab’-SH, F(ab’)2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv and ds-scFv); and multispecific antibodies formed from antibody fragments, but are not limited thereto. Antibody fragments include antigen-binding fragments produced by modification of the whole antibody or de novo synthesized antigen-binding fragments using recombinant DNA methods (see, e.g., Kontermann and Dubel (Eds), Antibody Engineering, Vols. 1-2, 2 nd ed., Springer-Verlag, 2010).

[0076] Antibodies also include genetically engineered forms such as chimeric antibodies (such as humanized mouse antibodies) and heteroconjugate antibodies (such as bispecific antibodies).

[0077] An antibody can have one or more binding sites. When multiple binding sites are present, they may be identical to each other or different. For example, a naturally occurring immunoglobulin has two identical binding sites, a single-chain antibody or Fab fragment has one binding site, while a bispecific or bifunctional antibody has two different binding sites.

[0078] Typically, naturally occurring immunoglobulins have heavy (H) and light (L) chains interconnected by disulfide bonds. Immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as a wide variety of immunoglobulin variable domain genes. There are two types of light chains: lambda (λ) and kappa (κ). There are five major heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE.

[0079] Each heavy chain and light chain contains a constant region (or constant domain) and a variable region (or variable domain). Together, the variable regions of the heavy chain and light chain bind specifically to the antigen.

[0080] "V H References to "VH" or "VH" refer to the variable region of the antibody heavy chain, which includes the variable region of the antigen-binding fragment, such as Fv, scFv, dsFv, or Fab. L References to "VL" or "VL" refer to the variable domains of the antibody light chain, including the variable domains of Fv, scFv, ds-scFv, or Fab.

[0081] V H and V L This refers to the "complementarity-determining region" or "CDR" (e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5 th It contains a “framework” region interrupted by three hypervariable regions (see ed., NIH Publication No. 91-3242, Public Health Service, National Institutes of Health, USD Department of Health and Human Services, 1991). The sequences of framework regions of different light chains or heavy chains are relatively conserved within a species. The framework region of an antibody, i.e., the combined framework region of the constituent light and heavy chains, helps to position and align the CDR in three-dimensional space.

[0082] CDRs are primarily responsible for binding antigens to epitopes. The amino acid sequence boundaries of a given CDR were determined by Kabat et al. (Sequences of Proteins of Immunological Interest, 5 th It can be easily determined using one of several schemes, including those described by Al-Lazikani et al. ("Standard conformations for the canonical structures of immunoglobulins", J.Mol.Bio., 273(4):927-948, 1997; "Chothia" numbering scheme) and Lefranc et al. ("IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains", Dev.Comp.Immunol., 27(1):55-77, 2003; "IMGT" numbering scheme). The CDRs of each strand are typically called CDR1, CDR2, and CDR3 (N-terminus to C-terminus) and are also typically identified by the strand on which a particular CDR is located. H CDR3 is the antibody in which it is found. H While it originates from CDR3, V L CDR1 is the antibody in which it is found. L This is the origin of CDR1. Light chain CDRs are sometimes called LCDR1, LCDR2, and LCDR3. Heavy chain CDRs are sometimes called HCDR1, HCDR2, and HCDR3.

[0083] In some examples, the disclosed humanized monoclonal antibodies include heterologous constant domains. For instance, the antibody includes a constant domain that differs from the native constant domain, e.g., one or more modifications (such as "LS" mutations) to increase the half-life.

[0084] A “monoclonal antibody” is an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies within the population are identical and / or bind to the same epitope, except for variant antibodies that may contain, for example, naturally occurring mutations or that may arise during the production of the monoclonal antibody preparation, and such variants are generally present in small amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each antibody in a monoclonal antibody preparation is against a single determinant on an antigen. Therefore, the modifier “monoclonal” indicates the characteristic of an antibody obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring the antibody product by a specific method. For example, monoclonal antibodies can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, and other exemplary methods for producing monoclonal antibodies described herein. In some examples, monoclonal antibodies are isolated from a subject, e.g., a mammal, e.g., a human. Monoclonal antibodies may have conserved amino acid substitutions that do not substantially affect antigen binding or other immunoglobulin functions. (e.g., Greenfield (Ed.), Antibodies: A Laboratory Manual, 2) nd (See ed. New York: Cold Spring Harbor Laboratory Press, 2014.)

[0085] A “humanized” antibody or antigen-binding fragment typically contains amino acid substitutions within the human framework region compared to the corresponding non-human (e.g., mouse, rat, or synthetic) antibody or antigen-binding fragment. In some examples of this disclosure, the non-human antibody or antigen-binding fragment providing the CDR is referred to as the “donor,” and the human antibody or antigen-binding fragment providing the framework is referred to as the “acceptor.” In some examples of this disclosure, many of all CDR and framework residues are derived from the donor immunoglobulin, but some of the framework residues at specific locations are substituted with amino acids from the corresponding locations in the human framework region. A constant region is not required to be present, but if present, it may be substantially identical to the human immunoglobulin constant region, e.g., at least about 85–90%, e.g., about 95% or more identical.

[0086] A "chimeric antibody" is an antibody that typically contains sequences derived from two different antibodies of different species. In some examples, a chimeric antibody contains one or more CDRs and / or framework regions from one antibody (e.g., a mouse antibody) and CDRs and / or framework regions from another antibody (e.g., a human antibody).

[0087] A "fully human antibody" or "human antibody" is an antibody that contains sequences from (or derived from) the human genome and does not contain sequences from another species. Human antibodies may, for example, contain CDRs, framework regions, and (if present) Fc regions from (or derived from) the human genome. Human antibodies are produced using techniques for creating antibodies based on sequences derived from the human genome, for example, by phage display or by transgenic animals (e.g., Barbas et al., Phage display: A Laboratory Manual.1). stIt can be identified and isolated using (see Ed. New York: Cold Spring Harbor Laboratory Press, 2004. Print.; Lonberg, Nat. Biotech., 23:1117-1125, 2005; Lonberg, Curr. Opin. Immunol., 20:450-459, 2008).

[0088] Biological specimen: A specimen obtained from a subject. Biological specimens include, but are not limited to, all clinical specimens useful for detecting disease or tumors in a subject (e.g., head and neck cancer, breast cancer, pancreatic cancer, colorectal cancer, bladder cancer, or glioma), including cells, tissues, and bodily fluids such as blood, blood derivatives and fractions (e.g., serum), cerebrospinal fluid, and tissues removed by biopsy or surgery (e.g., unfixed, frozen, or fixed in formalin or paraffin). In certain cases, biological specimens are obtained from subjects who have or are suspected of having tumors such as head and neck cancer, breast cancer, pancreatic cancer, colorectal cancer, glioma, or bladder cancer.

[0089] Bispecific antibodies are recombinant molecules composed of two distinct antigen-binding domains that consequently bind to two different antigenic epitopes. Bispecific antibodies contain a molecule with two chemically or genetically linked antigen-binding domains. The antigen-binding domains may be linked using a linker. The antigen-binding domains may be monoclonal antibodies, antigen-binding fragments (e.g., Fab, scFv, ds-scFv), or combinations thereof. Bispecific antibodies may contain one or more constant domains, but do not necessarily contain any constant domains.

[0090] Cancer: A malignant tumor containing transformed epithelial cells. Non-exclusive examples of cancer include adenocarcinoma, squamous cell carcinoma, anaplastic carcinoma, and large cell and small cell carcinoma. In some cases, cancer is breast cancer, head and neck cancer, pancreatic cancer, colorectal cancer, glioma cancer, or bladder cancer.

[0091] Chemotherapy agents: Any chemical agent that has therapeutic utility in the treatment of a disease characterized by abnormal cell proliferation. For example, chemotherapeutic agents are useful in the treatment of cancers including, but not limited to, head and neck cancer, breast cancer, pancreatic cancer, colorectal cancer, bladder cancer, and glioma. In a non-limiting example, a chemotherapeutic agent is a drug used to treat cancer. Specific examples of further therapeutic agents that may be used in combination with the disclosed humanized antibodies include one or more of microtubule binders, DNA intercalators or crosslinkers, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors. In a non-limiting example, a chemotherapeutic agent is a radioactive compound. Other examples include the use of the antineoplastic agent 5-fluorouracil (5-FU) and IRT.Those involved can easily identify the chemotherapy agents to be used (e.g., Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch.17 in Abeloff, Clinical Oncology 2nd ed., (c)2000 Churchill Livingstone, Inc; Baltzer, L., Berkerey, R. (eds): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer, DS, Knobf, MF, Durivage, HJ (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993; Chabner and Longo, Cancer Chemotherapy and Biotherapy: Principles and Practice (4th (See: ed.).Philadelphia: Lippincott Williams & Wilkins, 2005; Skeel, Handbook of Cancer Chemotherapy (6th ed.). Lippincott Williams & Wilkins, 2003). Combination chemotherapy is the administration of two or more drugs to treat cancer, such as one or more humanized antibodies and another chemotherapeutic agent. Such drugs may be administered sequentially or simultaneously.

[0092] Chimeric antigen receptors (CARs): Engineered T cell receptors having extracellular antibody-derived targeting domains (such as scFv) bound to one or more intracellular signaling domains of the T cell receptor. "Chimeric antigen receptor T cells" are T cells that express CARs and have antigen specificity determined by the antibody-derived targeting domain of the CAR. Methods for producing CARs (e.g., for cancer treatment) have been described (see, for example, Park et al., Trends Biotechnol., 29:550-557, 2011; Grupp et al., N Engl J Med., 368:1509-1518, 2013; Han et al., J. Hematol Oncol., 6:47, 2013; International Publication No. 2012 / 079000, International Publication No. 2013 / 059593; and U.S. Patent Application Publication No. 2012 / 0213783).

[0093] Conditions sufficient for immune complex formation: Conditions that allow an antibody or antigen-binding fragment to bind to its homologous epitope to a detectably greater extent than its binding to substantially all other epitopes, and / or substantially exclude all other epitopes. Conditions sufficient for immune complex formation depend on the form of the binding reaction and are typically the conditions used in immunoassay protocols or the conditions encountered in vivo. For a description of immunoassay forms and conditions, see Harlow & Lane, Antibodies: A Laboratory Manual, 2 nd This method is described in ed. Cold Spring Harbor Publications, New York (2013). The conditions used in this method are “physiological conditions” that include reference to conditions typical inside living mammals or mammalian cells (e.g., temperature, volumetric osmolality, pH). Although some organs are recognized to be exposed to extreme conditions, the in vivo and intracellular environment is typically around pH 7 (e.g., pH 6.0–pH 8.0, more typically pH 6.5–pH 7.5), contains water as the primary solvent, and exists at temperatures above 0°C and below 50°C. The volumetric osmolality is within a range that supports cell viability and proliferation.

[0094] Conjugate: A complex of two molecules that are linked to each other, for example, linked to each other by a covalent bond. In one example, an antibody is linked to an effector molecule, for example, the antigen-binding fragment described herein is covalently linked to an effector molecule. The linkage can be by chemical means or recombinant means. In one embodiment, the linkage is chemical, and the reaction between the antibody portion and the effector molecule produces a covalent bond formed between the two molecules to form one molecule. A peptide linker (short peptide sequence) can be included between the antibody and the effector molecule as needed. Since a conjugate can be prepared from two molecules having distinct functional groups such as an antibody and an effector molecule, it may also be referred to as a "chimeric molecule".

[0095] Conservative variant: A sequence variant by conservative substitution. A "conservative" amino acid substitution does not substantially affect or decrease the function of a protein, such as the ability of the protein to interact with a target protein. For example, the EGFRvIII-specific and / or gene-amplified EGFR-specific humanized monoclonal antibodies disclosed herein may contain up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10 conservative substitutions compared to the reference antibody sequence, and each may retain specific binding activity against EGFRvIII or gene-amplified EGFR. The term conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid.

[0096] Substitutions, deletions or additions that change, add or delete a single amino acid or a small percentage of amino acids (e.g., less than 5%, less than 3%, less than 1%) in an individual, encoded sequence are conservative mutations that result in substitution of an amino acid by a chemically similar amino acid.

[0097] The following six groups are examples of amino acids that are considered to be conservative substitutions for each other: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0098] Non-conservative substitutions are those that decrease the ability of an antibody to bind specifically to EGFRvIII and / or gene amplified EGFR, or to bind to cancer cells expressing EGFRvIII and / or gene amplified EGFR, for example, the activity or function of the antibody. For example, if an amino acid residue is essential for the function of a protein, even a conservative substitution in other respects may inhibit its activity. Thus, conservative substitutions do not alter the basic function of the protein of interest.

[0099] Contact: Arrangement in a direct physical association; including both solid and liquid forms that can occur either in vivo or in vitro. Contact includes contact between amino acids on the surface of one polypeptide, such as a peptide that contacts another molecule, for example, another polypeptide. Contact can also include contacting a cell, for example, by physically associating a polypeptide directly with the cell.

[0100] Control: A reference standard. In some examples, the control is a negative control sample obtained from a subject (e.g., a person without a tumor) or a subject without a tumor expressing EGFRvIII or gene amplified EGFR. In other examples, the control is a positive control sample obtained from a subject diagnosed with a tumor expressing EGFRvIII and / or gene amplified EGFR, or a recombinantly produced purified EGFRvIII. In still other examples, the control is a historical control or a standard reference value or range of values (e.g., a previously tested control sample, e.g., a group of subjects with a known prognosis or outcome, or a group of samples representing baseline or normal values). One skilled in the art can readily determine an appropriate control.

[0101] The difference between the test sample and the control may increase or decrease. This difference may be qualitative or quantitative, and may be statistically significant, for example. In some cases, the difference is an increase or decrease of at least about 5%, e.g., at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, or greater than 500% compared to the control. In some cases, tumor growth, volume, and / or metastasis decrease after treatment with the disclosed antibody.

[0102] Reduction or Reduction: Reducing quality, quantity, or intensity, e.g., reduction of tumor volume. In one example, treatment reduces a tumor (e.g., tumor size, number of tumors, tumor metastases, or a combination thereof) or one or more symptoms associated with a tumor, compared to, for example, the response in the absence of treatment. In a particular example, treatment reduces tumor size, number of tumors, tumor metastases, or a combination thereof after treatment, such as a reduction of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. Such reductions may be measured using the methods disclosed herein.

[0103] Degenerate Variants: In the context of this disclosure, “degenerate variant” refers to a polynucleotide sequence variant resulting from redundancy in the gene code (which consists of 20 native amino acids, most of which are identified by two or more codons). Thus, the nucleotide sequence of a degenerate variant changes, but the encoded peptide remains the same.

[0104] Detectable markers: Detectable molecules (also known as labels) that are directly or indirectly conjugated to a second molecule, such as a humanized monoclonal antibody, to facilitate the detection of the second molecule. For example, detectable markers can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or imaging techniques (CT scans, MRI, ultrasound, fiber optic examinations, laparoscopy, etc.). Specific non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzyme linkages, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). Methods and guidelines for using detectable markers in selecting detectable markers suitable for various purposes can be found, for example, in Green and Sambrook (Molecular Cloning: A Laboratory Manual, 4). th This has been discussed in (ed., New York: Cold Spring Harbor Laboratory Press, 2012) and Ausubel et al. (Eds.) (Current Protocols in Molecular Biology, New York: John Wiley and Sons, including supplements, 2017).

[0105] Detection: The act of identifying the existence, presence, or fact of something.

[0106] Effective dose: The amount of a particular substance sufficient to achieve the desired effect in the subject to which it is administered, for example, a therapeutic effective dose for the treatment of a disease. For example, this could be the amount of humanized monoclonal antibody required to inhibit tumor growth and / or metastasis, or to measurably alter the outward manifestations of a tumor.

[0107] In some embodiments, administration of an effective amount of a disclosed humanized monoclonal antibody or antigen-binding fragment that binds to EGFRvIII and / or gene-amplified EGFR reduces or inhibits tumor growth, tumor metastasis, or tumor volume by a desired amount, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (tumor elimination) compared to a suitable control.

[0108] The effective dose of humanized monoclonal antibody or antigen-binding fragment administered to a subject, which specifically binds to EGFRvIII and / or gene-amplified EGFR, may vary depending on several factors related to the subject, such as the subject's overall health and / or body weight. The effective dose can be determined by varying the dose and measuring the resulting response, such as a reduction in tumor load. The effective dose can also be determined by various in vitro, in vivo, or in situ immunoassays or clinical trials.

[0109] The effective dose includes the partial dose that contributes in combination with prior or subsequent administration to achieve an effective response. For example, the effective dose of the drug may be administered as a single dose or in several doses, for example, daily, during a series of treatments lasting several days or several weeks. However, the effective dose may depend on the subject being treated, the severity and type of the condition being treated, and the mode of administration. The unit dosage form of the drug may be packaged in a certain amount or in multiples of the effective dose, for example, in a vial with sterile components (e.g., with a puncturable lid) or syringe.

[0110] Effector molecule: A molecule intended to have or produce a desired effect. For example, the desired effect of an effector molecule on cells that are targeted. Effector molecules include molecules such as chemical compounds, polypeptides, radioisotopes, and small molecules. Non-limiting examples of effector molecules include toxins, chemotherapeutic agents, and anti-angiogenic agents. Those skilled in the art will understand that some effector molecules may have or produce two or more desired effects. In one example, the effector molecule comprises a disclosed humanized monoclonal antibody or a fragment thereof. In another example, the effector molecule is a chimeric molecule comprising a disclosed humanized monoclonal antibody or a fragment thereof, and the chimeric molecule is intended to have a desired effect on cells that it targets.

[0111] Epidermal growth factor receptor (EGFR): EGFR (also known as HER1 or ERBB1) is a receptor belonging to the ERBB family of receptor thymidine kinases (RTKs). In vivo, ligand binding by EGF leads to activation of the RTK / RAS / PI(3)K pathway via receptor phosphorylation, resulting in increased cell proliferation, angiogenesis, and local tissue invasion, as well as resistance to apoptosis. The nucleic acid sequences of human EGFR can be found in GENBANK® accession number NM_005228.5, June 18, 2019 and GENBANK® accession number NC_000007.14 (EGFR in chromosomes), June 14, 2019, both of which are incorporated herein by reference. EGFR has a 621-amino acid external domain (ECD), a 23-amino acid single-pass transmembrane domain (TM), and a 542-amino acid enzymatically active intracellular domain (ICD). Ligand binding results in receptor dimerization and activation of the kinase domain, which signals to one of several pathways that can promote the proliferation, survival, and expansion of mammalian cells. In tumors, loss of exons 2–7 produces constitutively active EGFR variant III (EGFRvIII). The cDNA sequence of EGFRvIII can be found in GENBANK® accession number NM_001346941 dated June 18, 2019, incorporated herein by reference, and the amino acid sequence can be found in NP_001333870.1 dated June 14, 2019, incorporated herein by reference. Activation of wild-type EGFR involves dimerization, which requires monomer-to-dimer transfer accompanied by ligand binding and associated changes in receptor conformation. Activating mutations can occur in either ECD or ICD. Loss of exon 19 produces a constitutively active enzyme variant. EGFR can be overexpressed by either gene amplification or reduction of transcriptional regulation. High levels of expression (e.g., more than approximately 50,000 receptors per cell) can lead to either receptor misfolding or mutations in one or more gene copies. Overexpression can result in a doubling or more of EGFR levels in cells compared to wild-type controls.One structural element that is not sterically available under normal conditions is the 287–302 (numbering in the mature receptor – or 301–326 in the full-length receptor) disulfide restriction loop. This loop is exposed in EGFRvIII and may be exposed when receptor expression is very high or when ECD mutations alter the wild-type structure.

[0112] Overexpressed EGFR and EGFRvIII are also known to occur in cancers of non-human animals (see, e.g., Cho et al., Oncology, 58(4):674-682, 2021). Non-human primate EGFR has the same reactive loop sequence as human EGFR to which the antibodies disclosed herein can bind (EGFR 287-302 It has a loop. In some cases, the antibodies disclosed herein bind to mammalian EGFRvIII or gene-amplified EGFR. In some cases, the antibodies disclosed herein bind to human, non-human primate, dog or cat EGFRvIII or gene-amplified EGFR. In non-limiting cases, the antibodies disclosed herein bind to human, non-human primate, dog or cat EGFRvIII.

[0113] Epitope: Antigen-determining group. These are specific chemical groups or peptide sequences on a molecule that are antigenic and therefore trigger a specific immune response. For example, an epitope is a region of an antigen to which B cells and / or T cells respond. Antibodies can bind to specific antigenic epitopes, such as epitopes on EGFRvIII.

[0114] Expression: The transcription or translation of a nucleic acid sequence. For example, a gene is expressed when its DNA is transcribed into RNA or an RNA fragment, and in some cases, it may be processed to become mRNA. A gene may also be expressed when its mRNA is translated into an amino acid sequence, such as a protein or a protein fragment. In certain cases, a heterologous gene is expressed when it is transcribed into RNA. In other cases, a heterologous gene is expressed when its RNA is translated into an amino acid sequence. The term “expression” is used herein to refer to either transcription or translation. Regulation of expression can include transcription, translation, RNA transport and processing, degradation of intermediate molecules such as mRNA, or control by activation, inactivation, compartmentalization, or degradation of specific protein molecules after they have been produced.

[0115] Regulatory sequences are nucleic acid sequences that regulate the expression of a functionally linked heterologous nucleic acid sequence. Regulatory sequences are functionally linked to a nucleic acid sequence if they control and regulate the transcription and, if necessary, the translation of that nucleic acid sequence. Thus, examples of regulatory sequences include appropriate promoters, enhancers, transcriptional terminators, pre-start codons (ATGs) of protein-coding genes, intron splicing signals, maintenance of the correct reading frame of that gene to enable proper translation of mRNA, and stop codons. The term "regulatory sequence" is intended to include at least a component whose presence can influence expression, and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences. Regulatory sequences may include promoters.

[0116] A promoter is the smallest sequence sufficient to direct transcription. It also includes promoter elements sufficient to make promoter-dependent gene expression cell-type specific, tissue-specific, or controllable by external signals or drugs. Such elements may be located in the 5' or 3' region of the gene. Both constitutive and inductive promoters are included (see, e.g., Bitter et al., Methods in Enzymology, 153:516-544, 1987). For example, when cloning in bacterial systems, exemplary inductive promoters include, and may be used, the bacteriophage lambda pL, plac, ptrp, ptac (ptrp-lac hybrid promoter) and similar ones. When cloning in mammalian cell systems, non-exclusive exemplary promoters include those derived from the genome of mammalian cells (e.g., metallothionein promoter) or mammalian viruses (e.g., retroviral long-terminal repeat sequences; adenovirus late promoter; vaccinia virus 7.5K promoter). Promoters produced by recombinant DNA or synthetic techniques may also be used to provide transcription of nucleic acid sequences.

[0117] Polynucleotides can be inserted into expression vectors that contain promoter sequences that facilitate the transcription of gene sequences inserted into host cells. Expression vectors typically include an origin of replication, a promoter, and specific nucleic acid sequences that enable phenotypic selection of transformed cells.

[0118] Expression vector: A vector containing an expression control sequence (e.g., promoter, enhancer, terminator, etc.) operably ligated to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression, and other elements for expression may be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, e.g., cosmids, plasmids (e.g., naked or liposome-containing) and viral vectors (e.g., lentiviruses, retroviruses, adenoviruses and adeno-associated viruses).

[0119] Fc region: The constant region of an antibody excluding the first heavy chain constant domain. The Fc region generally refers to the last two heavy chain constant domains of IgA, IgD, and IgG, and the last three heavy chain constant domains of IgE and IgM. The Fc region may also include part or all of the flexible hinge N-terminus for these domains. In the case of IgA and IgM, the Fc region may or may not include the tailpiece and may or may not be bound by the J chain. In the case of IgG, the Fc region is typically understood to include the immunoglobulin domains Cγ2 and Cγ3, and optionally the lower part of the hinge between Cγ1 and Cγ2. The boundaries of the Fc region can vary, but the human IgG heavy chain Fc region is usually defined to include the residues following C226 or P230 relative to the Fc carboxyl terminus, and the numbering follows Kabat. In the case of IgA, the Fc region includes the immunoglobulin domains Cα2 and Cα3, and optionally the lower part of the hinge between Cα1 and Cα2.

[0120] Framework region: The amino acid sequence intervening between the CDRs of the variable region of the heavy chain or the variable region of the light chain of an antibody. Includes variable light and variable heavy framework regions. The framework region serves to hold the CDRs in an appropriate orientation.

[0121] Gene amplification EGFR: An increase in the copy number of the EGFR gene in a cell (e.g., a tumor cell) compared to a normal cell (e.g., a non-tumor cell) that results in overexpression of EGFR. Typically, a cell (e.g., a tumor cell) containing more than 6 copies of EGFR is considered to have gene amplification EGFR. Gene amplification EGFR is described, for example, in French et al., "Defining EGFR amplification status" Neuro-Oncology, 21(19):1263-1272, 2019.

[0122] In some examples, amplified EGFR gene numbers include more than 6 EGFR gene copies, e.g., at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, or more EGFR gene copies. In non-limiting examples, amplified EGFR gene numbers include at least 7 EGFR gene copies. In some cases, amplified EGFR may contain 6 to 200 copies of the EGFR gene, e.g., 6 to 150, 6 to 100, 6 to 75, 6 to 50, 6 to 25, 6 to 10, 7 to 150, 7 to 100, 7 to 75, 7 to 50, 7 to 50, 7 to 25, 8 to 150, 8 to 100, 8 to 75, 8 to 50, 8 to 25, 8 to 10, 10 to 150, 10 to 100, 10 to 75, 10 to 50, 10 to 25, 15 to 150, 15 to 100, 15 to 75, 15 to 50, 15 to 25, 20 to 150, 20 to 100, 20 to 75, or 20 to 50 copies of the EGFR gene. In non-limiting cases, amplified EGFR may contain 7 to 100 copies of the EGFR gene. Gene amplification results in overexpression of EGFR in cells (e.g., tumor cells). In some cases, at least one copy of EGFR contains an activating mutation (e.g., a substitution, insertion, or deletion that activates EGFR).

[0123] Heterogeneous: Derived from a different genetic source. A heterogeneous nucleic acid molecule to a cell that originates from a genetic source other than that of that cell. In specific, non-limiting examples, heterogeneous nucleic acid molecules encoding proteins, such as humanized monoclonals or fragments thereof, are expressed in cells such as mammalian cells. Methods for introducing heterogeneous nucleic acid molecules into cells or organisms are known in the art and include, for example, nucleic acid transformation, including electroporation, lipofection, particle gun acceleration, and homologous recombination.

[0124] Host cell: A cell capable of growing a vector and expressing its DNA. The cell can be a prokaryote or a eukaryote. This term also includes any offspring of the target host cell. It is understood that not all offspring may be identical to the parent cell due to the possibility of mutations occurring during replication. However, such offspring are included when the term “host cell” is used.

[0125] IgG: A polypeptide belonging to a class or isotype of antibody substantially encoded by a recognized immunoglobulin gamma gene. In humans, this class includes IgG1, IgG2, IgG3, and IgG4.

[0126] Immune complexes: The binding of antibodies or antigen-binding fragments (such as scFv) to soluble antigens forms immune complexes. The formation of immune complexes can be detected by conventional methods, such as immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, ELISA, immunoblotting (e.g., Western blotting), magnetic resonance imaging, CT scans, radiography, and affinity chromatography.

[0127] Immune response: The response of cells in the immune system, such as B cells, T cells, or monocytes, to a stimulus. The response can be specific to a particular antigen ("antigen-specific response"). In some cases, the immune response is a T-cell response, such as a CD4+ response or a CD8+ response. In other cases, the response is a B-cell response, resulting in the production of specific antibodies.

[0128] Immunogen: A compound, composition, or substance that can stimulate antibody production or T cell response in animals, and which is injected into or absorbed by animals, for example, a composition comprising EGFRvIII conjugated to a carrier. An immunogen can be used to produce antibodies, such as those disclosed herein.

[0129] Tumor inhibition or treatment: Therapeutic interventions that reduce the signs or symptoms of the tumor (e.g., administration of a therapeutically effective dose of a humanized monoclonal antibody disclosed herein). In some cases, the treatment reduces the size or number of tumors, or induces remission. In certain cases, the treatment includes inhibiting metastasis.

[0130] The term "reduce" is a relative term used when a drug reduces a disease or condition quantitatively after administration, or when the disease or condition is reduced after administration compared to a reference drug. Relief of signs or symptoms refers to any observable beneficial effect of treatment. Relief of tumor-related signs or symptoms may be demonstrated, for example, by delay in the onset of clinical symptoms of the disease in a susceptible subject (such as a subject with a tumor that has not yet metastasized), reduction in the severity of some or all clinical symptoms of the disease, delay in disease progression (e.g., by extending the lifespan of a subject with a tumor), reduction in the number of tumors or the time between tumor removal and tumor recurrence, improvement in the subject's overall health or well-being, or other known parameters specific to a particular tumor.

[0131] "Prophylactic" treatment is administered to subjects who do not show signs of tumor but have a genetic predisposition to tumors, or who show only early signs such as precancerous lesions, with the aim of reducing the risk of developing a tumor. The term "preventive" does not necessarily mean that a drug completely eliminates a disease or condition, as long as at least one characteristic of the disease or condition is eliminated. Therefore, tumor-reducing or preventive compositions can prevent the risk of developing a tumor, although not necessarily completely.

[0132] Isolated: A biological component (e.g., nucleic acid, peptide, protein, or protein complex, e.g., antibody) that is substantially separated from the biological components of a cell or sample, produced separately, or purified. Therefore, isolated nucleic acids, peptides, and proteins include nucleic acids and proteins purified by standard purification methods. This term also includes nucleic acids, peptides, and proteins prepared by recombinant expression in host cells, as well as chemically synthesized nucleic acids. Isolated nucleic acids, peptides, or proteins, e.g., antibodies, may be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure.

[0133] Kabat et al. (Sequences of Proteins of Immunological Interest, 5 th The position of residues in an amino acid sequence according to the numbering rules described by Edition, Department of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda, NIH Publication No. 91-3242, 1991.

[0134] Linker: A bifunctional molecule that can be used to link two molecules to one adjacent molecule, for example, to link an effector molecule to an antibody or a detectable marker to an antibody. Non-limiting examples of peptide linkers include glycine-serine linkers.

[0135] The terms “conjugate,” “join,” “bond,” or “link” refer to the process of combining two molecules into a single, continuous molecule; for example, linking two polypeptides into a single, continuous polypeptide, or covalently attaching an effector molecule, a detectable marker radionuclide, or another molecule to a polypeptide such as scFv. Linking may be by either chemical or recombinant means. “Chemical means” refers to a reaction between an antibody moiety and an effector molecule such that a covalent bond is formed between the two molecules to form a single molecule.

[0136] Neoplasm, cancer, or tumor: A neoplasm is an abnormal proliferation of tissue or cells resulting from excessive cell division. Neoplasmic proliferation can lead to tumors. The amount of tumor in an individual is called the "tumor burden," which can be measured as the number, volume, or weight of tumors. Tumors that do not metastasize are called "benign." Tumors that can invade surrounding tissues or metastasize (or both) are called "malignant."

[0137] Tumors of the same histological type are primary tumors that originate in a specific organ or part of the body (such as the head and neck, breast, pancreas, colon or rectum, central nervous system (CNS), or bladder). Tumors of the same histological type can be classified into different subtypes. For example, lung carcinoma can be classified into adenocarcinoma, small cell carcinoma, squamous cell carcinoma, or non-small cell carcinoma.

[0138] Examples of solid tumors, such as sarcomas (connective tissue carcinomas) and carcinomas (epithelial cell carcinomas), include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synoviomas, mesotheliomas, Ewing's tumors, leiomyosarcomas, rhabdomyosarcomas, colorectal cancers, lymphoid malignancies, pancreatic cancers, breast cancers, head and neck cancers, lung cancers, ovarian cancers, prostate cancers, hepatocellular carcinomas, squamous cell carcinomas, basal cell carcinomas, adenocarcinomas, sweat gland carcinomas, and thyroid cancers. This includes medullary carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver carcinoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular cancer, seminoma, bladder cancer, and CNS tumors (such as glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma).

[0139] Nucleic acids: polymers composed of nucleotide units (e.g., ribonucleotides, deoxyribonucleotides) linked via phosphodiester bonds, associated naturally occurring structural variants, and their synthetic non-natural analogs. Therefore, this term includes nucleotide polymers in which nucleotides and the links between them are not naturally occurring synthetic analogs, such as, but are not limited to, phosphorothioates, phosphoramides, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, peptide-nucleic acid (PNA), etc. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term “oligonucleotide” typically refers to short polynucleotides, generally about 50 nucleotides or less. When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), it will be understood that this also includes RNA sequences (i.e., A, U, G, C) in which “U” replaces “T”.

[0140] A "nucleotide" includes, but is not limited to, monomers containing a base linked to a sugar, such as pyrimidines, purines, or their synthetic analogs, or a base linked to an amino acid, such as peptide nucleic acid (PNA). A nucleotide is a single monomer in a polynucleotide. A nucleotide sequence refers to the sequence of bases in a polynucleotide.

[0141] Conventional notation is used in this specification to describe nucleotide sequences. The left end of a single-stranded nucleotide sequence is the 5' end. The leftward direction of a double-stranded nucleotide sequence is called the 5' direction. The direction of nucleotide addition from 5' to 3' into a nascent RNA transcript is called the transcription direction. The DNA strand having the same sequence as the mRNA is called the "coding strand." The sequence on the DNA strand that has the same sequence as the mRNA transcribed from that DNA and is located at the 5' to 5' end of the RNA transcript is called the "upstream sequence." The sequence on the DNA strand that has the same sequence as the RNA and is at the 3' to 3' end of the coding RNA transcript is called the "downstream sequence."

[0142] "cDNA" refers to DNA that is complementary to or identical to mRNA, either in single-stranded or double-stranded form.

[0143] "Code" refers to the inherent properties of a particular sequence of nucleotides in a polynucleotide such as a gene, cDNA, or mRNA, as well as the biological properties arising therefrom, that act as a template for the synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids. Thus, a gene codes for a protein if the transcription and translation of the mRNA produced by that gene produces a protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is typically provided in sequence listings, and the non-coding strand, used as a template for the transcription of the gene or cDNA, can be said to code for a protein or other product of that gene or cDNA. Unless otherwise specified, "nucleotide sequences that code for an amino acid sequence" include all nucleotide sequences that are degenerate variants of each other and code for the same amino acid sequence. Nucleotide sequences that code for proteins and RNA may contain introns.

[0144] When a polynucleotide whose sequence is the first sequence specifically hybridizes with a polynucleotide whose sequence is the second sequence, the first sequence is "antisense" with respect to the second sequence.

[0145] Operable linking: When a first nucleic acid sequence has a functional relationship with a second nucleic acid sequence, the first nucleic acid sequence is operably linked to the second nucleic acid sequence. For example, when a promoter, such as the CMV promoter, affects the transcription or expression of a coding sequence, the promoter is operably linked to the coding sequence. Generally, operably linked DNA is contiguous and, when two protein-coding regions need to be linked, they are within the same reading frame.

[0146] Pharmacopoeia-acceptable carriers: The pharmaceutically acceptable carriers used are conventional ones. Remington's Pharmaceutical Sciences, by EW Martin, Mack Publishing Co., Easton, PA, 19th Edition, 1995, describes compositions and formulations suitable for the pharmaceutical delivery of the disclosed humanized monoclonal antibodies and their antigen-binding fragments.

[0147] The properties of the carrier may depend on the specific mode of administration employed. For example, parenteral formulations typically contain injectable fluids that include pharmaceutically and physiologically acceptable fluids as excipients, such as water, saline, equilibrium salt solutions, aqueous dextrose, and glycerol. In the case of solid compositions (e.g., in the form of powders, pills, tablets, or capsules), conventional non-toxic solid carriers include, for example, pharmaceutical-grade mannitol, lactose, starch, or magnesium stearate. In addition to a biologically neutral carrier, the administered pharmaceutical composition may contain small amounts of non-toxic auxiliary substances, such as humectants or emulsifiers, preservatives, and pH buffers, such as sodium acetate or sorbitan monolaurate. In certain examples, the carrier is sterile and / or contained in a unit dosage form containing one or more measured doses of the composition, suspended or otherwise. The unit dosage form may be, for example, a sealed vial containing sterile contents, or a syringe for injection into a subject, or it may be lyophilized for subsequent solubilization and administration, or it may be solid or in controlled-release doses.

[0148] Polypeptide: Any chain of amino acids, regardless of length or post-translational modifications (e.g., glycosylation or phosphorylation). “Polypeptide” includes natural and non-natural amino acid polymers, including those in which one or more amino acid residues are non-natural amino acids, for example, artificial chemical mimics of corresponding natural amino acids. “Residue” refers to an amino acid or amino acid mimic incorporated into the polypeptide by an amide bond or amide bond mimic. Polypeptides have an amino terminus (N-terminus) and a carboxyl terminus (C-terminus). “Polypeptide” is used interchangeably with peptide or protein and is used herein to refer to a polymer of amino acid residues.

[0149] Polypeptide Modification: Polypeptides and peptides, such as antibodies, disclosed herein can be modified by various chemical techniques to produce derivatives that have essentially the same activity as unmodified peptides and, if necessary, other desirable properties. For example, the carboxylic acid group of a protein may be provided in the form of a pharmaceutically acceptable cation salt, whether at the carboxyl terminus or side chain, or esterified to C1-C 16 They may form esters, or the formula NR1R2 (wherein R1 and R2 are each independently H or C1-C 16 The amino group of the peptide may be converted to an amide (which is alkyl) or combined to form a heterocycle such as a five-membered or six-membered ring. The amino group of the peptide may be in the form of a pharmaceutically acceptable acid addition salt such as HCl, HBr, acetic acid, benzoic acid, toluenesulfonic acid, maleic acid, tartaric acid and other organic salts, regardless of whether it is at the amino terminus or side chain, or C1-C 16 It can be modified with alkyl or dialkylamino compounds, or further converted to amide compounds.

[0150] The hydroxyl group of the peptide side chain is C1-C using a well-known technique. 16 Alkyl or C1-C 16 It can be converted to an ester. The phenyl and phenol rings of the peptide side chains are one or more halogen atoms, such as F, Cl, Br or I, or C1-C 16 Alkyl, C1-C 16The peptide side chains may be substituted with alkoxys, carboxylic acids and their esters, or amides of such carboxylic acids. The methylene group of the peptide side chain may be extended to homologous C2-C4 alkylenes. The thiol may be protected with one of many well-known protecting groups, such as an acetamide group. Methods are described for introducing cyclic structures into peptides to select and provide conformational constraints that result in, for example, improved stability. For example, a C-terminal or N-terminal cysteine ​​may be added to the peptide, resulting in a peptide containing a disulfide bond upon oxidation, thus forming a cyclic peptide. Other peptide cyclization methods include the formation of thioethers as well as carboxyl-terminal and amino-terminal amides and esters.

[0151] Purified: The term "purified" is intended to be relative, not requiring absolute purity. Therefore, for example, a purified peptide preparation is one in which the peptide or protein (such as an antibody or a fragment thereof) is more enriched than that peptide or protein in its natural intracellular environment. In one embodiment, a preparation is purified so that the protein or peptide accounts for at least 50% of the total peptide or protein content of the preparation, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or more. In a non-limiting example, a preparation is purified so that the protein (e.g., an antibody disclosed herein) accounts for at least 70% of the total protein content of the preparation.

[0152] Recombinant: Recombinant nucleic acids have sequences that do not exist in nature or sequences that are created by artificially combining two sequence segments that are otherwise separated. This artificial combination can be achieved, for example, by chemical synthesis or by artificially manipulating isolated segments of nucleic acid molecules using genetic engineering techniques. Recombinant proteins have sequences that do not exist in nature or sequences that are created by artificially combining two sequence segments that are otherwise separated. In some examples, recombinant proteins are encoded by heterologous (e.g., recombinant) nucleic acids introduced into host cells, such as bacteria or eukaryotic cells. Nucleic acids can be introduced, for example, into an expression vector having a signal that can express the protein encoded by the introduced nucleic acid, or the nucleic acid can be incorporated into the host cell chromosome.

[0153] Sequence identity: The degree of similarity between amino acid or nucleic acid sequences. Sequence identity is frequently measured in terms of identity percentage (or similarity or homology). A higher percentage indicates that the two sequences are more similar. Homologous polypeptides, homologous species, or variants exhibit a relatively high degree of sequence identity when aligned using standard methods.

[0154] Methods for aligning sequences for comparison are known. Various programs and alignment algorithms exist, for example: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al., Computer Appls. in the Biosciences. This is described in 8,155-65,1992; and Pearson et al., Meth.Mol.Bio.24:307-31,1994. Altschul et al., J.Mol.Bio.215:403-10,1990, present a detailed examination of sequence alignment methods and homology calculations.

[0155] When aligned, the number of matches is determined by counting the number of positions where identical nucleotides or amino acid residues exist in both sequences. Percent sequence identity is determined by dividing the number of matches by the length of the sequence described in the identified sequence or the concatenated length (e.g., 100 consecutive nucleotides or amino acid residues from the sequence described in the identified sequence), and then multiplying the resulting value by 100. For example, a peptide sequence with 1166 matches when aligned with a test sequence having 1554 amino acids is 75.0% identical to the test sequence (1166 ÷ 1554 * 100 = 75.0). Percent sequence identity values ​​are rounded to two decimal places. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, and 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. Length values ​​are always integers.

[0156] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol.Biol.215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Maryland) and the internet, for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Instructions on how to determine sequence identity using this program are available on the NCBI website. In some examples, sequence identity is determined by using the BLAST program with default parameters.

[0157] Polypeptide homologs and variants are typically characterized by having at least about 75% sequence identity, for example, at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, when counted over the full-length alignment with the amino acid sequence of interest. Proteins with greater similarity to the reference sequence will show an increase in the percentage of identity when evaluated by this method, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When a smaller sequence than the entire sequence is compared for sequence identity, homologs and variants typically have at least 80% sequence identity over a short window of 10–20 amino acids and may have at least 85% or at least 90% or 95% sequence identity depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available on the NCBI website. Those involved will understand that these sequence identity ranges are provided for guidance purposes only. It is entirely possible that highly significant homologs may be obtained outside the provided ranges.

[0158] For nucleic acid sequence comparison, typically one sequence acts as the reference sequence compared to the test sequence. When using a sequence comparison algorithm, the test and reference sequences are entered into a computer, subsequence coordinates are specified as needed, and sequence algorithm program parameters are specified. Default program parameters are usually used.

[0159] As used herein, a reference to “at least 80% identity” means “at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity” to a particular reference sequence. As used herein, a reference to “at least 90% identity” means “at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity” to a particular reference sequence.

[0160] Specific binding: When referring to antibodies or antigen-binding fragments, this refers to a binding reaction that determines the presence of a target protein in the presence of a heterogeneous population of proteins and other biological agents. Therefore, under specified conditions, an antibody preferentially binds to a specific target protein, peptide, or polysaccharide (e.g., an antigen present on the surface of tumor cells, e.g., EGFRvIII) and does not bind in significant amounts to other proteins present in the sample or subject, including wild-type EGFR expressed on wild-type (non-tumor) cells from the same tissue. Antibodies such as those disclosed herein can specifically bind to EGFRvIII and / or forms of EGFR overexpressed on tumor cells (e.g., gene-amplified EGFR), but not to wild-type EGFR expressed on wild-type (non-tumor) cells from the same tissue. Specific binding can be determined by standard methods. For a description of immunoassay formats and conditions that can be used to measure specific immunoreactivity, see Harlow & Lane, Antibodies, A Laboratory Manual, 2 nd See ed. Cold Spring Harbor Publications, New York (2013).

[0161] Regarding antibody-antigen complexes, the specific binding of antigen and antibody is approximately 10 -7 Less than molar concentration, for example, about 10 -8 Less than molar concentration, 10 -9 Less than molar concentration, or even about 10 -10 K below molar concentration D It has. K D This refers to the dissociation constant of a given interaction, such as polypeptide-ligand interactions or antibody-antigen interactions. For example, in the case of a bimolecular interaction between an antibody or antigen-binding fragment and an antigen, it is the concentration of the individual components of the bimolecular interaction divided by the concentration of the complex.

[0162] Humanized monoclonal antibodies that specifically bind to epitopes on EGFRvIII are This refers to EGFRvIII proteins, substrates to which EGFRvIII is bound, or humanized monoclonal antibodies that substantially bind to EGFRvIII in or isolated from biological specimens, including cells or tissues expressing EGFRvIII. Of course, it is recognized that some degree of nonspecific interaction can occur between the antibody and non-targets (e.g., cells of the same tissue type that do not express wild-type EGFR). Typically, specific binding results in a much stronger association between the antibody and the protein or cell containing the antigen than between the antibody and the protein or cell lacking the antigen. Specific binding typically results in more than a twofold, e.g., more than fivefold, more than tenfold, or more than a hundredfold increase in the amount of binding antibody (per unit time) to the protein containing the target epitope or to cells or tissues expressing the target epitope, compared to proteins or cells or tissues lacking this epitope. Specific binding to a protein under such conditions requires an antibody selected for its specificity to a particular protein. Various immunoassay formats are suitable for selecting antibodies or other ligands that are specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies that are specifically immunoreactive to proteins.

[0163] Subjects: A category including living multicellular vertebrate organisms, humans, and non-human mammals. In some cases, subjects are mammalian subjects, e.g., humans, non-human primates, dogs, or cats. In non-restrictive cases, subjects are humans. In specific cases, subjects have cancer. In further cases, subjects requiring inhibition of tumor growth or metastasis are selected. For example, a subject may be diagnosed with a tumor expressing EGFRvIII and / or gene-amplified EGFR, such as head and neck cancer, breast cancer, pancreatic cancer, colon cancer, CNS cancer, or bladder cancer, and may require treatment.

[0164] T cells: White blood cells that are important for the immune response. T cells are CD4 + T cells and CD8 + Includes, but is not limited to, T cells. CD4+ T lymphocytes are immune cells that express CD4 on their surface. Also known as helper T cells, these cells help integrate immune responses, including antibody and killer T cell responses. Th1 and Th2 cells are functional subsets of helper T cells. Th1 cells secrete a set of cytokines, including interferon-gamma, and their primary function is to stimulate phagocytic defense against infection, particularly infections associated with intracellular microorganisms. Th2 cells secrete a set of cytokines, including interleukin (IL)-4 and IL-5, and their primary function is to stimulate IgE and eosinophil / mast cell-mediated immune responses and downregulate the Th1 response.

[0165] Therapeutic agents: Used in a general sense, including treatment agents, prophylactic agents, and replacement agents. Therapeutic agents are used to alleviate a specific set of conditions in an object with a disease or disorder, such as cancer.

[0166] Toxins: Effector molecules that induce cytotoxicity upon contact with cells. Specific and non-exclusive examples of toxins include, but are not limited to, abrins, lysines, auristatins (e.g., monomethyl auristatin E (MMAE; see, e.g., Francisco et al., Blood, 102:1458-1465, 2003)) and monomethyl auristatin F (MMAF; see, e.g., Doronina et al., BioConjugate Chem., 17:114-124, 2006), maytansinoids (e.g., DM1; see, e.g., Phillips et al., Cancer Res., 68:9280-9290, 2008), pseudomonas exotoxins (PE, e.g., PE35, PE37, PE38, and PE40), diphtheria toxin (DT), botulinum toxin, saporins, restrictosin or geronin, or modified toxins thereof, or other toxic agents that directly or indirectly inhibit cell proliferation or kill cells. For example, PE and DT are highly toxic compounds that typically result in death due to hepatotoxicity. However, PE and DT can be modified into forms suitable for use as immunotoxins by removing the toxin's natural targeting components (such as domain Ia of PE and the B chain of DT) and replacing them with different targeting moieties, such as antibodies.

[0167] Transformed: Transformed cells are cells into which nucleic acid molecules have been introduced by molecular biology techniques. As used herein, the terms transformation, etc. (e.g., transformation, transfection, transduction, etc.) encompass all techniques by which nucleic acid molecules can be introduced into such cells, including transduction by viral vectors, transformation by plasmid vectors, and DNA introduction by electroporation, lipofection, and particle gun acceleration.

[0168] Treatment or prevention of disease: for example, inhibiting the complete onset of a disease or condition in a subject at risk of or with a disease, such as a tumor. "Treatment" refers to a therapeutic intervention that relieves the signs or symptoms of a disease or pathological condition after it has begun to develop. The term "relief" with respect to a disease or pathological condition refers to the observable beneficial effect of the treatment. Beneficial effects may be demonstrated, for example, by delaying the onset of clinical symptoms of the disease in a susceptible subject, reducing the severity of some or all clinical symptoms of the disease, delaying disease progression, improving the subject's overall health or well-being, or other parameters specific to a particular disease. "Prophylactic" treatment is a treatment administered to a subject who shows no signs of the disease or only early signs, with the aim of reducing the risk of developing the condition.

[0169] A vector is a nucleic acid molecule (such as a DNA or RNA molecule) that has a promoter(s) that can be operationally linked to the coding sequence of a target protein and express the coding sequence. Non-limiting examples include naked or packaged (lipid and / or protein) DNA, naked or packaged RNA, a non-replicating subcomponent of a virus, bacterium, or other microorganism, or a virus, bacterium, or other microorganism that may be replicable. A vector is sometimes called a construct. A recombinant DNA vector is a vector that contains recombinant DNA. A vector may contain nucleic acid sequences that enable replication within a host cell, such as an origin of replication. A vector may also contain one or more selective marker genes and other genetic elements. A viral vector is a recombinant nucleic acid vector that contains at least several nucleic acid sequences derived from one or more viruses. In some examples, viral vectors contain nucleic acid molecules that encode a humanized monoclonal antibody or antigen-binding fragment of disclosure.

[0170] Under conditions sufficient for: A phrase used to describe any environment that makes the desired activity possible.

[0171] III. Overview of Some Aspects of the Disclosure Isolated humanized monoclonal antibodies and their antigen-binding fragments are provided that specifically bind to mutant / variant EGFR expressed by cancer / tumor cells (e.g., EGFRvIII and / or gene-amplified EGFR). In some embodiments, the humanized monoclonal antibodies or antigen-binding fragments disclosed herein specifically bind to EGFRvIII. In some embodiments, the humanized monoclonal antibodies or antigen-binding fragments disclosed herein specifically bind to gene-amplified EGFR. In some embodiments, the humanized monoclonal antibodies or antigen-binding fragments disclosed herein specifically bind to EGFRvIII but not to gene-amplified EGFR. In some embodiments, the humanized monoclonal antibodies or antigen-binding fragments disclosed herein specifically bind to gene-amplified EGFR but not to EGFRvIII. In some embodiments, the humanized monoclonal antibodies or antigen-binding fragments disclosed herein specifically bind to cells expressing EGFRvIII and / or gene-amplified EGFR but not to wild-type EGFR. In some embodiments, the antibodies disclosed herein bind to EGFR exposed on the cell surface. 287-302 It binds to cells that have loops (e.g., tumor cells).

[0172] In some embodiments, the antibodies disclosed herein bind to mammalian EGFRvIII and / or mammalian gene-amplified EGFR, for example, EGFRvIII and / or gene-amplified EGFR of humans, non-human primates (e.g., anthropoids (Simiiformes), chimpanzees (Pan troglodytes), bonobos (Pan paniscus)), dogs (e.g., Canis lupus familiaris), or cats (e.g., Felis catus). In non-limiting examples, the antibodies disclosed herein bind to human EGFRvIII and / or human gene-amplified EGFR.

[0173] In some embodiments, the monoclonal antibodies and antigen-binding fragments provided herein may be used to treat tumors expressing EGFRvIII, for example, but not limited to, head and neck cancer, breast cancer, pancreatic cancer, colorectal or rectal cancer, CNS cancer, or bladder cancer. In some embodiments, the monoclonal antibodies and antigen-binding fragments may be used to treat tumors expressing gene-amplified EGFR, for example, but not limited to, head and neck cancer, breast cancer, pancreatic cancer, colorectal or rectal cancer, CNS cancer, or bladder cancer. The monoclonal antibodies and antigen-binding fragments are useful for treating tumors in human or veterinary subjects (e.g., non-human primates, dogs, cats).

[0174] Compositions comprising antibodies or antigen-binding fragments disclosed herein and pharmaceutically acceptable carriers are also disclosed. Nucleic acids encoding antibodies or antigen-binding fragments, expression vectors comprising these nucleic acids, and isolated host cells expressing nucleic acids are also provided.

[0175] Compositions comprising monoclonal antibodies or antigen-binding fragments disclosed herein may be used, for example, for research, diagnostic, and / or therapeutic purposes. For example, monoclonal antibodies may be used to diagnose and / or treat subjects having tumors expressing EGFRvIII and / or amplified EGFR. Subjects may be human or veterinary subjects (e.g., non-human primates, dogs, cats).

[0176] A. Antibodies and antigen-binding fragments Isolated humanized monoclonal antibodies and antigen-binding fragments that specifically bind to the epitopes of EGFRvIII and / or gene-amplified EGFR are provided. These antibodies are humanized antibodies and / or variants of the parental monoclonal antibody 40H3 (see U.S. Patent Application Publication No. 2022-0380474). In some embodiments, the isolated humanized monoclonal antibodies or antigen-binding fragments disclosed herein specifically bind to the epitopes of EGFRvIII. In some embodiments, the isolated humanized monoclonal antibodies or antigen-binding fragments disclosed herein specifically bind to the epitopes of gene-amplified EGFR. The disclosed antibodies contain one or more humanized residues and therefore contain one or more changes in the framework region compared to the parental mouse monoclonal antibody 40H3. In some embodiments, the disclosed humanized monoclonal antibodies are less immunogenic than the parental monoclonal antibody 40H3. The disclosed monoclonal antibody, surprisingly, exhibits increased binding to EGFRvIII compared to the parent monoclonal antibody 40H3.

[0177] In some embodiments, the disclosed humanized antibodies and antigen-binding fragments can inhibit the biological function or properties of the EGFRvIII protein in vivo, including, but not limited to, reduction and / or inhibition of tumor growth or reduction and / or inhibition of tumor metastasis. The disclosed humanized monoclonal antibodies and antigen-binding fragments preferentially bind to EGFRvIII and overexpressed EGFR expressed on tumor cells (e.g., gene-amplified EGFR), but substantially do not bind to EGFR expressed on wild-type (non-cancerous) cells. In some embodiments, the disclosed humanized monoclonal antibodies and antigen-binding fragments bind to EGFR 287-302 Join to the loop.

[0178] One structural element of EGFR that is not sterically available under normal conditions (e.g., when expressed on healthy, non-cancerous cells) is the disulfide restriction loop at residues 287–302 (the numbering refers to residues 301–326 in the mature receptor, or full-length receptor). EGFR 287-302 The loop is exposed during EGFRvIII. In some embodiments, the disclosed humanized monoclonal antibody is EGFR 287-302 Join the loop. In some examples, EGFR 287-302 The loop includes R300. The disclosed antibody is humanized and therefore contains one or more changes in the framework region compared to the parental mouse monoclonal antibody 40H3.

[0179] The following discussion of monoclonal antibodies includes HCDR and LCDR, respectively. H and V L This refers to isolated monoclonal antibodies containing [specific amino acids]. Those involved will understand that various CDR numbering schemes (e.g., Kabat, Chothia, or IMGT numbering schemes) can be used to determine the CDR location. The amino acid sequences and CDR locations of the heavy and light chains of the disclosed monoclonal antibodies are shown herein using Kabat, IGMT, or Chothia numbering. However, those involved will readily understand the use of various CDR numbering schemes when referring to specific amino acids of the antibodies disclosed herein. Programs for identifying CDRs using Chothia, IGMT, or Kabat are publicly available.

[0180] The antibodies disclosed herein have a heavy chain variable region (V H ) and light chain variable region (V L ) includes. In some cases, the antibodies disclosed herein are V (except A1) as listed in Table 1. H and V L This includes combinations of the above. [Table 1] 1 MDA-MB-468 *Chimera: 40H3 antibody + human IgG1CH / human IgκCL derived mouse VH and VL

[0181] In some implementations, the antibodies disclosed herein are a) SEQ ID NO: 5 and SEQ ID NO: 8 (A10, VH3+VL3), respectively; b) Sequence ID 3 and Sequence ID 6 (A2, VH1 + VL1), respectively; c) Sequence ID 3 and Sequence ID 7 (A3, VH1+VL2), respectively; d) Sequence ID 3 and Sequence ID 8 (A4, VH1 + VL3), respectively; e) Sequence ID 4 and Sequence ID 6 (A5, VH2 + VL1), respectively; f) Sequence ID 4 and Sequence ID 7 (A6, VH2 + VL2), respectively; g) Sequence ID 4 and Sequence ID 8 (A7, VH2 + VL3), respectively; h) Sequence ID 5 and Sequence ID 6 (A8, VH3 + VL1), respectively; i) Sequence ID 5 and Sequence ID 7 (A9, VH3 + VL2), respectively; j) Sequence ID 1 and Sequence ID 9 (B1, 40H3 VH+VL-EG), respectively; k) Sequence ID 1 and Sequence ID 10 (B2, 40H3 VH+VL-DA), respectively; l) Sequence ID 4 and Sequence ID 11 (B3, VH2 + VL1 - DA), respectively; m) Sequence ID 4 and Sequence ID 12 (B4, VH2 + VL2 - DA), respectively; n) Sequence ID 5 and Sequence ID 11 (B5, VH3, and VL1-DA), respectively; o) Sequence ID 31 and Sequence ID 32 (C10), respectively; p) Sequence ID 5 and Sequence ID 14 (D2), respectively; or q) Sequence ID 5 and Sequence ID 24 (D3), respectively. The heavy chain variable region (V H ) and light chain variable region (V L ) includes.

[0182] Humanized monoclonal antibodies contain the amino acid shown as SEQ ID NO: 5 and SEQ ID NO: 8, respectively. H and V L May contain (A10, VH3+VL3). Humanized monoclonal antibodies contain the amino acids shown as SEQ ID NO: 3 and SEQ ID NO: 6, respectively. H and V L May contain (VH1+VL1). Humanized monoclonal antibodies contain the amino acids shown as SEQ ID NO: 3 and SEQ ID NO: 7, respectively. H and V L May contain (VH1+VL2). Humanized monoclonal antibodies contain the amino acids shown as SEQ ID NO: 3 and SEQ ID NO: 8, respectively. H and V L May contain (VH1+VL3). Humanized monoclonal antibodies contain the amino acids shown as SEQ ID NO: 4 and SEQ ID NO: 6, respectively. H and V L May contain (VH2+VL1). Humanized monoclonal antibodies contain the amino acids shown as SEQ ID NO: 4 and SEQ ID NO: 7, respectively. H and V L May contain (VH2+VL2). Humanized monoclonal antibodies contain the amino acids shown as SEQ ID NO: 4 and SEQ ID NO: 8, respectively. H and V L May contain (VH2+VL3). Humanized monoclonal antibodies contain the amino acids shown as SEQ ID NO: 5 and SEQ ID NO: 6, respectively. H and V L May contain (VH3+VL1). Humanized monoclonal antibodies contain the amino acids shown as SEQ ID NO: 5 and SEQ ID NO: 7, respectively. H and V L May contain (VH3+VL2). Humanized monoclonal antibodies contain the amino acids shown as SEQ ID NO: 1 and SEQ ID NO: 9, respectively. H and V Lmay include (40H3 VH+VL-EG). The humanized monoclonal antibodies each include V amino acids shown as SEQ ID NO: 1 and SEQ ID NO: 10 H and V L may include (40H3 VH+VL-DA). The humanized monoclonal antibodies each include V amino acids shown as SEQ ID NO: 4 and SEQ ID NO: 11 H and V L may include (VH2+VL1-DA). The humanized monoclonal antibodies each include V amino acids shown as SEQ ID NO: 4 and SEQ ID NO: 12 H and V L may include (VH2+VL2-DA). The humanized monoclonal antibodies each include V amino acids shown as SEQ ID NO: 5 and SEQ ID NO: 11 H and V L may include (VH3 and VL1-DA). The humanized monoclonal antibodies each include V amino acids shown as SEQ ID NO: 31 and SEQ ID NO: 32 H and V L may include (C10). The humanized monoclonal antibodies each include V amino acids shown as SEQ ID NO: 5 and SEQ ID NO: 14 H and V L may include (D2). The humanized monoclonal antibodies each include V amino acids shown as SEQ ID NO: 5 and SEQ ID NO: 24 H and V L may include (D3). In some embodiments, the antibodies disclosed herein bind to EGFRvIII and / or gene amplified EGFR. In some examples, the antibodies disclosed herein bind to EGFRvIII. In some examples, the antibodies disclosed herein bind to gene amplified EGFR.

[0183] The monoclonal antibodies and antigen-binding fragments of the present disclosure are humanized. The human framework regions, and mutations that can be made in the human antibody framework regions, can be introduced into the antibody using various methods (see, for example, U.S. Patent No. 5,585,089; Jones et al., Nature 321:522, 1986; Riechmann et al., Nature 332:323, 1988; Verhoeyen et al., Science 239:1534, 1988; Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285, 1992; Sandhu, Crit. Rev. Biotech. 12:437, 1992; and Singer et al., J. Immunol. 150:2844, 1993).

[0184] 1. Further description of antibodies and antigen-binding fragments The humanized monoclonal antibody can be of any isotype. The monoclonal antibody can be, for example, an IgM or IgG antibody, such as an IgG1, IgG2, IgG3, or IgG4. The class of the humanized monoclonal antibody disclosed herein can be switched to another. In one aspect, the nucleic acid molecule encoding V L or V H is isolated so as not to contain the nucleic acid sequence encoding the constant region of the light or heavy chain, respectively. Then, the nucleic acid molecule encoding V L or V H is operably linked to the nucleic acid sequence encoding C L or C H from a different class of immunoglobulin molecule. This can be achieved using a vector or nucleic acid molecule containing the C L or C H chain known in the art. For example, a humanized monoclonal antibody that was originally IgG can be class switched. Class switching can be used to convert one IgG subclass to another, for example, from IgG1 to IgG2, IgG3, or IgG4.

[0185] In some cases, the disclosed humanized antibodies may be prepared as antibody oligomers such as dimers, trimers, tetramers, pentamers, hexamers, heptamers, and octamers.

[0186] (a) Binding affinity In some aspects of this disclosure, the antibody or antigen-binding fragment is applied to EGFRvIII or gene-amplified EGFR, for example, 1.0 × 10⁶. -8 M or less, 5.0×10 -8 M or less, 1.0×10 -9 M or less, 5.0×10 -9 M or less, 1.0×10 -10 M or less, 5.0×10 -10 M or less, or 1.0 × 10 -11 Affinity below M (for example, K d It specifically binds with an affinity (measured by) of less than 6 nm. In non-limiting examples, the antibody or antigen binding fragments disclosed herein have an affinity (K) of less than 6 nm. d ) specifically binds to EGFRvIII or gene-amplified EGFR. In non-limiting examples, the antibodies or antigen-binding fragments disclosed herein have an affinity of less than 4 nm (K d ) specifically binds to the EGFRvIII protein. In some embodiments, the antibody or antigen-binding fragment disclosed herein binds to EGFRvIII or gene-amplified EGFR at an affinity (K) of 0.5 to 10 nanomoles, for example, 0.5 to 9 nm, 1 to 9 nm, 1 to 8 nm, 1 to 7 nm, 1 to 6 nm, 1 to 5 nm, 1 to 4 nm, 1 to 3 nm, 2 to 9 nm, 2 to 8 nm, 2 to 7 nm, 2 to 6 nm, 2 to 5 nm, 2 to 4 nm, 2 to 3 nm, 3 to 9 nm, 3 to 8 nm, 3 to 7 nm, 3 to 6 nm, 3 to 5 nm or 3 to 4 nm. d ) specifically binds. In non-limiting examples, the antibody or antigen-binding fragments disclosed herein have an affinity (K) of 1 nm to 6 nm. d ) specifically binds to EGFRvIII or gene-amplified EGFR. In non-limiting examples, the antibodies or antigen-binding fragments disclosed herein have an affinity (K) of 2 nm to 6 nm. dIt specifically binds to EGFRvIII or gene-amplified EGFR. In some examples, affinity is measured using Octet® Bio-Layer Interferometry (BLI) Platform. In some aspects of this disclosure, the antibody or antigen-binding fragment disclosed herein binds to the gene-amplified EGFR product, for example, at an affinity of 1.0 × 10⁻¹⁶. -8 M or less, 5.0×10 -8 M or less, 1.0×10 -9 M or less, 5.0×10 -9 M or less, 1.0×10 -10 M or less, 5.0×10 -10 M or less, or 1.0 × 10 -11 Affinity below M (for example, K d It binds specifically at (measured by ). In some examples, the binding affinity of the antibodies or antigen-binding fragments disclosed herein to EGFRvIII is 2 nm to 5 nm (e.g., 2 nm to 4 nm, 2 nm to 3 nm, 3 nm to 5 nm, or 3 nm to 4 nm).

[0187] In other examples, K d This can be measured, for example, by a radiolabeled antigen-binding assay (RIA) performed using the Fab version of the humanized monoclonal antibody of interest and its antigen, using known methods. In one assay, the solution-binding affinity of Fab to the antigen is measured in the presence of an unlabeled antigen in a titration series, with Fab at a minimum concentration. 125 I) The antigen is equilibrated with a labeled antigen, and then the bound antigen is captured on a plate coated with anti-Fab antibody (see, for example, Chen et al., J.Mol.Biol.293:865-881 (1999)). To establish the assay conditions, a MICROTITER® multiwell plate (Thermo Scientific) is coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), followed by blocking with 2% (w / v) bovine serum albumin in PBS at room temperature (approximately 23°C) for 2-5 hours. In a non-adsorbent plate (NUNC number 269620), 100 μM or 26 pM [125 I) Mix the antigen with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of anti-VEGF antibody Fab-12 in Presta et al., Cancer Res. 57:4593-4599 (1997)). Then incubate the Fab of interest overnight. However, incubation can be continued for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Then transfer the mixture to a capture plate for incubation at room temperature (e.g., 1 hour). Then remove the solution and wash the plate eight times with 0.1% polysorbate 20 (TWEEN®-20) in PBS. Once the plate is dry, add 150 μl / well of scintillant (MICROSCINT-20; Packard) and count the plate with a TOPCOUNT® gamma counter (Packard) for 10 minutes. Select concentrations of each Fab that give less than 20% of maximum binding for use in competitive binding assays.

[0188] In another assay, K dThis can be measured at 25°C using a surface plasmon resonance assay with approximately 10 response units (RU) of immobilized antigen using a CM5 chip, with BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc. ((BIAcore, Inc., Piscataway, NJ))). In short, the carboxymethylated dextran biosensor chip (CM5, BIACORE®, Inc.) is immobilized with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-Hy-(Hy-(Hy-Hy-Hy-Hy-Hy-Hy-Hy-Hy-Hy-Hy-Hy-Hy- It is activated with droxisuccinimide (NHS). The antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 l / min to obtain a coupled protein of approximately 10 response units (RU). Following antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic analysis, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected at a flow rate of approximately 25 l / min at 25°C into PBS containing 0.05% polysorbate 20 (TWEEN®-20) surfactant (PBST). The association rate (k on ) and dissociation rate (k off The equilibrium dissociation constant (Kd) is calculated using a simple one-to-one Langmuir coupled model (BIACORE® evaluation software version 3.2) by simultaneously fitting association and dissociation sensorgrams. off / k on It is calculated as follows. For example, see Chen et al., J.Mol.Biol.293:865-881(1999). The above surface plasmon resonance assay yields an on-rate of 10 6 M -1 s -1If it exceeds this, the on-rate can be determined by using fluorescence quenching techniques to measure the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2 at 25°C in the presence of increasing concentrations of antigen, as measured by a spectrometer such as a stop-flow equipped spectrophometer (Aviv Instruments) or an 8000 series SLM-AMINCO (Trademark) spectrophotometer (ThermoSpectronic).

[0189] (b) Multispecific antibody In some embodiments, the disclosed humanized monoclonal antibodies or antigen-binding fragments are included in multispecific antibodies, such as bispecific antibodies. Such multispecific antibodies can be produced by known methods, e.g., crosslinking two or more antibodies, or by antigen-binding fragments of the same or different types (e.g., scFv). Exemplary methods for producing multispecific antibodies include those described in PCT Publication No. WO2013 / 163427. Suitable crosslinkers include heterobifunctional crosslinkers having two distinct reactive groups separated by a suitable spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl severate). Such linkers are available from Pierce Chemical Company in Rockford, Illinois.

[0190] In some embodiments, the disclosed humanized monoclonal antibody or antigen-binding fragment is contained in a bispecific antibody that specifically binds to EGFRvIII and / or gene-amplified EGFR, and further specifically binds to a second tumor antigen, e.g., Her-2, or a checkpoint inhibitor, e.g., programmed death (PD)-1, or a PD ligand (PD-L1) or PD-L2. In some examples, the bispecific antibody further binds to a MET oncogene antigen (see, e.g., Comoglio et al., "Known and novel roles of the MET oncogene in cancer: a coherent approach to targeted therapy," Nature Reviews Cancer, 18:341-358, 2018).

[0191] Various types of multispecific antibodies are known. Bispecific single-chain antibodies can be encoded by a single nucleic acid molecule. Examples of bispecific single-chain antibodies, as well as methods for constructing such antibodies, are described (see, for example, U.S. Patents Nos. 8,076,459, 8,017,748, 8,007,796, 7,919,089, 7,820,166, 7,635,472, 7,575,923, 7,435,549, 7,332,168, 7,323,440, 7,235,641, 7,229,760, 7,112,324, and 6,723,538). Further examples of bispecific single-chain antibodies can be found in PCT application number WO99 / 54440; Mack, J.Immunol., 158:3965-3970, 1997; Mack, PNAS, 92:7021-7025, 1995; Kufer, Cancer Immunol.Immunother., 45:193-197, 1997; Loffler, Blood, 95:2098-2103, 2000; and Bruhl et al., J.Immunol., 166:2420-2426, 2001. The production of bispecific Fab-scFv ("bibody") molecules has been described, for example, by Schoonjans et al. (J. Immunol., 165:7050-57, 2000) and Willems et al. (J. Chromatogr. B Analyt. Technol. Biomed Life Sci. 786:161-76, 2003). In the case of biobodies, the scFv molecule can be fused to either the VL-CL(L) chain or the VH-CH1 chain to create a biobody in which, for example, the scFv is fused to the C-terminus of the Fab chain.

[0192] (c) Antigen-binding fragment Antigen-binding fragments (e.g., Fab or scFv) of any monoclonal antibody (e.g., A10) disclosed herein are also disclosed. The antigen-binding fragments disclosed herein comprise heavy chain and light chain variable regions and specifically bind to EGFRvIII and / or gene-amplified EGFR. The antibody fragments retain the ability to selectively bind to antigens (e.g., EGFRvIII and / or gene-amplified EGFR), for example: (1) Fab, which is a fragment containing a monovalent antigen-binding fragment of an antibody molecule, can be produced by digesting the entire antibody with the enzyme papain to obtain an intact light chain and a portion of one heavy chain. (2) Fab', which is a fragment of the antibody molecule, can be obtained by treating the entire antibody with pepsin and then reducing it to obtain intact light and heavy chain fragments. Two Fab' fragments are obtained for each antibody molecule. (3)(Fab')2, a fragment of antibody that can be obtained by treating the entire antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds. (4) Fv, a genetically engineered fragment containing a variable region of the light chain and a variable region of the heavy chain expressed as two strands, and its disulfide-linked form (dsFV); and (5) Single-chain antibodies (such as scFv) are defined as genetically modified molecules containing a light chain variable region and a heavy chain variable region, linked by a polypeptide linker suitable as a gene fusion single-chain molecule. scFv is a fusion protein in which the light chain variable region and the heavy chain variable region of an immunoglobulin are linked by a linker (see, for example, Ahmad et al., Clin.Dev.Immunol., 2012, doi:10.1155 / 2012 / 980250; Marbry, IDrugs, 13:543-549, 2010). V in scFv H Domain and V L The intramolecular orientation of the domain is not definitive for the antibody provided (e.g., for the multispecific antibody provided). Therefore, both possible configurations (V HDomain-Linker Domain-V L Domain; V L Domain-Linker Domain-V H You can use scFv with a domain. Other forms such as ds-scFv are also useful. (6) A dimer of a single-chain antibody (scFV2), defined as a dimer of scFV. This is also called a "mini-antibody." Methods for preparing these fragments are described (e.g., Harlow and Lane, Antibodies: A Laboratory Manual, 2 nd (See Cold Spring Harbor Laboratory, New York, 2013).

[0193] In a further embodiment, the antibody-binding fragment may be an Fv antibody, which is typically about 25 kDa and contains a complete antigen-binding site with three CDRs per heavy chain and each light chain. V In order to produce antibodies, V H and V L It can be expressed from two individual nucleic acid constructs in the host cell. H and V L When expressed non-adjacent to each other, the Fv antibody chains are typically held together by non-covalent interactions. However, since these chains tend to dissociate upon dilution, methods have been developed to crosslink the chains via glutaraldehyde, intermolecular disulfides, or peptide linkers. Thus, in one example, Fv may be a disulfide-stabilized Fv (dsFv) in which the heavy-chain variable region and the light-chain variable region are chemically linked by disulfide bonds.

[0194] In a further example, the Fv fragment is linked to V by a peptide linker. H Chain and V L These single-chain antigen-binding proteins (scFv) are linked by an oligonucleotide. H Domain and V LIt is prepared by constructing a nucleic acid molecule that encodes the domain. The nucleic acid molecule is inserted into an expression vector and then introduced into a host cell, such as a mammalian cell. The recombinant host cell synthesizes a single polypeptide chain having a linker peptide that cross-links the two V domains. Methods for producing scFv have been described to date (see, for example, Whitlow et al., Methods: a Companion to Methods in Enzymology, Vol. 2, page 97, 1991; Bird et al., Science 242:423, 1988; U.S. Patent No. 4,946,778; Pack et al., Bio / Technology 11:1271, 1993; Ahmad et al., Clin. Dev. Immunol., 2012, doi:10.1155 / 2012 / 980250; Marbry, IDrugs, 13:543-549, 2010). Dimers of single-chain antibodies (scFV2) are also intended.

[0195] Antigen-binding fragments can be prepared by proteolytic hydrolysis of antibodies or by expression of the DNA encoding the fragment in host cells (e.g., E. coli cells). Antigen-binding fragments can also be obtained by pepsin or papain digestion of the whole antibody by conventional methods. For example, an antigen-binding fragment can be generated by enzymatic cleavage of an antibody with pepsin to provide a 5S fragment represented by F(ab')2. This fragment can be further cleaved using a thiol reducing agent and, optionally, a blocking group for the sulfhydryl group resulting from the cleavage of the disulfide bond to produce a 3.5S Fab' monovalent fragment. Alternatively, enzymatic cleavage using pepsin directly produces two monovalent Fab' fragments and one Fc fragment (see, for example, U.S. Patent Nos. 4,036,945 and 4,331,647; Nisonhoff et al., Arch. Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959; Edelman et al., Methods in Enzymology, Vol. 1, page 422, Academic Press, 1967; and Coligan et al., sections 2.8.1-2.8.10 and 2.10.1-2.10.4).

[0196] Other methods of cleaving the antibody, such as separation of the heavy chain to form monovalent light-heavy chain fragments, further cleavage of the fragment, or other enzymatic, chemical, or genetic techniques, can also be used, as long as the fragment binds to the antigen recognized by the intact antibody.

[0197] Antigen-binding single-V antibodies called domain antibodies (dAbs) H The domain was also amplified from the genomic DNA of immune mice. HThese have been identified from gene libraries (Ward et al., Nature 341:544-546, 1989). Human monoimmunoglobulin variable domain polypeptides capable of binding to antigens with high affinity have also been described (see, for example, PCT Public International Publication No. 2005 / 035572 and International Publication No. 2003 / 002609). The CDRs disclosed herein may also be included in dAb.

[0198] In some embodiments, one or more heavy chain and / or light chain complementarity-determining regions (CDRs) derived from the disclosed humanized monoclonal antibody are expressed on the surface of another protein, such as a scaffold protein. Expression of antibody domains on the surface of a scaffold protein has been described (see, e.g., Liu et al., J. Virology 85(17):8467-8476, 2011). Such expression creates a chimeric protein that retains binding to EGFRvIII and / or gene-amplified EGFR. In certain non-limiting examples, one or more heavy chain CDRs, e.g., one or more of heavy chain CDR1, CDR2, and / or CDR3, are grafted onto the scaffold protein. One or more CDRs may also be contained in a diabody or another type of single-chain antibody molecule.

[0199] In some embodiments, the antigen-binding fragment disclosed herein is an scFv. In some examples, the antigen-binding fragment comprises an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 100%) sequence identity with SEQ ID NO: 43 or 45. In some examples, the antigen-binding fragment comprises or consists of SEQ ID NO: 43 or 45. In some embodiments, the antigen-binding fragment disclosed herein is a Fab. In some examples, the antigen-binding fragment comprises an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 100%) sequence identity with SEQ ID NO: 47 and / or 49. In some examples, the antigen-binding fragment comprises or consists of SEQ ID NO: 47 and / or 49.

[0200] (d) Variant The amino acid sequence variants of humanized antibodies provided herein are included in this disclosure. For example, it may be desirable to further improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of humanized monoclonal antibodies can be prepared by introducing appropriate modifications to the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions and substitutions may be performed to arrive at the final construct, as long as the final construct has the desired features, for example, specific binding to EGFRvIII and / or gene-amplified EGFR.

[0201] In certain embodiments, antibody variants having one or more amino acid substitutions are provided. The target sites for substitutional mutagenesis include CDR and framework regions. The amino acid substitutions are introduced into the humanized monoclonal antibody of interest, and the product can be screened for desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0202] Variants are typically V H Region and V L It retains the amino acid residues necessary for correct folding and stabilization between regions, and preserves the charge properties of the residues to maintain a low pI and low toxicity of the molecule. To increase yield, V H Region and V L Amino acid substitutions can be performed within a given region. A table of conserved amino acid substitutions that provides functionally similar amino acids is known; for example, the following six groups are examples of amino acids that are considered to be conserved substitutions with respect to one another. 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), leucine (L), methionine (M), valine (V); and 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0203] In some embodiments, the antibody heavy chain contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conserved amino acid substitutions) compared to the amino acid sequence shown as any of SEQ ID NOs: 3, 4, 5 and / or 31. In some embodiments, the antibody light chain contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conserved amino acid substitutions) compared to the amino acid sequence shown as any of SEQ ID NOs: 6, 7, 8, 9, 10, 11, 12, 14, 24, or 32.

[0204] In some embodiments, the antibody or antigen-binding fragment may contain up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions) in the framework region of the antibody heavy chain and / or the framework region of the antibody light chain, or in the framework regions of the antibody heavy and light chains, compared to known framework regions or compared to the framework regions of antibodies disclosed herein, and may maintain specific binding activity to EGFRvIII proteins and / or gene-amplified EGFR products.

[0205] In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, provided that such changes do not substantially reduce the antibody's ability to bind to the target antigen (e.g., EGFRvIII and / or gene-amplified EGFR). For example, conservative changes that do not substantially reduce binding affinity (e.g., conservative substitutions provided herein) may be made in the CDRs. In certain examples of the variant VH and VL sequences provided above, each CDR is either unchanged or contains one, two, or three or fewer amino acid substitutions. In some examples, amino acid modifications may be made to the L-CDR2.

[0206] To increase the binding affinity of the antibody, V L Segments and V H The segments can be randomly mutated within the H-CDR3 or L-CDR3 region, etc., in a process similar to the in vivo somatic mutation process responsible for antibody affinity maturation during the innate immune response. Therefore, in vitro affinity maturation can be performed using PCR primers complementary to H-CDR3 or L-CDR3, respectively. H and V L This can be achieved by amplifying the region. In this process, the primers are used, and the resulting PCR product has random mutations. H and / or V L V introduced in the CDR3 area H and V L The segment is "spiked" with a random mixture of four nucleotide bases at specific positions to encode the segment. These randomly mutated V H and V L The binding affinity to EGFRvIII and / or gene-amplified EGFR products can be determined by testing the segments. Methods for in vitro affinity maturation are described (see, for example, Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)).

[0207] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions may be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. In substitution, or in addition, the crystal structure of the antigen-antibody complex is used to identify contact points between the antibody and antigen. Such contact residues and adjacent residues may be targeted or excluded as candidates for substitution. Variants can be screened to determine whether they contain the desired properties.

[0208] In certain embodiments, a humanized monoclonal antibody or antigen-binding fragment is modified to increase or decrease the degree of glycosylation. The addition or deletion of glycosylation sites can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0209] If an antibody contains an Fc region, the carbohydrate bound to it can change. Native antibodies produced by mammalian cells typically contain branched oligosaccharides commonly bound to Asn297 of the CH2 domain of the Fc region by N-linking. See, for example, Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose bound to GlcNAc in the "stem" of the branched oligosaccharide structure. In some embodiments, modification of oligosaccharides in humanized monoclonal antibodies may be performed to create variants with specific improved properties.

[0210] One embodiment provides an antibody variant having a carbohydrate structure lacking fucose (directly or indirectly) bound to the Fc region. For example, the amount of fucose in such an antibody may be 1%–80%, 1%–65%, 5%–65%, or 20%–40%. The amount of fucose is determined by calculating the average amount of fucose in the glycans at Asn297 relative to the total amount of all sugar structures attached to Asn297 (e.g., complex hybrid and high-mannose structures) as measured by MALDI-TOF mass spectrometry, as described, for example, in International Publication No. 2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 of the Fc region. However, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to slight sequence variations in the antibody. Such fucosylated variants may have improved ADCC function. See, for example, U.S. Patent Publication No. 2003 / 0157108 (Presta, L.); U.S. Patent Publication No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include U.S. Patent Publication No. 2003 / 0157108; International Publication No. 2000 / 61739; International Publication No. 2001 / 29246; U.S. Patent Publication No. 2003 / 0115614; U.S. Patent Publication No. 2002 / 0164328; U.S. Patent Publication No. 2004 / 0093621; U.S. Patent Publication No. 2004 / 0132140; U.S. Patent Publication No. 2004 / 0110704; U.S. Patent Publication No. 2004 / 0110282 Including U.S. Patent Publication No. 2004 / 0109865; International Publication No. 2003 / 085119; International Publication No. 2003 / 084570; International Publication No. 2005 / 035586; International Publication No. 2005 / 035778; International Publication No. 2005 / 053742; International Publication No. 2002 / 031140; Okazaki et al., J.Mol.Biol.,336:1239-1249(2004); Yamane-Ohnuki et al., Biotech.Bioeng.87:614(2004).Examples of cell lines capable of producing defucosylated antibodies include Lec 13 CHO cells lacking protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. 2003 / 0157108, Presta, L., and International Publication No. 2004 / 056312, Adam et al., particularly Example 11), and knockout cell lines, e.g., alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng., 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and International Publication No. 2003 / 085107).

[0211] Antibody variants are further provided, for example, bisected oligosaccharides in which a branched oligosaccharide bound to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in International Publication 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and U.S. Patent Publication 2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide bound to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in International Publication 1997 / 30087 (Patel et al.); International Publication 1998 / 58964 (Raju, S.); and International Publication 1999 / 22764 (Raju, S.).

[0212] In some embodiments, the antibody contains one or more amino acid substitutions to optimize the antibody's in vivo half-life. The serum half-life of IgG Abs is regulated by the neonatal Fc receptor (FcRn). Therefore, in some embodiments, the antibody contains amino acid substitutions that increase binding to FcRn. Several such substitutions are known, e.g., the IgG constant region T250Q and M428L (e.g., Hinton et al., J Immunol., 176:346-356, 2006); M428L and N434S ("LS" mutation, e.g., Zalevsky, et al., Nature) See Biotechnology, 28:157-159, 2010; N434A (see, e.g., Petkova et al., Int.Immunol., 18:1759-1769, 2006); T307A, E380A, and N434A (see, e.g., Petkova et al., Int.Immunol., 18:1759-1769, 2006); and M252Y, S254T, and T256E (see, e.g., Dall'Acqua et al., J.Biol.Chem., 281:23514-23524, 2006).

[0213] In some embodiments, the constant region of an antibody contains one or more amino acid substitutions to optimize antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC is mediated primarily via a closely related set of Fcγ receptors. In some embodiments, the antibody contains one or more amino acid substitutions that increase binding to FcγRIIIa. Several such substitutions are known, for example, the substitutions S239D and I332E in the IgG constant region (see, e.g., Lazar et al., Proc. Natl., Acad. Sci. USA, 103:4005-4010, 2006); and S239D, A330L, and I332E (see, e.g., Lazar et al., Proc. Natl., Acad. Sci. USA, 103:4005-4010, 2006).

[0214] The above substitution combinations are also included to generate an IgG constant region with increased binding to FcRn and FcγRIIIa. These combinations increase the antibody half-life and ADCC. For example, such combinations may include antibodies having the following amino acid substitutions in the Fc region: (1) S239D / I332E and T250Q / M428L; (2) S239D / I332E and M428L / N434S; (3) S239D / I332E and N434A; (4) S239D / I332E and T307A / E380A / N434A; (5) S239D / I332E and M252Y / S254T / T256E; (6) S239D / A330L / I3 32E and T250Q / M428L; (7) S239D / A330L / I332E and M428L / N434S; (8) S239D / A330L / I332E and N434A; (9) S239D / A330L / I332E and T307A / E380A / N434A; or (10) S239D / A330L / I332E and M252Y / S254T / T256E.

[0215] In some cases, antibodies or their antigen-binding fragments are modified to be directly cytotoxic to infected cells, or to utilize natural defenses such as complement, antibody-dependent cytotoxicity (ADCC), or macrophage phagocytosis.

[0216] Humanized monoclonal antibodies may be further modified to include additional non-proteinoid moieties. Moieties suitable for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propropylene glycol homopolymers, prolipropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may offer advantages in production due to its stability in water. The polymers may have any molecular weight and may be branched or unbranched. The number of polymers bound to the antibody may vary, and if two or more polymers are bound, they may be the same or different molecules. In general, the number and / or types of polymers used in derivatization may be determined based on considerations including, but not limited to, the specific properties or functions of the antibody being improved, and whether the antibody derivative will be used therapeutically under defined conditions.

[0217] Humanized antibodies or antigen-binding fragments can be derivatized or linked to another molecule (such as another peptide or protein). Generally, antibodies or antigen-binding fragments are derivatized so that binding to the desired target is not adversely affected by derivatization or labeling. For example, a humanized antibody or antigen-binding fragment can be functionally linked (by chemical coupling, gene fusion, non-covalent bonding or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or diabody), a detectable marker, an effector molecule, or a protein or peptide that can mediate the association of the humanized antibody or antibody moiety with another molecule (e.g., a streptavidin core region or polyhistidine tag).

[0218] The disclosed herein also includes humanized antibodies that bind to the same epitope on EGFRvIII to which the humanized monoclonal antibodies provided herein bind. 287-302 Humanized monoclonal antibodies that bind to such epitopes on the loop can be identified in a binding assay (e.g., as described in the examples) based on their ability to cross-compete (e.g., competitively inhibit binding in a statistically significant manner) with the specific antibodies provided herein. 6 ×K D In the presence of higher competitive antibody concentrations, the competitive antibody is superior to the EGFR of the humanized monoclonal antibody of this disclosure. 287-302 If loop binding is inhibited by more than 50%, it is considered "competitive" for binding. In a non-limiting example, the same EGFR as the antibody of this disclosure. 287-302 Humanized antibodies that bind to epitopes on a loop are human monoclonal antibodies. Such humanized monoclonal antibodies can be prepared and isolated as described herein.

[0219] B. Conjugate Humanized monoclonal antibodies or antigen-binding fragments disclosed herein may be conjugated to drugs such as effector molecules or detectable markers using any number of means known in the art. Covalent or non-covalent bonding may be used. The conjugate includes, but is not limited to, molecules on which the effector molecule or detectable marker has covalently bonded to the humanized monoclonal antibodies or antigen-binding fragments disclosed herein. Agents may use chemotherapeutic agents, anti-angiogenic agents, toxins, 125 I, 32 P, 14 C, 3 H and 35 It will be understood that a variety of effector molecules and detectable markers, including (but not limited to) radioactive agents such as S, as well as other labels, target moieties, and ligands, may be used.

[0220] The selection of a specific effector molecule or detectable marker depends on the specific target molecule or cell and the desired biological effect. For example, an effector molecule could be a cytotoxic used to induce death in a specific target cell (such as tumor cells expressing EGFRvIII and / or gene-amplified EGFR).

[0221] The procedure for attaching an effector molecule or detectable marker to an antibody or antigen-binding fragment varies depending on the chemical structure of the effector. Polypeptides typically contain a variety of functional groups. For example, carboxylic acid (COOH), free amine (-NH2), or sulfhydryl (-SH) groups are available for reaction with suitable functional groups on the antibody to result in the binding of the effector molecule or detectable marker. Alternatively, the antibody or antigen-binding fragment may be derivatized to expose or bind additional reactive functional groups. Derivatization may involve the binding of several known linker molecules, such as those available from Pierce Chemical Company, Rockford, IL. The linker can be any molecule used to bind the humanized antibody or antigen-binding fragment to the effector molecule or detectable marker. The linker can form a covalent bond to both the humanized antibody or antigen-binding fragment and the effector molecule or detectable marker. Suitable linkers are known and include, but are not limited to, linear or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers. When the humanized antibody or antigen-binding fragment and the effector molecule or detectable marker are polypeptides, the linker can be attached to the constituent amino acids via their side chain groups (e.g., via disulfide bonds to cysteine) or via the amino and carboxyl groups of the alpha carbon of the terminal amino acids.

[0222] Furthermore, the linker may include a spacer element, which, if present, increases the size of the linker so as to increase the distance between the effector molecule or detectable marker and the humanized antibody or antigen-binding fragment. Exemplary spacers are known, as specified in U.S. Patents 7,964,5667, 498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725, and 5,530,097. This includes those listed in U.S. Patent Publications No. 5,521,284, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444, and 4,486,414, as well as those listed in U.S. Patent Publications No. 20110212088 and 20110070248.

[0223] In some examples, the conjugate includes a linker that ligates an effector molecule or detectable marker to a humanized monoclonal antibody or antigen-binding fragment disclosed herein. In some examples, the linker is cleavable under intracellular conditions such that cleavage of the linker releases the effector molecule or detectable marker from the humanized antibody or antigen-binding fragment in the intracellular environment. In other examples, the linker is not cleavable, and the effector molecule or detectable marker is released, for example, by antibody degradation. In some examples, the linker is cleavable by a cleaving agent present in the intracellular environment (e.g., within lysosomes, endosomes, or caveoleas). The linker may be a peptide linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, lysosome or endosomal proteases. In some embodiments, the peptide linker is at least two amino acid long or at least three amino acid long. In some examples, linkers are 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid lengths, e.g., 1-2, 1-3, 2-5, 3-10, 3-15, 1-5, 1-10, 1-15 amino acid lengths. Proteases may include cathepsins B and D, as well as plasmin, all of which are known to hydrolyze dipeptide drug derivatives, resulting in the release of active drugs within target cells (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). For example, peptide linkers cleavable by the thiol-dependent protease cathepsin-B may be used (e.g., phenylalanine-leucine or glycine-phenylalanine-leucine-glycine linkers). Other examples of linkers are described, e.g., in U.S. Patent No. 6,214,345. In certain cases, the peptide linkers that can be cleaved by intracellular proteases are valine-citrulline linkers or phenylalanine-lysine linkers (see, for example, U.S. Patent No. 6,214,345, which describes the synthesis of doxorubicin using a valine-citrulline linker).

[0224] In other embodiments, cleavable linkers are pH-sensitive, i.e., susceptible to hydrolysis at specific pH values. Typically, pH-sensitive linkers are hydrolyzable under acidic conditions. For example, acid-unstable linkers hydrolyzable within lysosomes (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitamides, orthoesters, acetals, ketals, or similar) may be used (see, for example, U.S. Patents 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661). Such linkers are relatively stable under neutral pH conditions, such as those in blood, but unstable below pH 5.5 or 5.0, which is the approximate pH of lysosomes. In certain cases, the hydrolyzable linker is a thioether linker (e.g., a thioether bonded to the therapeutic agent via an acylhydrazone linkage (e.g., U.S. Patent No. 5,622,929)).

[0225] In yet another embodiment, the linker can be cleaved under reducing conditions (e.g., a disulfide linker). Various disulfide linkers are known in this technology, including, for example, SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyldithio)toluene), SPDB and SMPT (e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (CWVogel ed., Oxford U. Press, 1987); Phillips et al., Cancer This includes those that can be formed using (see Res.68:92809290,2008). See also U.S. Patent No. 4,880,935.

[0226] In other examples, the linker is a malonic acid linker (Johnson et al., 1995, Anticancer Res. 15: 1387-93), a maleimide benzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1305-12).

[0227] In some cases, linkers are resistant to cleavage in the extracellular environment. For example, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 3% or less, or about 1% or less of the linker in a sample of the conjugates disclosed herein will be cleaved when the conjugate is present in the extracellular environment (e.g., in plasma). Whether a linker is resistant to cleavage in the extracellular environment can be determined, for example, by incubating a conjugate containing the linker of interest with plasma for a predetermined time (e.g., 2, 4, 8, 16, or 24 hours) and then quantifying the amount of free effector molecules or detectable markers present in the plasma. Various exemplary linkers that may be used in conjugates are described in International Publication No. 2004-010957, U.S. Patent Application Publication No. 2006 / 0074008, U.S. Patent Application Publication No. 2005 / 0238649, and U.S. Patent Application Publication No. 2006 / 0024317.

[0228] Humanized monoclonal antibodies or antigen-binding fragments disclosed herein may be derivatized, for example, by crosslinking two or more antibodies (of the same or different types, e.g., for the production of bispecific antibodies). Suitable crosslinkers include heterobifunctional (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide) or homobifunctional (e.g., disuccinimidyl verate) linkers having two distinct reactive groups separated by a suitable spacer. Such linkers are commercially available.

[0229] The humanized antibodies or antigen-binding fragments disclosed herein may be conjugated with low molecular weight drugs, such as monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), maytansine, maytansine derivatives including maytansine derivatives known as DM1 (also known as meltansine), deruxtecan, or other chemotherapeutic agents for producing antibody-drug conjugates (ADCs). The chemotherapeutic agents described herein can be conjugated with the provided antibodies to produce ADCs.

[0230] The humanized monoclonal antibodies or antigen-binding fragments disclosed herein may be conjugated with one or more small molecule toxins such as calicheamicin, mytansinoids, drastatin, auristatin, trichothecene, and CC1065, as well as derivatives of these toxins having toxic activity. Maytansin compounds suitable for use as the mytansinoid toxin moiety are available and may be isolated from natural sources according to known methods, produced using genetic engineering techniques (see Yu et al. (2002) PNAS99:7968-7973), or synthetically prepared mytansinol and mytansinol analogs according to known methods. Mytansinoids are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansin was first isolated from the East African shrub Meitenus serrata (U.S. Patent No. 3,896,111). Subsequently, it was discovered that certain microorganisms also produce mytansinoids, such as mytansinol and C-3 mytansinol ester (U.S. Patent No. 4,151,042). Synthetic mytansinol and its derivatives and analogs are, for example, U.S. Patents No. 4,137,230, No. 4,248,870, No. 4,256,746, No. 4,260,608, No. 4,265,814, No. 4,294,757, No. 4,307,016, No. 4,308,268, No. 4,308,269, No. 4,309, These are disclosed in U.S. Patent Nos. 428, 4,313,946, 4,315,929, 4,317,821, 4,322,348, 4,331,598, 4,361,650, 4,364,866, 4,424,219, 4,450,254, 4,362,663, and 4,371,533. Conjugates containing mytansinoids, methods for preparing them, and their therapeutic uses are disclosed, for example, in U.S. Patent Nos. 5,208,020, 5,416,064, 6,441,163 and European Patent No. 0425235.

[0231] Considering the numerous methods reported for conjugating various radiodiagnostic compounds, radiotherapeutic compounds, labels (such as enzymes or fluorescent molecules), toxins, and other drugs to antibodies, practitioners can determine a suitable method for conjugating a given drug to a humanized monoclonal antibody or antigen-binding fragment disclosed herein.

[0232] In specific, non-limiting examples, a conjugate may include a humanized monoclonal antibody that specifically binds to an EGFRvIII or gene-amplified EGFR product (or its antigen-binding fragment) as disclosed herein, a non-reducing thioester linker, and a mytansinoid toxin DM1. For example, a conjugate may include the structure shown below (where "mAb" refers to a humanized monoclonal antibody or its antigen-binding fragment): [ka]

[0233] In some examples, the effector molecule is auristatin, such as auristatin E (also known as a derivative of dorastatin-10) or its derivatives. Auristatin can be, for example, an ester formed between auristatin E and a keto acid. For instance, auristatin E can be reacted with paraacetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other exemplary auristatins include AFP, MMAF, and MMAE. Exemplary auristatin synthesis and structure are described in U.S. Patent Application Publication No. 2003 / 0083263, International Publication No. 04 / 010957, International Publication No. 02 / 088172, and U.S. Patents No. 7,498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, and 5 It is listed in Nos. 635,483, 5,599,902, 5,554,725, 5,530,097, 5,521,284, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444, and 4,486,414. Further descriptions of antibody-drug conjugates containing auristatin MMAE and methods for preparing such conjugates are provided, for example, in U.S. Patent Application Publications 2011 / 0268751, 2008 / 0305044, and 2007 / 0258987. Auristatin has been shown to interfere with microtubule dynamics as well as nuclear and cell division and possesses anticancer activity. Auristatin can bind to tubulin and exert cytotoxic or cell proliferation inhibitory effects on cells. There are several different assays known in the art that can be used to determine whether auristatin or the resulting conjugate exerts cell proliferation inhibitory or cytotoxic effects on a desired cell line.

[0234] For example, the conjugate comprises a humanized monoclonal antibody (or its antigen-binding fragment) that specifically binds to the EGFRvIII or gene-amplified EGFR product disclosed herein, a cleavable linker containing a valine-citrulline (Val-Cit) peptide cleavage site, a spacer, and a toxin MMAE. For example, the conjugate may include the structure shown below (where "mAb" refers to the monoclonal antibody or its antigen-binding fragment): [ka]

[0235] I In another non-limiting example, a conjugate is: [ka] The formula is as follows: n is an integer (such as an even integer) between 0 and 10 (e.g., 0 to 8, 0 to 4, 2 to 4, 2 to 8, 1 to 10, 1 to 8, or 1 to 4, or 2, 4, 6, or 8), A is a humanized monoclonal antibody or its antigen-binding fragment disclosed herein, and S is a sulfur atom derived from the humanized antibody. In one example, preferably, n is an even integer between 0 and 8, for example, an even integer between 0 and 4. The S portion may be exposed by reduction or partial reduction of the interchain disulfide of the humanized antibody (e.g., by treatment with a reducing agent such as DTT or TCEP).

[0236] In one non-restrictive example, a conjugate is, [ka] The formula is such that n is 4 and A is a monoclonal antibody or its antigen-binding fragment disclosed herein.

[0237] Further toxins may be used in conjunction with humanized monoclonal antibodies that specifically bind to EGFRvIII and / or gene-amplified EGFR, as well as antigen-binding fragments of these antibodies. Exemplary toxins include Pseudomonas exotoxin (PE), lysine, abrin, diphtheria toxin and its subunits, ribotoxin, ribonuclease, saporin and calicheamicin, and botulinum toxins A-F. These toxins are known and many are readily available from commercial sources (e.g., Sigma Chemical Company, St. Louis, Missouri). The intended toxins also include variants of the toxins (see, for example, U.S. Patents 5,079,163 and 4,689,401). In some embodiments, the conjugates disclosed herein are used for the treatment of carcinomas, such as head and neck carcinomas, breast carcinomas, pancreatic carcinomas, colorectal or rectal carcinomas, CNS carcinomas, or bladder carcinomas.

[0238] Saporins are toxins derived from Saponaria officinalis that disrupt protein synthesis by inactivating the 60S portion of ribosome complexes (Stirpe et al., Bio / Technology, 10:405-412, 1992). However, the toxins do not have a mechanism for specific cell entry and therefore require conjugate to antibodies or antigen-binding fragments that recognize internalized cell surface proteins in order to be efficiently taken up by cells.

[0239] Diphtheria toxin is isolated from Corynebacterium diphtheriae. Typically, diphtheria toxin for use as an immunotoxin is mutated to reduce or eliminate nonspecific toxicity. A variant known as CRM107, which has full enzymatic activity but significantly reduced nonspecific toxicity, has been known since the 1970s (Laird and Groman, J. Virol. 19:220, 1976) and has been used in human clinical trials. See U.S. Patents 5,792,458 and 5,208,021.

[0240] Lysine is the lectin RCA60 derived from Ricinus communis (castor bean). For examples of lysine, see U.S. Patent Nos. 5,079,163 and 4,689,401. Ricinus communis agglutinins (RCAs) are classified according to their molecular weights of approximately 65 and 120 kD, respectively. 60 and RCA 120 It occurs in two forms as specified (Nicholson and Blaustein, J. Biochim. Biophys. Acta 266:543, 1972). Chain A is responsible for inactivating protein synthesis and cell death. Chain B binds lysine to galactose residues on the cell surface and facilitates the transport of chain A into the cytosol (Olsnes et al., Nature 249:627-631, 1974 and U.S. Patent Nos. 3,060,165).

[0241] Ribonucleases have also been conjugated into targeted molecules for use as immunotoxins (see Suzuki et al., Nat. Biotech. 17:265-70, 1999). Exemplary ribotoxins such as α-salsin and restrictosin are discussed, for example, in Rathore et al., Gene 190:31-5, 1997, and Goyal and Batra, Biochem. 345 Pt 2:247-54, 2000. Calicheamicin, first isolated from Micromonospora echinospora, is a member of the enediin antitumor antibiotic family that induces double-strand breaks in DNA leading to apoptosis (e.g., Lee et al., J. Antibiot. 42:1070-87, 1989). The drug is the toxic portion of immunotoxins in clinical trials (see, for example, Gillespie et al., Ann. Oncol. 11:735-41, 2000).

[0242] Abrin contains toxic lectins derived from Abrus precatorius. Abrin a, b, c, and d, which are responsible for the toxicity, have a molecular weight of approximately 63-67 kD and consist of two disulfide-linked polypeptide chains A and B. Chain A inhibits protein synthesis, and chain B (abrin-b) binds to D-galactose residues (see Funatsu et al., Agr. Biol. Chem. 52:1095, 1988, and Olsnes, Methods Enzymol. 50:330-335, 1978).

[0243] For example, the toxin is Pseudomonas exotoxin (PE) (U.S. Patent No. 5,602,095). As used herein, PE includes full-length natural (naturally occurring) PE or modified PE. Such modifications include, but are not limited to, the removal of domain Ia, various amino acid deletions in domains Ib, II, and III, single amino acid substitutions, and the addition of one or more sequences at the carboxyl terminus (see, e.g., Siegall et al., J. Biol. Chem. 264:14256-14261, 1989). PE used with the provided antibody may include natural sequences, cytotoxic fragments of natural sequences, and conservatively modified variants of natural PE and its cytotoxic fragments. Cytotoxic fragments of PE include those that are cytotoxic with or without subsequent proteolysis or other processing in target cells. Cytotoxic fragments of PE include PE25, PE40, PE38, and PE35. For further descriptions of PE and its variants, see, for example, U.S. Patent Nos. 4,892,827; 5,512,658; 5,602,095; 5,608,039; 5,821,238; and 5,854,044; PCT Public International Publication No. 99 / 51643; Pai et al., Proc. Natl. Acad. Sci. USA, 88:3358-3362, 1991; Kondo et al., J. Biol. Chem., 263:9470-9475, 1988; Pastan et al., Biochim. Biophys. Acta, 1333:C1-C6, 1997.

[0244] Protease-resistant PE variants and immunogenic PE variants, such as PE-LR, PE-6X, PE-8X, PE-LR / 6X, and PE-LR / 8X, are also intended, though not limited to these (see, e.g., Weldon et al., Blood 113(16):3792-3800, 2009; Onda et al., Proc. Natl. Acad. Sci. USA, 105(32):11311-11316, 2008; and PCT Public International Publication No. 2007 / 016150, International Publication No. 2009 / 032954, and International Publication No. 2011 / 032022). The PE variant may be PE25 (see Weldon et al., Blood 2009;113:3792-3800).

[0245] In some cases, PE is a variant resistant to lysosomal degradation, such as PE-LR (Weldon et al., Blood 113(16):3792-3800, 2009; PCT Public Publication No. 2009 / 032954). In other cases, PE is a variant called PE-LR / 6X (PCT Public Publication No. 2011 / 032022). In yet another case, PE is a variant designated as PE-LR / 8M (PCT Public Publication No. 2011 / 032022).

[0246] Humanized monoclonal antibodies (or their antigen-binding fragments) that specifically bind to EGFRvIII and / or gene-amplified EGFR can also be conjugated with detectable markers; for example, detectable markers that can be detected by ELISA, spectrophotometry, flow cytometry, microscopy or imaging techniques (such as computed tomography (CT), computed axial tomography (CAT) scans, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance imaging (MTR), ultrasound, fiber optics, and laparoscopy). Specific non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzyme conjugates, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable markers include fluorescent compounds such as fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, and lanthanide phosphors. Bioluminescent markers such as luciferase, green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), and cyan fluorescent protein (CFP) are also useful. Humanized monoclonal antibodies or antigen-binding fragments can also be conjugated with enzymes useful for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, and glucose oxidase. When humanized monoclonal antibodies or antigen-binding fragments are conjugated with detectable enzymes, they can be detected by adding further reagents that the enzymes use to produce identifiable reaction products. For example, in the presence of the active substance horseradish peroxidase, the addition of hydrogen peroxide and diaminobenzidine results in a visually detectable colored reaction product. Humanized monoclonal antibodies or antigen-binding fragments can also be conjugated with biotin and can be detected by indirect measurement of avidin or streptavidin binding. Avidin itself can also be conjugated with enzymes or detectable markers.

[0247] Humanized monoclonal antibodies or antigen-binding fragments can be conjugated with paramagnetic agents such as gadolinium. Paramagnetic agents such as superparamagnetic iron oxide are also used as labels. Antibodies can also be conjugated with lanthanides (e.g., europium and dysprosium) and manganese. Humanized monoclonal antibodies or antigen-binding fragments can also be labeled with a predetermined polypeptide epitope that is recognized by a secondary reporter (e.g., leucine zipper pair sequence, secondary antibody binding site, metal-binding domain, epitope tag).

[0248] Humanized monoclonal antibodies or antigen-binding fragments can also be conjugated with radiolabeled amino acids. Radiolabeling can be used for both diagnostic and therapeutic purposes. For example, radiolabeling can be used to detect EGFRvIII and / or amplified EGFR, as well as cells expressing EGFRvIII and / or amplified EGFR, by X-ray, emission spectroscopy, or other diagnostic techniques. Furthermore, radiolabeling can be used therapeutically as a toxin for the treatment of tumors in a target, for example, for the treatment of any tumor expressing EGFRvIII and / or amplified EGFR, such as cancer, e.g., head and neck cancer, breast cancer, pancreatic cancer, colorectal cancer, CNS cancer, or bladder cancer. Examples of labeling for polypeptides include, but are not limited to, the following radioisotopes or radionucleotides: 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I.

[0249] Means for detecting detectable markers are also described; for example, radioactive labels can be detected using photographic film or a scintillation counter, and fluorescent markers can be detected using a photodetector to detect luminescence. Enzyme labels are typically detected by providing a substrate to an enzyme and detecting the reaction product produced by the enzyme's action on the substrate, and colorimetric labels are detected by simply visualizing the colored label.

[0250] The humanized monoclonal antibodies or antigen-binding fragments disclosed herein may also be derivatized with chemical groups such as polyethylene glycol (PEG), methyl or ethyl groups, or carbohydrate groups. These groups may be useful to improve the biological properties of the humanized antibodies or antigen-binding fragments, for example, to increase their serum half-life or to increase their tissue binding.

[0251] The average number of effector molecules or detectable marker moieties per humanized monoclonal antibody or antigen-binding fragment in a conjugate may range, for example, from 1 to 20 moieties per antibody or antigen-binding fragment. For some conjugates, the average number of effector molecules or detectable marker moieties per antibody or antigen-binding fragment may be limited by the number of binding sites on the humanized monoclonal antibody or antigen-binding fragment. For example, if the binding is cysteinethiol, the humanized monoclonal antibody or antigen-binding fragment may have only one or more cysteinethiol groups, or only one or more sufficiently reactive thiol groups to which a linker can bind. In certain examples, the average number of effector molecules or detectable marker moieties per humanized monoclonal antibody or antigen-binding fragment in the conjugate ranges from 1 to 10, for example, 2 to 6, 2 to 8, 3 to 5, 3 to 4, 3.1 to 3.9, 3.2 to 3.8, 3.2 to 3.7, 3.2 to 3.6, 3.3 to 3.8, or 3.3 to 3.7. In further examples, the average number of effector molecules or detectable marker moieties per humanized monoclonal antibody or antigen-binding fragment is approximately 1, approximately 2, approximately 2.5, approximately 3, approximately 3.5, approximately 4, approximately 4.5, approximately 5, approximately 5.5, approximately 6, approximately 6.5, approximately 7, approximately 7.5, approximately 8, approximately 8.5, or approximately 9. The average number of effector molecules or detectable marker moieties per humanized monoclonal antibody or antigen-binding fragment in the conjugate preparation can be characterized by conventional means such as mass spectrometry or ELISA assay.The conjugate load (e.g., effector molecule / antibody ratio) can be controlled in various ways, for example, by (i) limiting the molar excess of the effector molecule-linker intermediate or linker reagent relative to the antibody, (ii) limiting the conjugation reaction time or temperature, (iii) partial or restrictive reduction conditions for cysteine ​​thiol modification, or (iv) manipulating the amino acid sequence of the humanized antibody by recombinant techniques (e.g., thioMab or thioFab prepared as disclosed in International Publication No. 2006 / 03448) so that the number and position of cysteine ​​residues are modified for control of the number or position of linker-effector molecule attachments.

[0252] C. Chimeric antigen receptor (CAR) Chimeric antigen receptors (CARs) containing antibodies disclosed herein (e.g., humanized monoclonal antibodies or antigen-binding fragments specific to EGFRvIII and / or gene-amplified EGFR) are also disclosed herein. A CAR is an artificially constructed chimeric receptor protein comprising a transmembrane domain linked to one or more intracellular T cell signaling domains, an extracellular antigen-binding domain (e.g., a single-strand variable fragment (scFv)) that specifically binds to a target (e.g., EGFRvIII). Features of the disclosed CARs include the ability to redirect T cell specificity and reactivity to EGFRvIII and / or gene-amplified EGFR-expressing cells in a non-MHC-restricted manner. Non-MHC-restricted EGFRvIII and / or gene-amplified EGFR recognition gives immune cells (e.g., T cells) expressing the disclosed CARs the ability to recognize antigens independently of antigen processing.

[0253] Intracellular T cell signaling domains may include, for example, T cell receptor signaling domains, T cell costimulatory signaling domains, or both. A T cell receptor signaling domain refers to a portion of a CAR that includes the intracellular domain of a T cell receptor, such as the intracellular portion of the CD3 zeta protein. A costimulatory signaling domain refers to a portion of a CAR that includes the intracellular domain of a costimulatory molecule, which is a cell surface molecule other than the antigen receptor or its ligand necessary for the efficient response of lymphocytes to an antigen.

[0254] 1. Extracellular region The CAR comprises an antigen-binding domain that specifically binds to EGFRvIII and / or gene-amplified EGFR. For example, the antigen-binding domain may be an scFv comprising the heavy chain variable region and light chain variable region of any humanized monoclonal antibody or its antigen-binding fragment disclosed herein.

[0255] In some embodiments, EGFRvIII and / or gene-amplified EGFR-specific antibodies have a variable heavy chain region (V H ) and variable light chain region (V L ) contains and specifically binds to EGFRvIII and / or gene-amplified EGFR. In some cases, the heavy and light chains contained in the CAR are a) Sequence ID 5 and Sequence ID 8 (A10, VH3 + VL3), respectively; b) Sequence ID 3 and Sequence ID 6 (A2, VH1 + VL1), respectively; c) Sequence ID 3 and Sequence ID 7 (A3, VH1+VL2), respectively; d) Sequence ID 3 and Sequence ID 8 (A4, VH1 + VL3), respectively; e) Sequence ID 4 and Sequence ID 6 (A5, VH2 + VL1), respectively; f) Sequence ID 4 and Sequence ID 7 (A6, VH2 + VL2), respectively; g) Sequence ID 4 and Sequence ID 8 (A7, VH2 + VL3), respectively; h) Sequence ID 5 and Sequence ID 6 (A8, VH3 + VL1), respectively; i) Sequence ID 5 and Sequence ID 7 (A9, VH3 + VL2), respectively; j) Sequence ID 1 and Sequence ID 9 (B1, 40H3 VH+VL-EG), respectively; k) Sequence ID 1 and Sequence ID 10 (B2, 40H3 VH+VL-DA), respectively; l) Sequence ID 4 and Sequence ID 11 (B3, VH2 + VL1 - DA), respectively; m) Sequence ID 4 and Sequence ID 12 (B4, VH2 + VL2 - DA), respectively; n) Sequence ID 5 and Sequence ID 11 (B5, VH3, and VL1-DA), respectively; o) Sequence ID 31 and Sequence ID 32 (C10), respectively; p) Sequence ID 5 and Sequence ID 14 (D2), respectively; or q) Sequence ID 5 and Sequence ID 24 (D3), respectively. It contains the amino acid sequence described below.

[0256] Any antibody or antigen-binding fragment disclosed herein may be used in a CAR. The disclosed antibody or antigen-binding fragment is humanized. In a non-limiting example, the heavy and light chains contained in the CAR include SEQ ID NO: 5 and SEQ ID NO: 8, respectively.

[0257] In some embodiments, the antigen-binding fragment is an scFv. In some embodiments, the scFv includes a heavy-chain variable region and a light-chain variable region, which are linked by a peptide linker, such as a linker containing an amino acid sequence represented as GGGGSGGGGSGGGGS (SEQ ID NO: 33).

[0258] A CAR may, for example, contain a signal peptide sequence located at the N-terminus of its antigen-binding domain. The signal peptide sequence can be any suitable signal peptide sequence. For example, the signal peptide sequence may be an amino acid sequence containing or consisting of a human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor sequence, e.g., LLVTSLLLCELPHPAFLLIPDT (SEQ ID NO: 34). While the signal peptide sequence may promote CAR expression on the cell surface, the presence of the signal peptide sequence in the expressed CAR is not required for the CAR to function. When a CAR is expressed on the cell surface, the signal peptide sequence may be cleaved from the CAR. Therefore, in some examples, CARs lack a signal peptide sequence.

[0259] A spacer domain containing a polypeptide sequence may exist between the antigen-binding domain and the transmembrane domain of the CAR. The spacer domain may contain up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids. In some embodiments, the spacer domain may contain an immunoglobulin domain, such as a human immunoglobulin sequence. In one example, the immunoglobulin domain contains immunoglobulin CH2 and CH3 immunoglobulin G (IgG1) domain sequences (CH2CH3). In this regard, the spacer domain may contain or comprise an immunoglobulin domain comprising an amino acid sequence represented as Sequence ID No. 35:EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPK.

[0260] While not bound by any particular theory, the CH2CH3 domain is thought to potentially mimic the size and domain structure of native TCRs more accurately by moving the antigen-binding domain of the CAR away from the membrane of the CAR-expressing cell.

[0261] 2. Transmembrane domain Regarding the transmembrane domain, CARs can be designed to include a transmembrane domain fused to the extracellular domain of the CAR. For example, a transmembrane domain that naturally associates with one of the domains in the CAR is used.

[0262] The transmembrane domain may be derived from either a natural or synthetic source. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Exemplary transmembrane domains for use in the disclosed CAR may include at least the transmembrane region of the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CDDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. Alternatively, the transmembrane domain may be synthetic, in which case it mainly consists of hydrophobic residues such as leucine and valine. In some examples, a triplet of phenylalanine, tryptophan, and valine is found at each end of the synthetic transmembrane domain.

[0263] If necessary, a short oligopeptide linker or polypeptide linker, preferably 2 to 10 amino acids in length, may form a linkage between the transmembrane domain of the CAR and the intracellular T cell signaling domain and / or T cell costimulation domain. An exemplary linker sequence comprises one or more glycine-serine diresidues.

[0264] In some examples, the transmembrane domain includes the transmembrane domain of a T cell receptor, such as the CD8 transmembrane domain. Therefore, the CAR may include or consist of a CD8 transmembrane domain containing SEQ ID NO: 36:TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC.

[0265] In other examples, the transmembrane domain includes the transmembrane domain of a T cell costimulatory molecule, such as CD137 or CD28. Therefore, CAR may include or consist of a CD28 transmembrane domain containing SEQ ID NO: 37:IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVR.

[0266] 3. Intracellular region The intracellular domain of a CAR includes one or more intracellular T cell signaling domains involved in the activation of at least one of the normal effector functions of T cells in which the CAR is expressed or located. Exemplary T cell signaling domains are provided and described herein.

[0267] While the entire intracellular T cell signaling domain can be used in a CAR, it is often not necessary to use the entire chain. As long as a cleavage portion of the intracellular T cell signaling domain is used, such a cleavage portion can be used in place of the intact chain, provided that it transmits the relevant T cell effector functional signal.

[0268] Examples of intracellular T cell signaling domains for use in CAR include cytoplasmic sequences of T cell receptors (TCRs), costimulatory molecules that act in coordination to initiate signaling after antigen receptor engagement, as well as any derivatives or variants of these sequences, and any synthetic sequences having the same functional capabilities.

[0269] T cell receptor signaling domains modulate primary activation of the T cell receptor complex either stimulatively or inhibitorily. The disclosed CARs may contain primary cytoplasmic signaling sequences that act stimulatively, which may contain signaling motifs known as immune receptor tyrosine activation motifs or ITAMs. Examples of ITAM-containing primary cytoplasmic signaling sequences that may be contained in the disclosed CARs include those derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, and CD66d proteins. In some examples, the cytoplasmic signaling molecules in the CARs include intracellular T cell signaling domains derived from CD3 zeta.

[0270] The intracellular domain of a CAR may contain an ITAM-containing primary cytoplasmic signaling domain (e.g., CD3-zeta) alone or in combination with any other desired cytoplasmic domain useful in relation to the CAR. For example, the cytoplasmic domain of a CAR may include a CD3-zeta chain portion and an intracellular costimulatory signaling domain. The costimulatory signaling domain refers to the portion of the CAR that contains the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands necessary for the efficient response of lymphocytes to antigens. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3. A further example of a signaling domain that may be included in the disclosed CAR is the tumor necrosis factor receptor superfamily member 18 (TNFRSF18; glucocorticoid-inducible TNFR-related protein, also known as GITR) signaling domain.

[0271] In some examples, the CAR contains a CD3-zeta signaling domain, a CD8 signaling domain, a CD28 signaling domain, a CD137 signaling domain, or a combination of two or more of these. In another example, the cytoplasmic domain contains a CD3-zeta signaling domain and a CD28 signaling domain. In yet another example, the cytoplasmic domain contains a CD3-zeta signaling domain and a CD137 signaling domain. In yet another example, the cytoplasmic domain contains a CD3-zeta signaling domain as well as CD28 and CD137 signaling domains. The order of one or more T cell signaling domains on the CAR may be modified as needed by the operator. Exemplary amino acid sequences of such T cell signaling domains are provided. For example, the CD3 zeta signaling domain contains or may consist of the amino acid sequence shown as SEQ ID NO: 38:RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR. The CD8 signaling domain contains or may consist of the amino acid sequence shown as SEQ ID NO: 39:FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNR. The CD28 signaling domain contains or may consist of the amino acid sequence shown as SEQ ID NO: 40:SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS. The CD137 signaling domain contains, or may consist of, the amino acid sequence shown as SEQ ID NO: 41:KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL or SEQ ID NO: 42:RFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0272] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR disclosed herein may be linked to one another randomly or in a specified order. If necessary, short polypeptide linkers, preferably 2 to 10 amino acids long, may form the linkage. A glycine-serine diresidue provides a particularly suitable linker. Furthermore, a spacer domain containing a polypeptide sequence may be present between the signaling domain and the transmembrane domain of the CAR. The spacer domain may contain up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids.

[0273] 4. Further explanation of CAR Functional parts of CARs described herein are also provided. When used in relation to a CAR, the term “functional part” refers to any part or fragment of a CAR that retains the biological activity of the CAR from which it is a part (parent CAR). A functional part includes, for example, a part of a CAR that retains, to the same degree as, or to a greater degree than, the parent CAR, the ability to recognize target cells or to detect, treat or prevent disease. With respect to a parent CAR, a functional part may include, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more of the parent CAR.

[0274] CARs or their functional segments may contain additional amino acids at the amino-terminus, carboxy-terminus, or both ends, which are not present in the amino acid sequence of the parent CAR. Preferably, the additional amino acids do not interfere with the biological function of the CAR or functional segment, such as recognizing target cells, detecting cancer, or treating or preventing cancer. More preferably, the additional amino acids enhance the biological activity compared to that of the parent CAR.

[0275] Functional variants of the CARs described herein are also provided, which have substantial or significant sequence identity or similarity with the parent CAR, retain the biological activity of the CAR, and are variants of that CAR. Functional variants include, for example, variants of the CARs described herein (parent CARs) that retain the ability to recognize target cells to the same degree, the same degree, or a higher degree as the parent CAR. With respect to the parent CAR, functional variants may, for example, have amino acid sequences identical to the parent CAR by at least about 30%, about 50%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more.

[0276] A functional variant may, for example, include the amino acid sequence of a parent CAR having at least one conserved amino acid substitution. Alternatively, the functional variant may further include the amino acid sequence of a parent CAR having at least one non-conserved amino acid substitution. In this case, it is preferable that the non-conserved amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. The non-conserved amino acid substitution may enhance the biological activity of the functional variant such that its biological activity is increased compared to that of the parent CAR.

[0277] CARs (including functional parts and functional variants) can be of any length; that is, a CAR (or its functional part or functional variant) can contain any number of amino acids, as long as it retains its biological activity, such as the ability to specifically bind to an antigen, the ability to detect diseased cells in mammals, or the ability to treat or prevent a disease in mammals. In some examples, CARs are approximately 50 to 5000 amino acid long, e.g., 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more.

[0278] The CARs disclosed herein (including functional parts and functional variants of the CARs disclosed herein) may include synthetic amino acids instead of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-aminon-decanoic acid, homoserine, S-acetylaminomethylcysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, and β-phenylserine. Contains β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyllysine, N',N'-dibenzyllysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, oc-aminocycloheptanecarboxylic acid, -(2-amino-2-norbornane)-carboxylic acid, γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0279] The CARs disclosed herein (including functional parts and functional variants) may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via disulfide crosslinks, etc., or converted to acid addition salts, and / or dimerized, polymerized, or conjugated as needed.

[0280] Chimeric antigen receptors, methods for generating T cells containing such receptors, and their use (e.g., for the treatment of cancer) are known, for example, Brentjens et al., 2010, Molecular Therapy, 18:4, 666-668; Morgan et al., 2010, Molecular Therapy, published online February 23, 2010, pages 1-9; Till et al., 2008, Blood, 1 12:2261-2271; Park et al., Trends Biotechnol., 29:550-557, 2011; Grupp et al., N Engl J Med., 368:1509-1518, 2013; Han et al., J. Hematol Further details are provided in Oncol., 6:47, 2013; PCT Publication International Publication No. 2012 / 079000, International Publication No. 2013 / 126726; and U.S. Patent Application Publication No. 2012 / 0213783.

[0281] In some examples, the nucleic acid molecules encoding the CARs disclosed herein are included in an expression vector (such as a lentiviral vector) for expression in host cells such as T cells to produce the disclosed CARs. In some embodiments, the method using a chimeric antigen receptor comprises isolating immune cells from a subject (e.g., T cells), transforming the isolated cells with an expression vector encoding the CARs disclosed herein (e.g., a lentiviral or AAV vector), and administering the engineered cells expressing the CARs to the subject for treatment, for example, for treatment of a tumor in the subject.

[0282] D. Polynucleotides and Expression Provided herein are nucleic acids encoding humanized monoclonal antibodies, antibody-conjugate fragments, conjugates, or CARs that specifically bind to EGFRvIII and / or gene-amplified EGFR as disclosed herein. These nucleic acids encoding these molecules can be readily produced by practitioners using amino acid sequences provided herein (such as CDR sequences, heavy and light chain sequences), sequences available in the art (such as framework sequences), and genetic codes. Practitioners can readily use the genetic codes to produce nucleic acids encoding the same antibody sequence but with different sequences, or V L and / or V H It is possible to construct various functionally equivalent nucleic acids, such as nucleic acids that encode conjugate or fusion proteins containing nucleic acid sequences.

[0283] Disclosed humanized monoclonal antibodies, antibody-conjugate fragments, conjugates, and nucleic acid sequences encoding CARs that specifically bind to EGFRvIII and / or gene-amplified EGFR may be obtained, for example, by cloning a suitable sequence or by the phosphotriester method of Narang et al., Meth. Enzymol. 68:90-99, 1979; the phosphodiester method of Brown et al., Meth. Enzymol. 68:109-151, 1979; the diethylphosphoramidite method of Beaucage et al., Tetra. Lett. 22:1859-1862, 1981; for example, Needham-VanDevanter et al., Nucl. Acids DNA can be prepared by any suitable method, including direct chemical synthesis using automated synthesizers such as those described in Res.12:6159-6168,1984, for example, the solid-phase phosphoramidite triester method described in Beaucage & Caruthers, Tetra. Letts. 22(20):1859-1862,1981, and the solid support method of U.S. Patent No. 4,458,066. Chemical synthesis produces single-stranded oligonucleotides, which can be converted to double-stranded DNA by hybridization with a complementary sequence or by polymerization with DNA polymerase using the single strand as a template. Those involved will recognize that while the chemical synthesis of DNA is generally limited to sequences of about 100 bases, longer sequences can be obtained by ligation of shorter sequences.

[0284] The nucleic acids of this disclosure can be prepared using standard molecular biology techniques. Examples of appropriate cloning and sequencing techniques are known (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 4 thSee ed, Cold Spring Harbor, New York, 2012 and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, through supplement 104, 2013). Product information from manufacturers of biological reagents and laboratory equipment also provides useful information. Such manufacturers include SIGMA Chemical Company (St. Louis, Missouri), R&D Systems (Minneapolis, Minnesota), Pharmacia Amersham (Piscataway, New Jersey), CLONTECH Laboratories, Inc. (Palo Alto, California), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, Wisconsin), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaythersburg, Maryland), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Bux, Switzerland), Invitrogen (Carlsbad, California), and Applied Biosystems (Foster City, California), as well as many other commercially available sources.

[0285] Nucleic acids can also be prepared by amplification methods. Amplification methods include polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification systems (TAS), and autologous sequence replication systems (3SR). A wide variety of cloning methods, host cell methods, and in vitro amplification methods are well known.

[0286] In non-limiting examples, nucleic acid molecules encoding CARs disclosed herein are provided, which are expressed on immune cells, such as T cells, natural killer (NK) cells, natural killer T (NKT) cells, cytotoxic T lymphocytes (CTLs), or regulatory T cells. In some examples, the CARs disclosed herein are for expression in T cells, for example, to generate chimeric antigen receptor T cells (CAR-T cells). Nucleic acid molecules encoding CARs may be included in vectors (such as lentiviral vectors). Nucleic acid molecules encoding chimeric antigen receptors and methods for generating cells containing such receptors are known (e.g., Brentjens et al., 2010, Molecular Therapy, 18:4, 666-668; Morgan et al., 2010, Molecular Therapy, published online February 23, 2010, pages 1-9; Till et al., 2008, Blood, 1 12:2261-2271; Park et al., Trends Biotechnol., 29:550-557, 2011; Grupp et al., N Engl J Med., 368:1509-1518, 2013; Han et al., J. Hematol (See Oncol., 6:47, 2013; PCT Publication International Publication No. 2012 / 079000, International Publication No. 2013 / 126726; and U.S. Patent Application Publication No. 2012 / 0213783).

[0287] Nucleic acid molecules can be expressed in recombinant cells such as bacteria, plants, yeast, insects, and mammalian cells. Antibodies, antigen-binding fragments, and conjugates can be expressed in individual V H and / or V L It can be expressed as a chain (linked to an effector molecule or detectable marker as needed) or as a fusion protein. Methods for expressing and purifying antibody and antigen-binding fragments are known and further described herein (see, for example, Al-Rubeai (ed.), Antibody Expression and Production, Springer Press, 2011). Immunoadhesins can also be expressed. Therefore, in some examples, V H and VL A nucleic acid encoding a leader sequence, as well as an immunoadhesian, is provided. The nucleic acid sequence can optionally encode a leader sequence.

[0288] To create scFv, V H and V L A DNA fragment encoding V can be functionally linked to another fragment encoding a flexible linker, for example, another fragment encoding the amino acid sequence (Gly4-Ser)3, and as a result, V H and V L The array is V L and V H The domain can be expressed as a continuous single-strand protein joined by a flexible linker (e.g., Bird et al., Science 242:423-426, 1988; Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-5883, 1988; McCafferty et al., Nature, 348:552-554, 1990; Kontermann and Dubel (Ed), Antibody Engineering, Vols. 1-2, 2 nd Ed., Springer Press, 2010; Harlow and Lane, Antibodies: A Laboratory Manual, 2 nd (See Cold Spring Harbor Laboratory, New York, 2013). If necessary, the cleavage site may include a linker, such as a Fulin cleavage site.

[0289] V H and / or V L The nucleic acid encoding the immunoadhesin may optionally encode an Fc domain (immunoadhesin). The Fc domain may be an IgA, IgM, or IgG Fc domain. The Fc domain may be an optimized Fc domain as described in U.S. Patent Application Publication 2010 / 093979. In one example, the immunoadhesin is IgG1Fc.

[0290] Single-chain antibodies are single V H and VL It is monovalent when only one is used, and has two V H and V L When used, it is divalent, or three or more V H and V L When used, it may be polyvalent. For example, a bispecific or polyvalent antibody that specifically binds to EGFRvIII and another antigen, such as CD3 (but not limited to), may be produced. Encoded V H and V L V H Domain and V L The domain may contain a Fulin cleavage site.

[0291] Numerous expression systems are known and available for expressing nucleic acids in hosts such as Escherichia coli, other bacterial hosts, yeast, or various higher eukaryotic cells, such as COS, CHO, HeLa, and myeloma cell lines.

[0292] One or more DNA sequences encoding a humanized monoclonal antibody, antibody-conjugated fragment, conjugate, or CAR disclosed may be expressed in vitro by DNA transfer into a suitable host cell. The cell may be prokaryotic or eukaryotic. This term also includes any offspring of the host cell of interest. It is understood that not all offspring may be identical to the parent cell due to the possibility of mutations occurring during replication. Stable transfer methods, meaning that the foreign DNA is continuously maintained within the host, are known in the art. Hybridomas expressing humanized monoclonal antibodies are also included in this disclosure.

[0293] The disclosed humanized monoclonal antibodies, antibody-binding fragments, conjugates, or polynucleotide sequences encoding CARs can be operably ligated to an expression regulatory sequence. The expression regulatory sequence operably ligated to the coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the expression regulatory sequence. The expression regulatory sequence includes, but is not limited to, promoters, enhancers, transcriptional terminators, pre-start codons of protein-coding genes (e.g., ATG), intron splicing signals, spacers to maintain the correct reading frame and enable proper translation of mRNA, and stop codons.

[0294] To obtain high levels of expression of a cloned gene, it is desirable to construct an expression cassette that includes, at a minimum, a strong promoter to direct transcription, a ribosome binding site (internal ribosome binding sequence) for translation initiation, and a transcription / translation terminator. In the case of E. coli, this includes a promoter such as T7, trp, lac, or lambda promoter, a ribosome binding site, and preferably a transcription termination signal. In the case of eukaryotic cells, the regulatory sequence may include, for example, an immunoglobulin gene, a promoter and / or enhancer derived from HTLV, SV40, or cytomegalovirus, as well as a polyadenylation sequence, and may further include splice donor and / or acceptor sequences (e.g., CMV and / or HTLV splice acceptor and donor sequences). The expression cassette can be transferred to selected host cells by known methods such as transformation or electroporation for E. coli, or calcium phosphate treatment, electroporation, or lipofection for mammalian cells. Cells transformed by an expression cassette or vector can be selected based on their resistance to antibiotics conferred by the genes contained in such expression cassettes or vectors, such as AMP, GPT, NEO, and HYG genes.

[0295] The disclosed humanized monoclonal antibodies, antigen-binding fragments, conjugates, or polynucleotide sequences encoding CARs may, but are not limited to, be manipulated to allow for sequence insertion or incorporation and may be inserted into expression vectors containing plasmids, viruses, or other vehicles that can be expressed in either prokaryotes or eukaryotes. Hosts may include microorganisms, yeasts, insects, and mammalian organisms. Methods for expressing DNA sequences containing eukaryotic or viral sequences in prokaryotes are known. Numerous viral and plasmid DNA vectors capable of expression and replication in host cells have been described, and many are commercially available.

[0296] When the host is a eukaryote, conventional mechanical procedures such as DNA transfection as calcium phosphate coprecipitate, microinjection, electroporation, insertion of liposome-encapsulated plasmids, or viral vectors may be used. Eukaryotic cells may also be co-transformed with, for example, a polynucleotide sequence encoding a humanized antibody or antigen-binding fragment disclosed herein, and, for example, a second exogenous DNA molecule encoding a selectable phenotype (e.g., a herpesthymidine kinase gene). Another method is to transiently infect or transform eukaryotic cells with eukaryotic viral vectors such as Simian virus 40 (SV40) or bovine papillomavirus to express proteins (see, e.g., Viral Expression Vectors, Springer press, Muzyczka ed., 2011). Workers can readily use expression systems such as plasmids and vectors used to produce proteins in cells, including higher eukaryotic cells such as COS, CHO, HeLa, and myeloma cell lines.

[0297] For the purpose of producing recombinant CARs, the host cell may be a mammalian cell. The host cell may be a human cell. In some cases, the host cell may be a peripheral blood lymphocyte (PBL), peripheral blood mononuclear cell (PBMC), or T cell. The T cell may be any T cell, e.g., cultured T cell, e.g., primary T cell, or T cell from a cultured T cell line, e.g., Jurkat, SupTl, etc., or T cell obtained from a mammal (e.g., T cell obtained from a human subject). When obtained from a mammal, the T cell may be obtained from a number of sources, including but not limited to blood, bone marrow, lymph nodes, thymus, or other tissues or body fluids. The T cell may also be concentrated or purified. The T cell may be a human T cell. The T cell may be a T cell isolated from a human. The T cell may be any type of T cell, but is not limited to CD4 + / CD8 + Double positive T cells, CD4 + Helper T cells, e.g., Th1 and Th2 cells, CD8 + This can include T cells (e.g., cytotoxic T cells), tumor-infiltrating cells, memory T cells, naive T cells, and other cells at any developmental stage. T cells are CD8 + T cells or CD4 + It could be a T cell.

[0298] Also provided herein is a population of cells comprising at least one host cell described herein. The population of cells may be a heterogeneous population comprising at least one other cell, e.g., a host cell not comprising any of the recombinant expression vectors (e.g., a T cell), or a non-T cell population, e.g., B cells, macrophages, neutrophils, erythrocytes, hepatocytes, endothelial cells, epithelial cells, muscle cells, brain cells, etc., in addition to a host cell comprising any of the recombinant expression vectors described herein. Alternatively, the population of cells may be substantially homogeneous, and the population comprises (e.g., essentially comprises) a host cell comprising a recombinant expression vector. The population may also be a clonal population of cells in which all cells in the population are clones of a single host cell comprising a recombinant expression vector, such that all cells in the population comprise a recombinant expression vector. In one example, the population of cells is a clonal population comprising a host cell comprising a recombinant expression vector described herein.

[0299] Modifications may be performed on nucleic acids encoding humanized monoclonal antibodies or antigen-binding fragments described herein without reducing their biological activity. Several modifications may be performed to facilitate the cloning, expression, or incorporation of targeted molecules into fusion proteins. Such modifications are known and include, for example, stop codons, methionine added to the amino terminus to provide an initiation, sites, additional amino acids placed at either end to create conveniently located restriction sites, or additional amino acids (such as polyHis) to assist in purification steps. In addition to recombination methods, the immunoconjugates, effector moieties, and antibodies of this disclosure can also be constructed whole or partially using standard peptide synthesis.

[0300] Once expressed, humanized monoclonal antibodies, antigen-binding fragments, and conjugates can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, and column chromatography (see generally Simpson ed., Basic methods in Protein Purification and Analysis: A Laboratory Manual, Cold Harbor Press, 2008). Antibodies, antigen-binding fragments, and conjugates do not need to be 100% pure. If used therapeutically after partial or homogeneous purification as desired, polypeptides should be substantially endotoxin-free.

[0301] Methods for the expression of humanized monoclonal antibodies, antigen-binding fragments, and conjugates from mammalian cells and bacteria, such as Escherichia coli, and / or for refolding them into appropriate active forms are described and applicable to the antibodies disclosed herein. For example, Harlow and Lane, Antibodies: A Laboratory Manual, 2 nd See Cold Spring Harbor Laboratory, New York, 2013, Simpson ed., Basic methods in Protein Purification and Analysis: A laboratory Manual, Cold Harbor Press, 2008, and Ward et al., Nature 341:544, 1989.

[0302] In many cases, functional heterologous proteins from E. coli or other bacteria are isolated from inclusion bodies and require solubilization with a strong denaturant and subsequent refolding. During the solubilization process, a reducing agent is present to separate the disulfide bonds. An exemplary buffer containing a reducing agent is 0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, and 0.3 M DTE (dithioerythritol). Reoxidation of disulfide bonds can occur in the presence of reduced and oxidized low molecular weight thiol reagents, as described by Saxena et al., Biochemistry 9:5015-5021, 1970, and especially by Buchner et al., cited above.

[0303] In addition to recombination methods, humanized monoclonal antibodies, antigen-binding fragments, and / or conjugates can also be constructed whole or partially using standard peptide synthesis. Solid-phase synthesis of polypeptides can be achieved by attaching the C-terminal amino acid of a sequence to an insoluble support, followed by the sequential addition of the remaining amino acids in the sequence. Techniques for solid-phase synthesis are described by Barany & Merrifield, The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A. pp. 3-284; Merrifield et al., J. Am. Chem. Soc. 85: 2149-2156, 1963; and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co., Rockford, Illinois, 1984. Longer proteins can be synthesized by condensation of the amino and carboxyl terms of shorter fragments. A method for forming peptide bonds by activating the carboxyl terminus (for example, by using the coupling reagent N,N'-dicyclohexylcarbodiimide) is known.

[0304] E. Detection Method A method is provided for detecting the presence of cells expressing EGFRvIII and / or gene-amplified EGFR in a subject, such as tumor cells expressing EGFRvIII and / or gene-amplified EGFR. In some embodiments, the method includes contacting cells derived from the subject (e.g., tumor cells) with one or more humanized monoclonal antibodies, or their conjugates, that specifically bind to EGFRvIII and / or gene-amplified EGFR, to form an immune complex. The presence (or absence) of the immune complex is then detected. The presence of the immune complex indicates the presence of cells expressing EGFRvIII and / or gene-amplified EGFR in the subject.

[0305] In some cases, the method involves contacting cells derived from a target (e.g., tumor cells) with one or more humanized monoclonal antibodies that specifically bind to EGFRvIII, or their conjugates, to form an immune complex. The presence (or absence) of the immune complex is then detected, and the presence of the immune complex indicates the presence of cells expressing EGFRvIII in the target.

[0306] The detection method may include in vivo or in vitro detection of immune complexes. In some embodiments, the detection of cells expressing EGFRvIII includes the detection of cell surface expression of EGFRvIII on tumor cells. In some embodiments of the provided method, tumors are detected by detecting cells expressing EGFRvIII and / or gene-amplified EGFR in the subject. In some non-limiting examples, the tumors are cancers such as head and neck cancer, breast cancer, pancreatic cancer, colorectal cancer, CNS cancer, or bladder cancer. In some examples, the above method detects tumor cells overexpressing EGFRvIII (e.g., gene-amplified EGFR). In some examples, a humanized monoclonal antibody is used to detect EGFR 287-302 Join to the loop.

[0307] Various formats are useful for detecting cells expressing EGFRvIII and / or gene-amplified EGFR (e.g., tumor cells). In some embodiments, subjects who have, are suspected of having, or are at risk of developing tumors, such as cancer, are selected. For example, subjects have, are suspected of having, or are at risk of developing head and neck cancer, breast cancer, pancreatic cancer, colorectal cancer, CNS cancer, or bladder cancer. In some cases, subjects have, are suspected of having, or are at risk of developing head and neck cancer, breast cancer, or bladder cancer. Therefore, the presence of cells expressing EGFRvIII and / or gene-amplified EGFR can be detected in these subjects.

[0308] The disclosed method can also detect EGFR overexpression. Overexpression can be measured so as to detect any cells having more than approximately 50,000 receptors, more than approximately 60,000 receptors, more than approximately 70,000 receptors, more than approximately 80,000 receptors, more than approximately 90,000 receptors, or more than approximately 100,000 receptors. For example, such a method involves contacting tumor cells in a biological sample derived from a subject with one or more of the conjugates or antibodies or their antigen-binding fragments provided herein to form an immune complex. The presence (or absence) of the immune complex is then detected and / or quantified. The presence (or amount) of the immune complex on cells derived from the subject indicates the presence of tumor cells overexpressing EGFR in the subject.

[0309] In one non-limiting example, a sample is obtained from a subject, and the presence of tumor cells expressing EGFRvIII is assessed in vitro. For example, such a method involves contacting tumor cells in a biological sample derived from the subject with one or more conjugates or humanized monoclonal antibodies provided herein that specifically bind to EGFRvIII or its antigen-binding fragments, thereby forming an immune complex. The presence (or absence) of the immune complex is then detected. The presence of the immune complex on cells derived from the subject indicates the presence of tumor cells expressing EGFRvIII in the subject. For example, an increase in the presence of immune complexes in the sample compared to the formation of immune complexes in a control sample indicates the presence of tumor cells expressing EGFRvIII in the subject. In some examples, a control is used. Using a similar method, the presence of tumor cells expressing gene-amplified EGFR may be detected using one or more of the humanized monoclonal antibodies or antigen-binding fragments disclosed herein, or their conjugates, that specifically bind to gene-amplified EGFR.

[0310] Biological specimens are typically obtained from the mammalian subject of interest, such as humans. Specimens can include, but are not limited to, tissues from biopsies, autopsies, and pathological specimens. Biological specimens also include tissue sections, such as frozen sections taken for histological purposes.

[0311] In some examples of the methods disclosed herein, the humanized monoclonal antibody or antigen-binding fragment disclosed herein is conjugated to a detectable marker. In some examples, the method further includes contacting the antibody disclosed herein, its antigen-binding fragment, or a second antibody that specifically binds to the conjugate for a sufficient time to form an immune complex, and detecting this immune complex. An increase in the presence of this immune complex in a biological sample derived from a selected target, compared to the presence of the immune complex in a control sample or other standard (as described above), detects the presence of endothelial cells expressing EGFRvIII and / or gene-amplified EGFR in the biological sample. In some examples, the second antibody is conjugated to a detectable marker.

[0312] Suitable detectable markers for humanized monoclonal antibodies or secondary antibodies are described and known to the practitioner. Examples include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, magnetic agents, and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Non-limiting examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Non-limiting examples of suitable fluorescent substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin. A non-limiting exemplary luminescent material is luminol. A non-limiting exemplary magnetic agent is gadolinium, and non-limiting exemplary radioactive labels are 125 I, 131 I, 35 S or 3 Includes H.

[0313] The humanized monoclonal antibodies and their conjugates disclosed herein may be used in immunohistochemical assays. These assays are known and described (see, for example, Harlow & Lane, Antibodies, A Laboratory Manual, 2nd ed., Cold Spring Harbor Publications, New York (2013)).

[0314] Humanized monoclonal antibodies disclosed herein may also be used to detect tumor cells expressing EGFRvIII and / or gene-amplified EGFR in vivo. In some examples, in vivo detection diagnoses the presence of a tumor in a subject. Thus, methods are disclosed for detecting pathological conditions in a subject, e.g., tumors, such as head and neck cancer, breast cancer, pancreatic cancer, colorectal cancer, CNS cancer, or bladder cancer. In one example, an effective amount of the disclosed humanized monoclonal antibody (or its antigen-binding fragment) or its conjugate is administered to the subject for a sufficient time for the humanized monoclonal antibody or antigen-binding fragment to form an immune complex, which can then be detected. Detection of the immune complex in the subject determines the presence of tumor cells expressing EGFRvIII and / or gene-amplified EGFR. In specific, non-limiting examples, detection of the immune complex is performed by immunoscintigraphy. Other specific, non-limiting examples of immune complex detection include radiolocalization, radioimaging, magnetic resonance imaging (e.g., using biotinylated antibodies and avidin-iron oxide), or fluorescence imaging (e.g., using luciferase or green fluorescent protein-labeled antibodies) (see, e.g., Paty et al., Transplantation., 77:1133-1137, 2004). In some examples, the disclosed methods detect, for example, head and neck cancer, breast cancer, pancreatic cancer, colorectal cancer, CNS cancer, or bladder cancer.

[0315] In magnetic resonance imaging (MMRI) settings, contrast agent detection can be significantly influenced by the magnetic field strength of the MMRI scanner. Increased magnetic field strength provides an order of magnitude improvement in the ability to detect contrast agents (Hu et al., Ann. Rev. Biomed. Eng., 6:157-184, 2004; Wedeking et al., Magn. Reson. Imaging., 17:569-575, 1999). For example, the detection limit for gadolinium at 2 Tesla (T) is approximately 30 μM. At 4 T, the detection limit is reduced to approximately 1 μM. Newly available 7-12 T scanners are expected to detect low (10-100) nM concentrations of this contrast agent. Similar sensitivities can also be identified using contrast agents such as iron oxide. Once detected, the test results can be used to assist or guide surgical or other resection of the tumor.

[0316] In one example, an effective amount of a humanized monoclonal antibody or antigen-binding fragment or conjugate thereof, which specifically binds to EGFRvIII as disclosed herein, is administered to a subject with a tumor after anticancer treatment. After sufficient time has elapsed to allow the administered antibody or antigen-binding fragment or conjugate to form immune complexes with EGFRvIII on tumor cells, these complexes are detected. For example, a humanized monoclonal antibody or its conjugate that specifically binds to EGFRvIII may be administered to a subject before or after treatment of a tumor. The tumor may be (but not limited to) head and neck cancer, breast cancer, pancreatic cancer, colorectal cancer, CNS cancer, or bladder cancer. The presence (or absence) of immune complexes indicates the effectiveness of the treatment. For example, an increase in immune complexes compared to a control performed before treatment indicates that the treatment is ineffective, while a decrease in immune complexes compared to a control performed before treatment indicates that the treatment is effective. Similar methods disclosed herein, using humanized monoclonal antibodies or antigen-binding fragments or conjugates thereof that specifically bind to gene-amplified EGFR, can be used to detect gene-amplified EGFR, and the presence (or absence) of an immune complex indicates the effectiveness of the treatment.

[0317] F. Treatment Method A therapeutically effective dose of any humanized monoclonal antibody or antigen-binding fragment disclosed herein, their conjugates, or CAR-expressing immune cells (e.g., CAR T cells or CAR NK cells) that specifically binds to EGFRvIII may be administered to a subject to treat EGFRvIII-expressing tumors, such as head and neck cancer, breast cancer, pancreatic cancer, colorectal or rectal cancer, CNS cancer, or bladder cancer. In some cases, administration of a therapeutically effective dose of any humanized monoclonal antibody or antigen-binding fragment disclosed herein that specifically binds to EGFRvIII, their conjugates, or CAR-expressing immune cells reduces the signs or symptoms of EGFRvIII-expressing tumors. Tumors may be cancers, such as head and neck cancer, breast cancer, pancreatic cancer, colorectal or rectal cancer, CNS cancer, or bladder cancer. Therefore, subjects may be selected for treatment if they have, are suspected of having, or are at risk of developing an EGFRvIII-expressing tumor. Therapeutic amounts of nucleic acid molecules and vectors disclosed herein may also be used. In other examples, tumors may overexpress EGFR and / or misfolded EGFR. In some examples, subjects are selected that have tumors overexpressing EGFR and / or misfolded EGFR. The compositions disclosed herein are useful for treating these tumors in subjects.

[0318] In a further example, a therapeutically effective amount of the humanized antibody or antigen-binding fragment disclosed herein may also be used in a method to inhibit tumor-overexpressed EGFR in a subject (e.g., gene-amplified EGFR). This method comprises administering a therapeutically effective amount of the humanized antibody, antigen-binding fragment, nucleic acid molecule, vector, T cell, or pharmaceutical composition disclosed herein to a subject having a tumor overexpressing EGFR. In a non-limiting example, an isolated humanized monoclonal antibody or antigen-binding fragment may also be used. a) Sequence ID 5 and Sequence ID 8 (A10, VH3 + VL3), respectively; b) Sequence ID 3 and Sequence ID 6 (A2, VH1 + VL1), respectively; c) Sequence ID 3 and Sequence ID 7 (A3, VH1+VL2), respectively; d) Sequence ID 3 and Sequence ID 8 (A4, VH1 + VL3), respectively; e) Sequence ID 4 and Sequence ID 6 (A5, VH2 + VL1), respectively; f) Sequence ID 4 and Sequence ID 7 (A6, VH2 + VL2), respectively; g) Sequence ID 4 and Sequence ID 8 (A7, VH2 + VL3), respectively; h) Sequence ID 5 and Sequence ID 6 (A8, VH3 + VL1), respectively; i) Sequence ID 5 and Sequence ID 7 (A9, VH3 + VL2), respectively; j) Sequence ID 1 and Sequence ID 9 (B1, 40H3 VH+VL-EG), respectively; k) Sequence ID 1 and Sequence ID 10 (B2, 40H3 VH+VL-DA), respectively; l) Sequence ID 4 and Sequence ID 11 (B3, VH2 + VL1 - DA), respectively; m) Sequence ID 4 and Sequence ID 12 (B4, VH2 + VL2 - DA), respectively; n) Sequence ID 5 and Sequence ID 11 (B5, VH3, and VL1-DA), respectively; o) Sequence ID 31 and Sequence ID 32 (C10), respectively; p) Sequence ID 5 and Sequence ID 14 (D2), respectively; or q) Sequence ID 5 and Sequence ID 24 (D3), respectively. V containing the amino acid sequence described H and V L Includes.

[0319] Immune cells expressing CARs containing nucleic acid molecules, vectors, and their antigen-binding fragments are also useful. Tumors can be cancers, such as head and neck cancer, breast cancer, pancreatic cancer, colorectal or rectal cancer, CNS cancer, or bladder cancer. Therefore, subjects who have, are suspected of having, or are at risk of developing tumors that overexpress EGFR (e.g., expressing gene-amplified EGFR) may be selected for treatment.

[0320] In some cases, the humanized monoclonal antibodies, antigen-binding fragments, CAR-expressing immune cells (e.g., CAR T cells), compositions, and conjugates disclosed herein may be administered to a subject to slow or inhibit tumor growth or metastasis, reduce tumor volume, or reduce metastasis. In these applications, a therapeutically effective dose of the humanized monoclonal antibodies, antigen-binding fragments, or conjugates, or CAR T cells or compositions disclosed herein that specifically bind to EGFRvIII and / or gene-amplified EGFR, is administered to the subject in an amount and under conditions sufficient to form an immune complex with EGFRvIII and / or gene-amplified EGFR, thereby slowing or inhibiting tumor growth or metastasis, reducing tumor volume, or inhibiting signs or symptoms of cancer. Examples of suitable subjects include subjects diagnosed with or suspected of having a tumor expressing EGFRvIII and / or gene-amplified EGFR, such as subjects with cancers including head and neck cancer, breast cancer, pancreatic cancer, colorectal cancer, CNS cancer, or bladder cancer.

[0321] The therapeutically effective dose depends on the severity of the disease and the patient's general health condition. The therapeutically effective dose is the amount that provides either a subjective relief of symptoms or an objectively identifiable improvement, as noted by a clinician or other qualified observer. For example, the therapeutically effective dose is the amount necessary to inhibit tumor growth (such as the growth of cancers like head and neck cancer, breast cancer, pancreatic cancer, colorectal cancer, CNS cancer, or bladder cancer), to inhibit metastasis, to reduce tumor volume, or to reduce the signs or symptoms of a tumor. The therapeutically effective dose of a drug administered may vary depending on the desired effect and the target being treated. In some cases, the therapeutic dose is the amount that eliminates or reduces the patient's tumor burden, prevents or reduces the growth of metastatic cells, or reduces the symptoms of a tumor.

[0322] Subjects that may benefit from the disclosed methods include human subjects and veterinary subjects (e.g., cats, dogs, non-human primates, etc.). In some examples, the subjects are mammalian subjects, e.g., humans, non-human primates, dogs, or cats. In some examples, the subjects are humans, non-human primates, dogs (Canis lupus familiaris), or cats (e.g., Felis catus). In non-limiting examples, the subjects are humans. Subjects may be screened before initiating the disclosed treatment, for example, to determine whether the subject has a tumor. The presence of a tumor expressing EGFRvIII and / or gene-amplified EGFR indicates that the composition may be treated using the methods provided herein.

[0323] Any method of administration, including local or systemic administration, may be used. For example, local, oral, intravascular (e.g., intravenous), intramuscular, intraperitoneal, intranasal, intradermal, intrathecal, intratumoral, and subcutaneous administration may be used. Specific modes of administration and drug regimens may be selected by the attending physician, taking into account the details of the case (e.g., the subject, the disease, the disease state involved, and whether the treatment is prophylactic). If two or more drugs or compositions are administered, one or more routes of administration may be used. For example, chemotherapeutic agents may be administered orally, or humanized monoclonal antibodies or antigen-binding fragments or conjugates or compositions may be administered intravenously. Methods of administration include injecting conjugates, humanized monoclonal antibodies, antigen-binding fragments, immune cells containing CARs, nucleic acid molecules, or compositions provided herein into a non-toxic, pharmaceutically acceptable carrier such as water, saline, Ringer's solution, dextrose solution, 5% human serum albumin, fixative oil, ethyl oleate, or liposomes. In some cases, topical administration may be used by applying, for example, a humanized antibody or antigen-binding fragment disclosed herein to an area of ​​tissue from which a tumor has been removed, or to an area suspected of being susceptible to tumor development. In some cases, sustained intratumoral (or proximal) release of a pharmaceutical preparation containing a therapeutically effective amount of a humanized antibody or antigen-binding fragment (or its conjugate) may be beneficial.

[0324] Compositions comprising humanized monoclonal antibodies or antigen-binding fragments or their conjugates, or immune cells containing CARs, disclosed herein may be formulated in unit dosage forms suitable for individual administration of precise doses. Furthermore, the compositions may be administered in single-dose or multi-dose schedules. A multi-dose schedule may consist of two or more separate doses, e.g., 1 to 10 doses, after which other doses may be given at subsequent time intervals as needed to maintain or enhance the effect of the composition. Treatment may involve daily or multi-day doses of the compound over a period of several days to several months, or even several years. Thus, the drug regimen is also determined, at least in part, based on the specific needs of the person being treated and depends on the judgment of the administering physician.

[0325] Exemplary doses of humanized monoclonal antibodies, antigen-binding fragments, conjugates, compositions, or further agents may range from about 0.01 to about 30 mg / kg (body weight of the subject), for example, from about 0.1 to about 10 mg / kg or from about 5 to about 15 mg / kg. In some cases, the dose is at least 0.1 mg / kg, at least 0.2 mg / kg, at least 0.3 mg / kg, at least 0.4 mg / kg, at least 0.5 mg / kg, at least 1 mg / kg, at least 4 mg / kg, at least 3 mg / kg, at least 5 mg / kg, at least 6 mg / kg, at least 7 mg / kg, at least about 8 mg / kg, at least about 9 mg / kg, at least 10 mg / kg, at least 11 mg / kg, at least 12 mg / kg, at least 13 mg / kg, at least 14 mg / kg, at least 15 mg / kg, at least 16 mg / kg, at least 17 mg / kg, at least 18 mg / kg, at least 19 mg / kg, at least 20 mg / kg, at least 21 mg / kg, at least 22 mg / kg, at least 23 mg / kg, at least 24 mg / kg, at least 25 mg / kg, at least 26 mg / kg, at least 27 mg / kg, at least 28 mg / kg, at least 29 mg / kg, or at least 30 mg / kg. In some cases, the dose is approximately 1 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, or 30 mg / kg or less. In some cases, the dose of the humanized monoclonal antibody, antigen-binding fragment, conjugate, composition, or further agent disclosed herein is 1 mg / kg to 20 mg / kg, for example, 2 mg / kg to 20 mg / kg, 2 mg / kg to 15 mg / kg, 2 mg / kg to 10 mg / kg, 2 mg / kg to 5 mg / kg, 5 mg / kg to 20 mg / kg, 5 mg / kg to 15 mg / kg, 5 mg / kg to 10 mg / kg, 8 mg / kg to 20 mg / kg, 8 mg / kg to 15 mg / kg, 8 mg / kg to 12 mg / kg, or 8 mg / kg to 10 mg / kg. In non-limiting examples, the doses of the humanized monoclonal antibodies or antigen-binding fragments disclosed herein are 5 mg / kg to 15 mg / kg.In another non-limiting example, the dose of the humanized monoclonal antibody or antigen-binding fragment disclosed herein is 8 mg / kg to 12 mg / kg.

[0326] In some examples, the dose of a humanized monoclonal antibody, antigen-binding fragment, conjugate, composition, or further drug disclosed herein is 100 mg / m². 2 ~700mg / m 2 For example, 200 mg / m² 2 ~700mg / m 2 , 200 mg / m² 2 ~600mg / m 2 , 200 mg / m² 2 ~500mg / m 2 , 200 mg / m² 2 ~400mg / m 2 , 300 mg / m² 2 ~700mg / m 2 , 300 mg / m² 2 ~600mg / m 2 , 300 mg / m² 2 ~500mg / m 2 , 300 mg / m² 2 ~400mg / m 2 , 350 mg / m² 2 ~700mg / m 2 , 350 mg / m² 2 ~600mg / m 2 , 350 mg / m² 2 ~500mg / m 2 , 350 mg / m² 2 ~400mg / m 2 This is within the range. In non-limiting examples, the dose of a humanized monoclonal antibody or antigen-binding fragment disclosed herein is 200 mg / m². 2 ~500mg / m 2 In another non-limiting example, the dose of a humanized monoclonal antibody or antigen-binding fragment disclosed herein is 300 mg / m². 2 ~400mg / m 2 That is the case.

[0327] In some embodiments, 10 CARs (e.g., CAR T cells) per kg of body weight of the subject 4~10 12 Individual immune cells, for example, 10 per kg 4 ~10 8 pieces, 10 6 ~10 7 pieces, 10 6 ~10 8 pieces, 10 6 ~10 10 pieces, 10 6 ~10 12 pieces, 10 8 ~10 10 pieces, or 10 8 ~10 12 Individual cells are administered to the subject. In some cases, 5 × 10⁶ cells per kg of the subject's body weight are administered. 6 ~5×10 8 Immune cells containing CARs (e.g., CAR T cells) are administered to the subject. In some cases, 1 × 10⁶ cells are administered per kg of the subject's body weight. 7 ~5×10 7 Immune cells containing CARs (e.g., CAR T cells) are administered to the target. In some cases, approximately 10 per kg. 6 ~10 8 Individual cells are administered to the target, for example, 0.2 to 5.0 × 10⁶ cells per kg. 6 0.1 to 2.5 per piece or per kg × 10 8 The number of doses administered to the subject is at least 10 per kg. In some cases, at least 10 4 , 10 5 , 10 6 , 10 7 1 or 10 8 A number of cells is administered to the target. In a further example, 10 per kg 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 or 10 10A small number of cells or fewer are administered to the target. Multiple doses may be administered; for example, immune cells including CARs (e.g., CAR T cells) may be administered daily, every other day, twice a week, weekly, bi-weekly, every three weeks, monthly, or less frequently. The dose and frequency of administration may be influenced by numerous factors, such as the condition being treated, the severity of the symptoms, the age of the subject, or their overall condition. Typically, the treatment regimen is determined by clinical trials.

[0328] Immune cells containing CARs (e.g., CAR T cells) can be administered using any appropriate route of administration. In some cases, immune cells containing CARs are administered parenterally, for example, intravenously. However, injection or infusion close to the target tumor (e.g., local administration), or administration into the peritoneal cavity may also be used. In some cases, immune cells containing CARs disclosed herein are delivered locally to the tumor by injection or catheter. The appropriate route of administration may be determined by the practitioner based on factors such as the condition being treated, the severity of the symptoms, the age or overall condition of the subject, or other factors.

[0329] In certain cases, a subject receives a therapeutic composition comprising one or more of the following: a conjugate, a humanized monoclonal antibody, an antigen-binding fragment, a composition, CAR T cells, or further agents, in multiple daily dosing schedules (e.g., at least two consecutive days, ten consecutive days, etc.) over a period of several weeks, months, or years. In one example, a subject receives the conjugate, antibody, composition, or further agent for at least 30 days, e.g., at least two months, at least four months, at least six months, at least twelve months, at least 24 months, or at least 36 months.

[0330] In some cases, the disclosed therapeutic agent is administered intravenously, subcutaneously, or in another manner multiple times daily or weekly over a period of time, followed by a no-treatment period, after which the cycle is repeated. In some cases, the initial treatment period (e.g., multiple daily or weekly administrations of the therapeutic agent) is 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks. In some cases, the no-treatment period lasts 3 days, 1 week, 2 weeks, 3 weeks, or 4 weeks. In certain cases, the treatment regimen may be daily for 3 days followed by a 3-day break, or daily or multiple times per week for 1 week followed by a 3-day or 1-week break, or daily or multiple times per week for 2 weeks followed by a 1 or 2-week break, or daily or multiple times per week for 3 weeks followed by a 1, 2, or 3-week break, or daily or multiple times per week for 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks followed by a 1, 2, 3, or 4-week break.

[0331] Administration of humanized monoclonal antibodies, antigen-binding fragments, conjugates, CARs, immune cells, or compositions may be accompanied by the administration of other anticancer or anti-angiogenic agents or therapeutic procedures (such as surgical resection of the tumor or radiotherapy). For example, before, during, or after administration of a therapeutic dose of antibody or conjugate, the subject may receive one or more further treatments. In one example, the subject receives one or more procedures to remove or reduce the tumor before administration of one or more therapeutic doses of agents for the treatment of the tumor. For example, the additional agents may include, but are not limited to, chemotherapeutic agents, anti-angiogenic agents, or combinations thereof. In another example, at least a portion of the tumor is surgically or otherwise resected, or its size or volume is reduced, before administration of a therapeutically effective dose of humanized antibody or antigen-binding fragment or conjugate.

[0332] Specific examples of further therapeutic agents that may be used include microtubule binding agents, DNA intercalators or crosslinking agents, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors. These agents (administered in therapeutically effective doses) and treatments may be used alone or in combination. For example, any suitable anticancer or anti-angiogenic agent may be administered in combination with the antibodies, conjugates disclosed herein. The methods and therapeutic doses of such agents are known and may be determined by the practitioner. In one example, the chemotherapeutic agent may include 5-FU or IRT, or both.

[0333] Microtubule-binding agents interact with tubulin to stabilize or destabilize microtubule formation, thereby inhibiting cell division. Examples of microtubule-binding agents that may be used in combination with the disclosed therapies include, but are not limited to, paclitaxel, docetaxel, vinblastine, vindesine, vinorelbine (navelbine), epothirone, colchicine, dorastatin 15, nocodazole, podophyllotoxin, and rhizoxin. Analogues and derivatives of such compounds may also be used. For example, suitable epothirone and epothirone analogs are described in International Publication No. 2004 / 018478. Taxoids (e.g., paclitaxel and docetaxel), as well as analogues of paclitaxel as taught by U.S. Patent Nos. 6,610,860; 5,530,020; and 5,912,264, may also be used.

[0334] Appropriate DNA and RNA transcription regulators, including but not limited to actinomycin D, daunorubicin, doxorubicin, and their derivatives and analogues, are also suitable for use in combination with the disclosed therapies. DNA intercalators and crosslinkers that can be administered to a subject include, but are not limited to, cisplatin, carboplatin, oxaliplatin, mitomycin, e.g., mitomycin C, bleomycin, chlorambucil, cyclophosphamide, and their derivatives and analogues. DNA synthesis inhibitors suitable for use as therapeutic agents include, but are not limited to, methotrexate, 5-fluoro-5'-deoxyuridine, 5-FU, and their analogues. Examples of suitable enzyme inhibitors include, but are not limited to, camptothecin, etoposide, formestane, trichostatin, and their derivatives and analogues. Suitable compounds that affect gene regulation include agents that result in increased or decreased expression of one or more genes, such as raloxifene, 5-azacitidine, 5-aza-2'-deoxycytidine, tamoxifen, 4-hydroxytamoxifen, mifepristone, and their derivatives and analogues.

[0335] Examples of commonly used chemotherapy drugs include Adriamycin, Alkeran, Ara-C, BiCNU, Busulfan, CCNU, Carboplatin, Cisplatin, Cytoxan, Daunorubicin, DTIC, 5-FU, Fludarabine, Hydrea, Idarubicin, Ifosfamide, Methotrexate, Mithramycin, Mitomycin, Mitoxantrone, Nitrogen Mustard, Taxol (or other taxanes such as Docetaxel), Verban, Vincristine, and VP-16. On the other hand, some newer drugs include gemcitabine (Gemzar), Herceptin, IRT (Camptosar, CPT-11), Leustatin, Navelbine, Rituxan STI-571, Taxotere, Topotecan (Hycamtin), Xeloda (Capecitabine), Zevelin, and Calcitriol.

[0336] Non-exclusive examples of immunomodulatory agents that may be used include AS-101 (Wyeth-Ayerst Labs.), bropyrimin (Upjohn), gamma interferon (Genentech), GM-CSF (granulocyte-macrophage colony-stimulating factor; Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immunoglobulin (Cutter Biological), IMREG (Imreg, New Orleans, Louisiana), SK&F 106528, and TNF (tumor necrosis factor; Genentech).

[0337] Therefore, non-limiting examples of chemotherapeutic agents for use in combination with antibodies, antigen-binding fragments, their conjugates, immune cells including CARs, and nucleic acid molecules disclosed herein include erlotinib (TARCEVA®, Genentech / OSI Pharm.), bortezomib (VELCADE®, Millennium) Pharm.), fulvestrant (FASLODEX®, AstraZeneca), sutent (SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis), PTK787 / ZK222584 (Novartis), oxaliplatin (Eloxatin®, Sanofi), 5-FU (5-fluorouracil), leucovorin, rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, GlaxoSmithKline), ronafarnib (SCH66336), sorafenib (BAY43-9006, Bayer Alkylating agents such as gefitinib (IRESSA®, AstraZeneca), AG1478, AG1571 (SU5271; Sugen), thiotepa and CYTOXAN® cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; pemetrexed (ALIMTA® Eli Antifolic acid antitumor agents such as Lilly; aziridines such as benzodopa, carbocon, metsuredopa, and uredopa; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylomelamamine; acetogenins (especially bratacin and bratacinone); camptothecin (including its synthetic analog topotecan); bryostatin; calisthotin; CC-1065 (including its synthetic analogs adzeresin, karzeresin, and bizeresin); cryptophycin (especially cryptophycin 1 and cryptophycin 8); dorastatin; duocalmycin (including its synthetic analogs KW-2189 and CB1-TM1); eleuterobin;Pancratistatin; sarcodicin; spongstatin; chlorambucil, chlornafadin, colophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobenbitin, fenestrine, prednimustine, trophosphamide, uracil mustard and other nitrogenous mustards; carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine and other nitrosoureas; engine antibiotics, calicheamicin, calicheamicin gamma 1I and caliche Antibiotics such as amycin omega I1; dynemicins including dynemicin A; bisphosphonates such as clodronate; esperamicin; and neocardinostatin chromophores and related chromoprotein enediin antibiotic chromophores, crasinomycin, actinomycin, anthramycin, azaserin, bleomycin, kakutinomycin, carabicin, carminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN (registered trademark) Standard doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin such as mitomycin C, mycophenolic acid, nogaramycin, olibomycin, peplomycin, potophyllomycin, puromycin, keramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zolbicin; methotrexate and Antimetabolites such as 5-fluorouracil (5-FU); folate analogs such as denopterin, methotrexate, pteropterin, and trimethrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and phloxuridine; androgens such as carsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testactone;Anti-adrenal agents such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as floric acid; acegraton; aldofsphamide glycoside; aminolevulinic acid; enyluracil; amsacrin; bestrabusil; bisanthren; edatraxate; defofamine; demecoltin; diazicon; elfornithine; eriptinium acetate; epotilon; etogluside; gallium nitrate; hydroxyurea; lentinan; ronidynin; mytansinoids such as meitansine and ansamitosine; mitogluazone; mitoxantrone; mopidammole; nitraerine; pentostatin; fenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK (registered trademark) polysaccharide complex (JHS Natural Products, Eugene, Oregon); Lazoxane; Rhizoxin; Schizophyllan; Spirogermanium; Tenuazonic Acid; Triadicone; 2,2',2''-Trichlorotriethylamine; Trichothecenes (especially T-2 toxin, Veraculine A, Loridine A and Anguidin); Urethanes; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacitosine; Arabinoside ("Ara-C"); Cyclophosphamide; Thiotepa; Taxoids, e.g., Paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, New Jersey), ABRAXANE® Cremophore-Free, Albumin, Nanoparticle Formulations of Paclitaxel (American Pharmaceutical Partners, Schaumburg, Illinois), and TAXOTERE® Docetaxel (Rhone-Poulenc Rorer, France, Antony); Chlorambucil; GEMZAR® gemcitabine; 6-thioguanine; Mercaptopurine; Methotrexate; Platinum analogs such as cisplatin and carboplatin; Vinblastine; Platinum; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Vincristine; NAVELBINE® vinorelbine; Novantrone; Teniposide; Edatraxate; Daunomycin; Aminopterin; Xeloda; Ibandronate; CPT-11;This includes topoisomerase inhibitors RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and any pharmaceutically acceptable salts, acids, or derivatives of the above.

[0338] Non-exclusive examples of anti-angiogenic agents include molecules such as proteins, enzymes, polysaccharides, oligonucleotides, DNA, RNA, and recombinant vectors, as well as small molecules that function to reduce or even inhibit angiogenesis. Examples of suitable angiogenic inhibitors, but not limited to, include angiostatins K1-3, staurosporine, genistein, fumagiline, medroxyprogesterone, suramin, interferon-alpha, metalloproteinase inhibitors, platelet factor 4, somatostatin, tromovospondin, endostatin, thalidomide, and their derivatives and analogs. For example, in some cases, the anti-angiogenic agent is an antibody that specifically binds to VEGF (e.g., AVASTIN®, Roche) or the VEGF receptor (e.g., VEGFR2 antibody). In one example, the anti-angiogenic agent includes a VEGFR2 antibody, or DMXAA (also known as bajimezan or ASA404; e.g., Sigma Corp., commercialized from St. Louis, Missouri), or both. Exemplary kinase inhibitors include GLEEVAC®, IRESSA®, and TARCEVA®, which prevent the phosphorylation and activation of growth factors. Antibodies that may be used include HERCEPTIN® and AVASTIN®, which block growth factors and angiogenic pathways.

[0339] In some cases, additional drugs include monoclonal antibodies, such as 3F8, avagovomab, adecatumumab, aftuzumab, alacizumab, alemtuzumab, artumomab penteate, anatumomab mafenatox, apolizumab, artumomab, bavituximab, bectomomab, belimumab, becilesomab, bevacizumab, vivacuzumab meltansine, blinatumomab, brentuximab vedotin, cantuzumab meltansine, capromab pendetide, catumakisomab, CC49, cet Ximab, Sitatuzumab Bogatox, Sixtumumab, Cribatuzumab Tetraxetan, Conatumumab, Dasetuzumab, Detumomab, Eclomeximab, Eculizumab, Edrecolomab, Epratuzumab, Erzmaxomab, Etalacizumab, Farletuzumab, Figitumumab, Galiximab, Gemtuzumab Ozogamicin, Dilentuximab, Glenbatumumab Vedotin, Ibritumomab Tiuxetan, Igobomab, Imsilomab, Intetumumab, Inotuzumab Ozogamicin, Ipilim Mab, Iratumumab, Labetuzumab, Lexatumumab, Lintuzumab, Rorbotuzumab Meltansine, Lucatumumab, Lumiliximab, Mapatumumab, Matuzumab, Mepolizumab, Meterimumab, Miratuzumab, Mitsumomab, Morolimmab, Nacolumumab Butafenatox, Naptumomab Estafenatox, Necitumumab, Nimotuzumab, Nofetumomab Merpentan, Ofatumumab, Olaratumumab, Oportuzumab Monatox, Olegobomab, Panitumumab, Pemtumomab, Pertuz These include mab, pintumomab, pritumumab, ramucirumab, rilotumumab, rituximab, lobatumumab, satumomab pendetide, sibrotuzumab, sonepcizumab, sorafenib, sunitinib, takatuzumab tetraxetan, tapritumomab paptox, tenatumomab, TGN1412, tisilimmab (tremelimumab), tigatuzumab, TNX-650, trastuzumab, tremelimumab, tucotzumab cermoloykin, beltuzumab, borosiximab, botumumab, and zaltumumab.

[0340] Another common treatment for some types of cancer is surgical intervention, such as surgical removal of the cancer or a part of it. Another example of treatment is radiotherapy, such as the administration of radioactive material or energy (external beam therapy, for example) to the tumor site, which helps to eradicate the tumor or shrink it before surgical removal.

[0341] Other therapeutic agents, such as antitumor agents, may or may not fall into one or more of the above classifications and are suitable for administration in combination with the disclosed treatments. Examples of such agents include doxorubicin, apigenin, rapamycin, zebralin, cimetidine, and their derivatives and analogues.

[0342] The preparation and administration schedule of additional drugs may be used in accordance with the manufacturer's instructions or to be determined empirically by the practitioner. Such chemotherapy preparations and administration schedules are also described, for example, in Chemotherapy Service, (1992) Ed., MCPerry, Williams & Wilkins, Baltimore, Md.

[0343] Combination therapy can provide a synergistic effect, and the effect may be demonstrated when the combined active ingredients are greater than the combined effect of the compounds used separately. A synergistic effect can be achieved when the active ingredients are: (1) co-formulated and administered or delivered simultaneously in a combined unit dosage form; (2) delivered alternately or in parallel as separate formulations; or (3) by some other regimen. When delivered alternately, a synergistic effect can be achieved when the compounds are administered or delivered sequentially, for example by different injections in separate syringes. Generally, during alternation, the effective dose of each active ingredient is administered sequentially, for example, consecutively, but in combination therapy, the effective doses of two or more active ingredients are administered together.

[0344] G. Composition This specification provides compositions comprising one or more humanized monoclonal antibodies, antigen-binding fragments, conjugates, immune cells including CARs (e.g., CAR T cells), or nucleic acid molecules on a carrier (e.g., a pharmaceutically acceptable carrier). In some examples, the compositions are pharmaceutical compositions. The compositions may be prepared in unit dosage forms for administration to a subject. The amount and timing of administration are at the discretion of the treating clinician to achieve the desired outcome. The compositions may be formulated for systemic (e.g., intravenous) or topical (e.g., intratumor) administration. In one example, the humanized monoclonal antibodies or their antigen-binding fragments, conjugates, nucleic acid molecules, or immune cells including CARs disclosed herein are formulated for parenteral administration, e.g., intravenous administration. The disclosed compositions are useful, for example, for the treatment and / or detection of tumors occurring in the head and / or neck, breast, pancreas, colon or rectum, central nervous system (CNS), or bladder. In some examples, the compositions are useful for the treatment or detection of carcinomas.

[0345] The compositions for administration to the subject may include a solution of a therapeutic agent dissolved (or suspended in cells) on a pharmaceutically acceptable carrier, such as an aqueous carrier. Various aqueous carriers, such as buffered saline, may be used. These solutions are sterile and generally free of undesirable substances. These compositions can be sterilized by conventional sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusters and buffers, toxicity adjusters, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate. The concentrations of humanized monoclonal antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, or immune cells containing CARs in these formulations may vary widely and are selected mainly based on body fluid volume, viscosity, body weight, etc., according to the specific mode of administration and the needs of the subject. Practical methods for preparing such dosage forms will be known or apparent to the practitioner.

[0346] Exemplary compositions for intravenous administration include, for example, an antibody or antigen-binding fragment or conjugate (or a corresponding dose of a conjugate containing a humanized antibody or antigen-binding fragment) at a dose of approximately 0.01 to approximately 30 mg / kg per subject per day. Practical methods for preparing administerable compositions are known or obvious to practitioners and are described in further detail in publications such as, for example, Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, PA (1995). In some examples, the composition is a liquid formulation containing one or more antibodies or antigen-binding fragments (such as humanized monoclonal antibodies or antigen-binding fragments that specifically bind to EGFRvIII) in a concentration range of approximately 0.1 mg / ml to approximately 20 mg / ml, or approximately 0.5 mg / ml to approximately 20 mg / ml, or approximately 1 mg / ml to approximately 20 mg / ml, or approximately 0.1 mg / ml to approximately 10 mg / ml, or approximately 0.5 mg / ml to approximately 10 mg / ml, or approximately 1 mg / ml to approximately 10 mg / ml.

[0347] The compositions disclosed herein are provided in lyophilized form and may be rehydrated with sterile water before administration, but are also provided in sterile solutions of known concentrations. In one example, a lyophilized humanized monoclonal antibody or antigen-binding fragment or conjugate is suspended and added to an infusion bag containing 0.9% sodium chloride, USP, and administered, possibly at a dose of 0.5–15 mg / kg body weight. Considerable experience in the administration of antibody or antigen-binding fragments and conjugate drugs is available in the art. For example, antibody drugs have been marketed in the United States since the approval of Rituxan® in 1997. Antibodies, antigen-binding fragments and conjugates may be administered by slow infusion rather than by intravenous push or bolus. In one example, a higher loading dose is administered, followed by a lower maintenance dose. For example, an initial loading dose of 4 mg / kg of antibody or antigen-binding fragment (or the corresponding dose of a conjugate containing a humanized monoclonal antibody or antigen-binding fragment) may be infused over approximately 90 minutes, followed by weekly maintenance doses of 2 mg / kg over 30 minutes for 4 to 8 weeks, provided the previous dose was well tolerated.

[0348] Any of the compositions disclosed herein can be formulated for controlled release. Controlled-release parenteral formulations can be prepared as implants, oily injections, or microparticle systems. For a comprehensive overview of protein delivery systems, see, for example, Banga, AJ, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA, (1995). Microparticle systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain a therapeutic protein, such as a cytotoxin or drug, as a central core. In microspheres, the therapeutic agent is dispersed throughout the particle. Particles smaller than about 1 μm, microspheres, and microcapsules are generally called nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of about 5 μm so that only nanoparticles are administered intravenously. Microparticles are typically about 100 μm in diameter and are administered subcutaneously or intramuscularly. See, for example, Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc., New York, NY, pp. 315-339 (1992).

[0349] Polymers may be used for the ion-controlled release of compositions disclosed herein. A variety of degradable and non-degradable polymer matrices are known and described for use in controlled drug delivery (see, e.g., Langer, Accounts Chem. Res. 26:537-542, 1993). For example, the block copolymer Polaxamer 407 exists as a viscous but mobile liquid at low temperatures, but forms a semi-solid gel at body temperature. It has been shown to be an effective vehicle for the formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. 9:425-434, 1992; and Pec et al., J.Parent. Sci. Tech. 44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for the controlled release of proteins (Ijntema et al., Int. J. Pharm. 112:215-224, 1994). In yet another embodiment, liposomes are used for controlled release and drug targeting of lipid-encapsulated drugs (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). Numerous additional systems are known for the controlled release of therapeutic proteins (see U.S. Patents 5,055,303, 5,188,837, 4,235,871, 4,501,728, 4,837,028, 4,957,735, 5,019,369, 5,055,303, 5,514,670, 5,413,797, 5,268,164, 5,004,697, 4,902,505, 5,506,206, 5,271,961, 5,254,342, and 5,534,496).

[0350] In some cases, subjects are administered, for example, using the subject's cellular mechanisms, with humanized monoclonal antibodies, their antigen-binding fragments, or DNA encoding a conjugate (such as a toxin) as disclosed herein, in order to provide in vivo antibody production. Immunization with nucleic acid constructs is known and taught, for example, in U.S. Patents 5,643,578, 5,593,972 and 5,817,637; and 5,880,103. This method may include liposome delivery of nucleic acids. Such a method may be applied to the production of humanized monoclonal antibodies or their antibody-binding fragments by an operator.

[0351] One approach to administering nucleic acids is the direct administration of a vector, such as a mammalian expression plasmid (or viral vector). A nucleotide sequence encoding a disclosed humanized monoclonal antibody or its antibody-conjugated fragment may be included in the vector and placed under the control of a promoter to increase expression.

[0352] In another approach using nucleic acids, the disclosed humanized monoclonal antibodies or antibody-conjugated fragments or their conjugates may also be expressed by attenuated viral hosts or vectors or bacterial vectors. Recombinant vaccinia virus, adeno-associated virus (AAV), herpesvirus, retrovirus, cytomegalovirus, or other viral vectors can be used to express humanized antibodies. For example, a useful protocol for a vaccinia virus vector and method is described in U.S. Patent No. 4,722,848. BCG (Bacillus Calmette Guerin) provides another vector for the expression of the disclosed humanized monoclonal antibodies (see Stover, Nature 351:456-460, 1991).

[0353] In one example, a nucleic acid encoding a disclosed humanized monoclonal antibody or its antibody-conjugated fragment is introduced directly into cells. For example, the nucleic acid may be loaded onto a gold microsphere by standard methods and introduced into the skin using a device such as Bio-Rad's HELIOS® Gene Gun. The nucleic acid may be a “naked” plasmid under the control of a strong promoter. Typically, DNA is also injected into the muscle, but it may be injected directly into other sites. Doses for injection are usually about 0.5 μg / kg to about 50 mg / kg, and typically about 0.005 mg / kg to about 5 mg / kg (see, for example, U.S. Patent No. 5,589,466).

[0354] H. Kit Kits are also provided. For example, kits are provided for detecting tumor cells expressing EGFRvIII and / or gene-amplified EGFR in a subject, and / or for treating tumors in a subject. The kits will typically include humanized monoclonal antibodies or antigen-binding fragments, and / or their conjugates, that specifically bind to EGFRvIII and / or gene-amplified EGFR, as disclosed herein. In some examples, the components of the kit are useful for both tumor detection and / or treatment.

[0355] A kit may include two or more of the conjugates, humanized antibodies, antigen-binding fragments, immune cells containing CARs, nucleic acids, or compositions disclosed herein. Therefore, a kit may include two or more humanized monoclonal antibodies that specifically bind to EGFRvIII and / or gene-amplified EGFR. A kit may include at least two humanized monoclonal antibodies or antigen-binding fragments that specifically bind to EGFRvIII and / or gene-amplified EGFR, their conjugates, or combinations thereof. In some examples, the kit includes antigen-binding fragments or conjugates containing antigen-binding fragments such as Fv fragments. In one example, for in vivo use, the fragment may be an scFv fragment.

[0356] A kit may include a container, a label on or associated with the container, or accompanying documentation. Suitable containers include, for example, bottles, vials, syringes, etc. Containers may be formed from a variety of materials, such as glass or plastic. Typically, a container holds a composition containing one or more of the disclosed antibodies, antigen-binding fragments, or conjugates. In some examples, a container may have a sterile access port (for example, a container may be a vial with a stopper that can be punctured by an intravenous solution bag or a subcutaneous needle). The label or accompanying documentation indicates that the composition is used to treat a particular condition.

[0357] The label or accompanying information typically further includes instructions for use of the kit components (e.g., antibodies or their fragments, conjugates, nucleic acids, CAR-containing immune cells, or compositions disclosed herein) in a method for treating, preventing, or detecting tumors. The accompanying information typically includes instructions that are usually included in the commercial packaging of a therapeutic product, which include information regarding indications, usage, dosage, administration, contraindications, and / or warnings relating to the use of such therapeutic product. Instructions for use may be written in electronic format (e.g., on a computer diskette or compact disk) or visual (e.g., a video file). The kit may also include additional components to facilitate the specific application for which the kit is designed. Thus, for example, the kit may further include means for detecting labels (e.g., enzyme substrates for enzyme labeling, filter sets for detecting fluorescent labels, appropriate secondary labels such as secondary antibodies). The kit may further include buffers and other reagents used to carry out a particular method. [Examples]

[0358] Examples The mouse monoclonal antibody 40H3 binds to human amplified EGFR when this receptor is overexpressed on cancer cells. 40H3 also binds to immobilized EGFRvIII or EGFRvIII when expressed on transfected cells. To utilize 40H3 as a clinical antibody, the variable portions of its heavy and light chains were humanized.

[0359] Each CDR was tentatively identified by alignment with KABAT or a similar database. The variable regions of the heavy and light chains of 40H3 were compared to the closest human immunoglobulin family, and residues were then modified to produce candidate humanized antibodies. The candidates contained a different number of human substituted amino acids in slightly different positions. Not only were several heavy and light chain candidates generated, but combinations were also generated in which a given humanized heavy chain matched several light chains, and vice versa. Thus, optimal pairs were established.

[0360] Humanization of mouse antibodies is a modifiable endeavor. Substituting multiple human residues into the mouse framework generates less immunogenic proteins, but carries the risk of loss of binding activity. All candidates were assayed for retention of binding activity to both immobilized EGFRvIII (by ELISA) or cells expressing high levels of EGFR or EGFRvIII (by flow cytometry).

[0361] To evaluate the binding activity of humanization candidates, either immobilized extracellular domains of EGFRvIII (assayed in ELISA or Octet format), various cancer cells expressing EGFR (assayed by flow cytometry), or cancer cells expressing transfected EGFRvIII (assayed by flow cytometry) were used. All disclosed humanization sequences retained binding activity to immobilized EGFRvIII, but only a subset of them bound to cells overexpressing EGFR or cells with transfected EGFRvIII. Antibodies with superior cell-binding activity were identified.

[0362] Example 1 material and method ELISA The binding of candidate humanized antibodies was evaluated by an ELISA-type assay. The extracellular domain (ECD) of human EGFRvIII with a C-terminal His tag (EGI-H52H4, ACROBiosystems, Newark, DE, USA) was conjugated at 3.5 μg / ml at room temperature for 1 hour in a clear nickel-coated 96-well plate (Thermo Fisher Scientific). The plate was washed three times with PBS-Tween® 20 (PBST) containing 0.05% TWEEN®. Primary antibodies of various concentrations were added to immobilized EGFRvIII on a shaking platform at room temperature for 1 hour. The antibodies were diluted to the desired concentration in PBS containing 5% BSA (Sigma-Aldrich). After the addition of the primary antibody, the plate was washed three times with PBST. Peroxidase-labeled AffiniPure donkey anti-human IgG (H+L) (catalog number 709035149; Jackson ImmunoResearch, Maine, USA) was diluted 1:20,000 and incubated at room temperature for 1 hour with shaking. Plates were washed three times with PBST, and horseradish peroxidase (HRP)-labeled secondary antibody was quantified by adding 3,3',5,5'-tetramethylbenzidine substrate solution (Thermo Fisher Scientific). The reaction was stopped by adding 1M sulfuric acid (Sigma-Aldrich). Horseradish peroxidase activity was analyzed using a VersaMax plate reader with SoftMax Pro software (Molecular Devices, California, USA).

[0363] Flow cytometry The antibody was used to test on suspended cells (2.5 × 10⁶) in 500 μl of FACS buffer consisting of PBS (KD Medical, Maryland, USA), 2 mM EDTA (KD Medical, Maryland, USA), 1% BSA (Sigma-Aldrich, Missouri, USA), and 0.1% sodium azide (Sigma-Aldrich, Missouri, USA). 5 Each tube was incubated with individual cells for 30 minutes on ice. The bound antibody was detected at a 1:250 dilution using R-phycoerythrin AffiniPure F(ab')2 fragment goat anti-human IgG(H+L) (Cat#109116088; Jackson ImmunoResearch, Maine, USA) for 30 minutes on ice. Subsequently, the survival dye efluor R780 (catalog number 65086514, Invitrogen) was added to each tube and incubated on ice for 15 minutes (dilution 1 / 700). Antibody binding was characterized using the BD FACSCANTO II System (BD Bioscience, San Jose, California, USA), and the data were analyzed using FlowJo (Tree Star, Inc., Ashland, Oregon, USA) and presented in histogram format with median fluorescence intensity plotted. EC for cell binding 50 The values ​​were calculated using GraphPad Prism Version 9.4.

[0364] Example 2 result When evaluated for binding to cells or immobilized EGFRvIII, the A10 antibody was the best conjugate (of all candidates), outperforming chimeric antibodies with the original mouse VH and mouse VL (see, e.g., Figures 1A–1B, 2A–2B, and 4A–4B for assay results). A10 showed increased binding affinity (i.e., tighter binding) to both cells and immobilized antigens. The A10 antibody is a combination of candidate light chain "VL3" and candidate heavy chain "VH3". Other combinations produced candidates with lower binding affinity but retained binding activity. The superior binding activity of all candidates could not be predicted by sequence analysis alone.

[0365] All antibody binding data are summarized in Figures 4A-4B, while results for specific cell lines are shown in separate figures. Figures 1A-1B show binding to EGFRvIII when transfected F98 cells were analyzed by flow cytometry at increasing concentrations of various antibodies. Figures 2A-2B show antibody binding to EGFR-amplified cell lines MDA-MB-468 (Figure 2A) and A431 (Figure 2B). 40H3 does not bind to WT EGFR. To confirm increased binding activity to WT EGFR, WI-38 and U87MG cells (expressing only WT EGFR) were incubated with several humanization candidates. Figures 3A-3B show no increased binding to WT EGFR by any of the humanization candidates. However, the presence of WT EGFR was confirmed by cetuximab binding.

[0366] Figures 4A-4B provide four series of humanized antibodies. Series A consists of VH1-3 candidates paired with VL1-3. All candidates except A1 have amino acid substitutions in the variable heavy chain and variable light chain framework portions. Candidate A1 is the original mouse VH1-3 fused with the human constant sequences of the CH1-3 and constant κ chain (Invivogen pFUSE vectors (pFUSE2ss-CLIg-hK and pFUSEss-hchg1)). H and V L That is the case.

[0367] The B series contained two light chain variants (DA and EG - see sequence information) that altered the last residue of the light chain CDR2. These alterations were intended to "protect" the antibody from potential post-translational modifications. These variants were not superior to the A10 candidate and were not further characterized.

[0368] The C series contained sequences generated using the BioPhi platform (Prihoda et al., MABs, 2022). Surprisingly, only C10 possessed beneficial properties regarding cell binding. C1-C9 did not show cell binding but bound to the antigen. The D series contained variable light chains derived from antibody C1 paired with VH3 from series A, or light chains derived from antibody C6 paired with VH3. D1 (VH and VL3 derived from C1) did not bind to cells, while D2 and D3 did.

[0369] For cell binding experiments, cetuximab is used as a positive control to detect all surface-expressed EGFR. While the 40H3 antibody (and all humanized versions of 40H3) binds to a subset of EGFR expressed only on cancer cells, cetuximab binds to all cells with surface-expressed EGFR (see, e.g., Figures 5A-5B). Using 40H3 and its humanized versions in vivo allows for specificity—binding to cancer cells without binding to normal cells—and thus reduces potential side effects.

[0370] Example 3 A10 crystal structure The crystal structure of EGFR loop-A10 fab was determined (see Figures 6A-6C). The crystallization conditions were as follows: 1357 Wizard G11 NaH2PO4 (0.8M) KH2PO4(1.2M) Sodium acetate (0.1M, pH 4.5) No freeze-protectant [Table 2] [Table 3]

[0371] Example 4 A10 CAR-T cells CAR vectors were prepared as shown in Figures 8A–8B. CAR T cells were generated by lentiviral transduction and activation of T cells with anti-CD3 and IL-2. Figure 9 shows the cell viability of the A431-luc cell line 24 or 48 hours after the indicated treatment. "CAR-1" and "CAR-2" refer to T cells containing / expressing the A10 scFv-CAR construct in Figure 8A or Figure 8B, respectively.

[0372] For in vivo administration, the cells are grown for one week after transformation, and then injected via IV, IP, or administered topically to the tumor site. Transduction of T cells is monitored by including surface markers (e.g., truncated EGFR) that can be detected using antibodies (e.g., cetuximab). T cell preparations containing 35-50% cetuximab are suitable for injection.

[0373] Example 5 A10 antibody-drug conjugate Purified A10 antibodies were conjugated to deruxtecan (A10-Dxd) or monomethyl auristatin E (A10-MMAE) payloads. The conjugation reaction was commercially performed by NJBio® (Princeton, New Jersey). Briefly, for conjugation via internal and reduced disulfides, the antibody preparations were subjected to mild reduction (TCEP in excess of 4.5 molars relative to the antibody in PBS). The samples were desalted using a Zeba® column (ThermoFisher® Scientific, New York, USA) according to the manufacturer's instructions and then conjugated to the linker-payload composition via a maleimide group. After the conjugation reaction, the ADCs were fractionated to remove both unmodified and overmodified antibodies. The drug-antibody ratio (DARS) for each ADC was determined. ADCs with 2 to 8 drugs per antibody (no agglutination) were used. Figure 10 shows the cell viability of MDA-MB-468 cells after treatment with A10-Dxd or A10-MMAE.

[0374] Example 6 Further research The antibodies or fragments thereof disclosed herein (see, for example, Table 1) can be further modified by conjugating a drug-based cytotoxic payload, manipulating a CAR construct containing the antibody or fragment, or combining it with a T-cell engager to produce novel antitumor agents. In all cases, cancer cells with amplified EGFR or EGFRvIII are killed. Furthermore, cells expressing low or normal levels of WT EGFR (non-cancerous cells) are not killed.

[0375] It will be apparent that the exact details of the methods or compositions described herein may be changed or modified without departing from the spirit of this disclosure. The inventors assert that all such modifications and variations fall within the scope of the following claims and spirit.

Claims

1. An isolated humanized monoclonal antibody or its antigen-binding fragment, a) Sequence ID 5 and Sequence ID 8 (A10, VH3 + VL3), respectively; b) Sequence ID 3 and Sequence ID 6 (A2, VH1 + VL1), respectively; c) Sequence ID 3 and Sequence ID 7 (A3, VH1 + VL2), respectively; d) Sequence ID 3 and Sequence ID 8 (A4, VH1 + VL3), respectively; e) Sequence ID 4 and Sequence ID 6 (A5, VH2 + VL1), respectively; f) Sequence ID 4 and Sequence ID 7 (A6, VH2 + VL2), respectively; g) Sequence ID 4 and Sequence ID 8 (A7, VH2 + VL3), respectively; h) Sequence ID 5 and Sequence ID 6 (A8, VH3 + VL1), respectively; i) Sequence ID 5 and Sequence ID 7 (A9, VH3 + VL2), respectively; j) Sequence ID 1 and Sequence ID 9 (B1, 40H3 VH+VL-EG), respectively; k) Sequence ID 1 and Sequence ID 10 (B2, 40H3 VH+VL-DA), respectively; l) Sequence ID 4 and Sequence ID 11 (B3, VH2 + VL1 - DA), respectively; m) Sequence ID 4 and Sequence ID 12 (B4, VH2 + VL2 - DA), respectively; n) Sequence ID 5 and Sequence ID 11 (B5, VH3, and VL1-DA), respectively; o) Sequence ID 31 and Sequence ID 32 (C10), respectively; p) Sequence ID 5 and Sequence ID 14 (D2), respectively; or q) Sequence ID 5 and Sequence ID 24 (D3), respectively The heavy chain variable region (V Ｈ ) and light chain variable region (V Ｌ Isolated humanized monoclonal antibodies or their antigen-binding fragments, including ).

2. The isolated humanized monoclonal antibody according to claim 1, wherein the antibody comprises a human constant domain.

3. The isolated humanized monoclonal antibody according to claim 1 or claim 2, wherein the antibody is IgG.

4. An isolated humanized monoclonal antibody according to any one of the preceding claims, comprising a recombinant constant domain having a modification that increases the half-life of the antibody.

5. An isolated humanized monoclonal antibody or antigen-binding fragment according to any one of the preceding claims, conjugated with a toxin or chemotherapeutic agent.

6. The isolated humanized monoclonal antibody or antigen-binding fragment according to claim 5, wherein the toxin is Pseudomonas exotoxin (PE), lysine, abrin, diphtheria toxin, ribotoxin, ribonuclease, saporin, calicheamicin, or botulinum toxin.

7. The isolated humanized monoclonal antibody or antigen-binding fragment according to claim 5 or 6, wherein the toxin is the PE, and the PE is PE25, PE38, or PE40.

8. The isolated humanized monoclonal antibody or antigen-binding fragment according to claim 5, wherein the chemotherapeutic agent is monomethyl auristatin E or a mytansinoid.

9. The antigen-binding fragment according to any one of the preceding claims.

10. The antigen-binding fragments are Fv, dsFV, ds-scvFV, Fab, F(ab') ２ , scFV or scFV ２ A fragment, which is an antigen-binding fragment according to any one of the preceding claims.

11. The antigen-binding fragment according to any one of the preceding claims, wherein the antigen-binding fragment is Fab or scFV.

12. An isolated humanized monoclonal antibody or antigen-binding fragment according to any one of the preceding claims, conjugated to a detectable marker.

13. A chimeric antigen receptor (CAR) comprising an antigen-binding fragment as described in any one of the preceding claims.

14. A bispecific antibody comprising a humanized monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 11.

15. A humanized antibody or antigen-binding fragment according to any one of claims 1 to 12, or the V of the antibody or antigen-binding fragment Ｈ Or V Ｌ an isolated nucleic acid molecule encoding the CAR described in claim 13, or the bispecific antibody described in claim 14.

16. The isolated nucleic acid molecule according to claim 15, wherein the nucleic acid molecule is a cDNA sequence.

17. An isolated nucleic acid molecule according to claim 15 or claim 16, operably linked to a promoter.

18. A vector comprising a nucleic acid molecule according to any one of claims 15 to 17.

19. The vector according to claim 18, wherein the vector is a viral vector.

20. An isolated host cell containing a nucleic acid molecule according to any one of claims 15 to 17, or a vector according to claim 18 or 19.

21. Isolated T cells expressing CAR as described in claim 13.

22. A pharmaceutical composition for use in the treatment of cancer expressing EGFRvIII, comprising an effective amount of a humanized monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 12, a bispecific antibody according to claim 14, a nucleic acid molecule according to any one of claims 15 to 17, a vector according to claim 18 or claim 19, a host cell according to claim 20, or a T cell according to claim 21, and a pharmaceutically acceptable carrier.

23. A method for producing a monoclonal antibody or antigen-binding fragment that specifically binds to EGFRvIII, or a bispecific antibody containing the monoclonal antibody or antigen-binding fragment, wherein the method is Expressing one or more nucleic acid molecules according to claims 15 to 17 in a host cell, A method comprising purifying the antibody, antigen-binding fragment, or bispecific antibody.

24. A method for detecting the presence of EGFRvIII in a biological sample derived from a target, The biological sample is contacted with an effective amount of a humanized monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 12 or a bispecific antibody according to claim 14, under conditions sufficient to form an immune complex. A method comprising detecting the presence of the immune complex in the biological sample, wherein the presence of the immune complex in the biological sample indicates the presence of EGFRvIII in the sample.

25. The method according to claim 24, wherein the biological sample is a biopsy from a glioma, head and neck cancer, breast cancer, pancreatic cancer, colorectal cancer, or bladder cancer.

26. A method for inhibiting a tumor expressing EGFRvIII and / or gene-amplified EGFR in a subject, comprising administering to the subject an effective amount of a humanized monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 12, a bispecific antibody according to claim 14, a nucleic acid molecule according to any one of claims 15 to 17, a vector according to claim 18 or claim 19, a host cell according to claim 20, a T cell according to claim 21, or a pharmaceutical composition according to claim 22, wherein the subject is a tumor expressing EGFRvIII or gene-amplified EGFR, respectively.

27. The method according to claim 26, wherein the method inhibits the tumor expressing EGFRvIII, and a subject having the tumor expressing EGFRvIII is selected for treatment.

28. A method for inhibiting tumors that overexpress EGFR in a target, A method comprising administering an effective amount of a humanized monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 12, a bispecific antibody according to claim 14, a nucleic acid molecule according to any one of claims 15 to 17, a vector according to claim 18 or claim 19, a T cell according to claim 21, or a pharmaceutical composition according to claim 22 to a subject having the tumor overexpressing EGFR, wherein the VH of the monoclonal antibody or antigen-binding fragment thereof comprises SEQ ID NO: 5, and the VL of the monoclonal antibody or antigen-binding fragment thereof comprises SEQ ID NO:

8.

29. The method according to any one of claims 24 to 28, wherein the tumor is a glioma, head and neck cancer, breast cancer, pancreatic cancer, colorectal cancer, or bladder cancer.

30. The method according to any one of claims 24 to 29, wherein the subject is a human.

31. The method according to any one of claims 26 to 33, comprising inhibiting the tumor to reduce the growth, size, or metastasis of the tumor.

32. Use of a humanized monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 12, a bispecific antibody according to claim 14, a nucleic acid molecule according to any one of claims 15 to 17, a vector according to claim 18 or claim 19, a T cell according to claim 21, or a pharmaceutical composition according to claim 22 for inhibiting a tumor expressing EGFRvIII in a target or for detecting the presence of EGFRvIII in a biological sample.

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