IGF1R antibody

Abstract

Described herein are antibodies that bind to insulin-like growth factor receptor 1 (IGF1R) and inhibit signal transduction through IGF1R. Described herein are IGF1R antibodies with high binding affinity and high biological inhibitory activity. Also described herein are IGF1R antibodies with an extended half-life and a low ADCC activity level. Such antibodies can be effectively incorporated into formulations for subcutaneous administration to improve the ease of administration to patients.

Description

Technical Field

[0001] Cross-reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 500,168, filed May 4, 2023, and U.S. Provisional Patent Application No. 63 / 351,077, filed Jun. 10, 2022, both of which are hereby incorporated by reference in their entirety.

Background Art

[0002] Subcutaneous administration of antibody treatments to patients is a more convenient and cost-effective delivery route than intravenous administration. This is because such treatments can be administered at home and do not require the presence of trained medical professionals. However, one obstacle to subcutaneous administration of antibodies is the substantial reduction in the volume of antibody formulations that will be used in prefilled syringes, on-body injectors, autoinjectors, etc. To address this, high-concentration antibody formulations are required, but these can be impractical for subcutaneous delivery due to viscosity issues, aggregation behavior, and other exacerbating factors for formulating large molecules. One solution is the use of novel antibodies that bind to targets with higher affinity than known antibodies or exhibit high biological activity. In addition, antibodies containing specific Fc region mutations can increase the in vivo half-life and further reduce the amount of antibody that will be included in subcutaneous formulations.

Summary of the Invention

Means for Solving the Problems

[0003] Described herein are IGF1R antibodies with high binding affinity and high biological inhibitory activity. Also described herein are IGF1R antibodies with an extended half-life and a low ADCC activity level. Such antibodies can be effectively incorporated into formulations for subcutaneous administration to improve the ease of administration to patients. Described herein, in one aspect, is an antibody or antigen-binding fragment thereof that binds to the insulin-like growth factor 1 receptor (IGF1R), the antibody or antigen-binding fragment thereof comprising: (a) an immunoglobulin heavy chain CDR1 (HCDR1) comprising the amino acid sequence SX1GMH, wherein X1 is H, Y, A, or T; (b) an immunoglobulin heavy chain CDR2 (HCDR2) comprising the amino acid sequence X1IX2X3DX4SX5TYYADSVRG, wherein X1 is I, T, or Y, X2 is W, N, or A, X3 is F, H, A, or G, X4 is G or A, and X5 is S or T; (c) an immunoglobulin heavy chain CDR3 (HCDR3) comprising the amino acid sequence ELX1RRYFDL, wherein X1 is G or N; (d) an immunoglobulin light chain CDR1 (LCDR1) comprising the amino acid sequence RASQSVSSX1LA, wherein X1 is Y, A, or T; (e) an immunoglobulin light chain CDR2 (LCDR2) comprising the amino acid sequence DASKRAT; and / or an immunoglobulin light chain CDR3 (LCDR3) comprising the amino acid sequence QQRX1KX2PPWT, wherein X1 is S or G and X2 is Y or W); the antibody or antigen-binding fragment thereof does not include an immunoglobulin heavy chain variable region identical to SEQ ID NO: 1 and / or an immunoglobulin light chain variable region identical to SEQ ID NO: 2).In one embodiment, it is an antibody or an antigen-binding fragment thereof that binds to the insulin-like growth factor 1 receptor (IGF1R), and the antibody or the antigen-binding fragment thereof comprises the following: (a) an immunoglobulin heavy chain CDR1 (HCDR1) comprising the amino acid sequence SX1GMH (wherein X1 is H, Y, A, or T); (b) an immunoglobulin heavy chain CDR2 (HCDR2) comprising the amino acid sequence X1IX2X3DX4SX5TYYADSVRG (wherein X1 is I, T, or Y, X2 is W, N, or A, X3 is F, H, A, or G, X4 is G or A, and X5 is S or T); (c) an immunoglobulin heavy chain CDR3 (HCDR3) comprising the amino acid sequence ELX1RRYFDL (wherein X1 is G or N); (d) an immunoglobulin light chain CDR1 (LCDR1) comprising the amino acid sequence RASQSVSSX1LA (wherein X1 is Y, A, or T); (e) an immunoglobulin light chain CDR2 (LCDR2) comprising the amino acid sequence DASKRAT; and / or an immunoglobulin light chain CDR3 (LCDR3) comprising the amino acid sequence QQRX1KX2PPWT (wherein X1 is S or G and X2 is Y or W); the antibody or the antigen-binding fragment thereof does not include an immunoglobulin heavy chain variable region identical to SEQ ID NO: 1 and an immunoglobulin light chain variable region identical to SEQ ID NO: 2). In certain embodiments, the antibody or the antigen-binding fragment thereof comprises an immunoglobulin heavy chain CDR1 (HCDR1), an immunoglobulin heavy chain CDR2 (HCDR2), an immunoglobulin heavy chain CDR3 (HCDR3), an immunoglobulin light chain CDR1 (LCDR1), an immunoglobulin light chain CDR2 (LCDR2), and / or an immunoglobulin light chain CDR3 (LCDR3): (a) HCDR1 comprises the amino acid sequence SHGMH; (b) HCDR2 comprises the amino acid sequence YIWFDGSSTYYADSVRG; (c) HCDR3 comprises the amino acid sequence ELGRRYFDL; (d) LCDR1 comprises the amino acid sequence RASQSVSSALA; (e) LCDR2 comprises the amino acid sequence DASKRAT; and / or (f) LCDR3 comprises the amino acid sequence QQRSKYPPWT.In certain embodiments, the antibody or antigen-binding fragment thereof comprises immunoglobulin heavy chain CDR1 (HCDR1), immunoglobulin heavy chain CDR2 (HCDR2), immunoglobulin heavy chain CDR3 (HCDR3), immunoglobulin light chain CDR1 (LCDR1), immunoglobulin light chain CDR2 (LCDR2), and / or immunoglobulin light chain CDR3 (LCDR3), wherein (a) HCDR1 comprises the amino acid sequence SYGMH; (b) HCDR2 comprises the amino acid sequence IIWFDGSSTYYADSVRG; (c) HCDR3 comprises the amino acid sequence ELGRRYFDL; (d) LCDR1 comprises the amino acid sequence RASQSVSSYLA; (e) LCDR2 comprises the amino acid sequence DASKRAT; and / or (f) LCDR3 comprises the amino acid sequence QQRSKYPPWT. In certain embodiments, the antibody or antigen-binding fragment thereof comprises immunoglobulin heavy chain CDR1 (HCDR1), immunoglobulin heavy chain CDR2 (HCDR2), immunoglobulin heavy chain CDR3 (HCDR3), immunoglobulin light chain CDR1 (LCDR1), immunoglobulin light chain CDR2 (LCDR2), and / or immunoglobulin light chain CDR3 (LCDR3), wherein (a) HCDR1 comprises the amino acid sequence SHGMH; (b) HCDR2 comprises the amino acid sequence IIAGDASTTYYADSVRG; (c) HCDR3 comprises the amino acid sequence ELGRRYFDL; (d) LCDR1 comprises the amino acid sequence RASQSVSSYLA; (e) LCDR2 comprises the amino acid sequence DASKRAT; and / or (f) LCDR3 comprises the amino acid sequence QQQRSKYPPWT. In certain embodiments, the CDRs are according to the Kabat definition. In certain embodiments, the CDRs are according to the Chothia definition. In certain embodiments, the CDRs are according to the IMGT definition. In certain embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, wherein the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 3; and the immunoglobulin light chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 4.In certain embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, wherein the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 5; and the immunoglobulin light chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 6. In certain embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, wherein the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 7; and the immunoglobulin light chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 8. In certain embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, wherein the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 9; and the immunoglobulin light chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 10. In certain embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, wherein the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 11; and the immunoglobulin light chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 12. In certain embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, wherein the immunoglobulin heavy chain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 13; and the immunoglobulin light chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 14.In certain embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, wherein the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 15; and the immunoglobulin light chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 16. In certain embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, wherein the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 17; and the immunoglobulin light chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 18. In certain embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, wherein the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 19; and the immunoglobulin light chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 20. In certain embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, wherein the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 21; and the immunoglobulin light chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 22. In certain embodiments, the antibody or antigen-binding fragment thereof is an IgG antibody. In certain embodiments, the antibody or antigen-binding fragment is a Fab, F(ab)2, or single-chain variable fragment (scFv). In certain embodiments, the antibody or antigen-binding fragment thereof is chimeric or humanized.In certain embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 51, and the immunoglobulin light chain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 52. In certain embodiments, the antibody has a half-life of 14 days or more in humans. In certain embodiments, the antibody has a half-life of 21 days or more in humans. In certain embodiments, the antibody has a half-life of 25 days or more in humans. In certain embodiments, the antibody has a half-life of 30 days or more in humans. In certain embodiments, the antibody or antigen-binding fragment thereof comprises the M252Y / S254T / T256E substitution according to EU numbering in one or both of the heavy chain constant regions. In certain embodiments. , the antibody inhibits signal transduction via IGF1R. In certain embodiments, the antibody has a K -9 of less than 5×10 D . In certain embodiments, the antibody has a K -9 of less than 1×10 D . In certain embodiments, the antibody has a K -10 of less than 5×10 D . Also described herein is a nucleic acid encoding the antibody or antigen-binding fragment. Also described herein is a cell line comprising a nucleic acid encoding the antibody or antigen-binding fragment thereof. In certain embodiments, the cell line is a Chinese hamster ovary cell line. Also described herein is a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof and a pharmaceutically acceptable excipient, carrier, or diluent. In certain embodiments, the pharmaceutical composition is formulated for intravenous administration. In certain embodiments, the pharmaceutical composition is formulated for subcutaneous administration. In certain embodiments, the antibody or antigen-binding fragment thereof or the pharmaceutical composition is used in a method of inhibiting IGF1R signal transduction in an individual.

[0004] The novel features described in this specification are particularly set out in the appended claims. A better understanding of the features and advantages described in this specification will be obtained by reference to the following detailed description which sets out illustrative examples in which the principles of the features described herein are utilized, and to the appended drawings.

Brief Description of the Drawings

[0005]

Figure 1

Figure 2

Figure 3

Modes for Carrying Out the Invention

[0006] In the following description, specific details are set forth in order to provide a thorough understanding of the various embodiments. However, one of ordinary skill in the art will understand that the provided embodiments may be practiced without these details. Unless the context requires otherwise, throughout the specification and the following claims, the word "comprise" and its variations, such as "comprises" and "comprising," are to be construed in an open, inclusive sense, that is, as "including, but not limited to." As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Note also that the term "or" is generally used in its inclusive sense, that is, "and / or," unless the content clearly dictates otherwise. Further, the headings provided in this specification are for convenience only and do not interpret the scope or meaning of the claimed embodiments.

[0007] As used herein, the term "about" refers to an amount that is within 10% less than the stated amount.

[0008] As used herein, the terms "individual," "patient," or "subject" refer to an individual who has been diagnosed with at least one disease for which the described compositions and methods are useful for treatment, is suspected of having the disease, or is at risk of developing the disease. In certain embodiments, the individual is a mammal. In certain embodiments, the mammal is a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, or yak. In certain embodiments, the individual is a human.

[0009] Among the antibodies provided are monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies and multireactive antibodies), and antibody fragments. Antibodies include antibody conjugates and molecules containing the antibody, such as chimeric molecules. Thus, antibodies include, but are not limited to, full-length antibodies and native antibodies, and fragments and portions thereof that retain their binding specificity, such as those having any of several immunoglobulin classes and / or isotypes (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE, and IgM); and any and all binding moieties, including but not limited to Fab, F(ab’)2, Fv, and scFv (single-chain or related entities), and biologically relevant (antigen-binding) fragments or specific binding portions thereof. Monoclonal antibodies are generally one of substantially homogeneous antibody compositions; thus, all individual antibodies contained in a monoclonal antibody composition are identical, except for naturally occurring mutations that may be present in small amounts. Monoclonal antibodies may include the human IgG1 constant region. Monoclonal antibodies may include the human IgG4 constant region.

[0010] The term "antibody" as used herein is used in the broadest sense and includes monoclonal antibodies, and fragment antigen-binding (Fab) fragments, F(ab’)2 fragments, Fab’ fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain antibody fragments (single-chain variable fragments (sFv or scFv), and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments), intact antibodies and functional (antigen-binding) antibody fragments thereof. The term includes immunoglobulins that have been genetically engineered and / or otherwise modified, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecificities, such as bispecific antibodies, diabodies, triabodies, and tetra-bodies, tandem di-scFv, tandem tri-scFv. Unless otherwise specified, it should be understood that the term "antibody" includes its functional antibody fragments. The term also includes intact or full-length antibodies, which include antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD. An antibody may include a human IgG1 constant region. An antibody may include a human IgG4 constant region.

[0011] The terms "complementary determining region" and "CDR", which are synonymous with "hypervariable region" or "HVR", are known in the art to refer to discontinuous sequences of amino acids within the variable region of an antibody that confer antigen specificity and / or binding affinity. Generally, there are three CDRs (CDR-H1, CDR-H2, CDR-H3) within each heavy chain variable region, and three CDRs (CDR-L1, CDR-L2, CDR-L3) within each light chain variable region. The terms "framework region" and "FR" are known in the art to refer to the non-CDR portions of the variable regions of the heavy and light chains. Generally, there are four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) within each full-length heavy chain variable region, and four FRs (FR-L1, FR-L2, FR-L3, and FR-L4) within each full-length light chain variable region.The exact amino acid sequence boundaries of a given CDR or FR can be readily determined using any of several well-known schemes, including those described below: Kabat et al. (1991), “Sequences of Proteins of Immunological Interest”, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (the “Kabat” numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 (the “Chothia” numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography”, J. Mol. Biol. 262, 732-745”. (the “Contact” numbering scheme); Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains”, Dev Comp Immunol, 2003 Jan;27(1):55-77 (the “IMGT” numbering scheme); Honegger A and Plueckthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool”, J Mol Biol, 2001 Jun 8;309(3):657-70, (the “Aho” numbering scheme); and Whitelegg NR and Rees AR, “WAM: an improved algorithm for modelling antibodies on the WEB”, Protein Eng. 2000 Dec;13(12):819-24 (the “AbM” numbering scheme).In certain embodiments, the CDRs of the antibodies described herein can be defined by methods selected from Kabat, Chothia, IMGT, Aho, AbM, or combinations thereof.

[0012] The boundaries of a given CDR or FR can vary depending on the scheme specifically used. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. The numbering of both the Kabat and Chothia schemes is based on the most common antibody region sequence lengths, with insertions being addressed by inserted letters, e.g., "30a", and deletions occurring in some antibodies. The two schemes place certain insertions and deletions ("indels") at different positions, resulting in different numberings. The Contact scheme is based on the analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme.

[0013] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The variable domains of the heavy and light chains of a native antibody (V H and V L ), respectively) generally have similar structures, and each domain contains four conserved framework regions (FRs) and three CDRs (see, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single V H or V L domain may be sufficient to confer antigen-binding specificity. Further, antibodies that bind a particular antigen can be isolated by screening libraries of V H or V L domains derived from antibodies that bind the antigen against libraries of complementary V L or V H domains (see, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

[0014] As used herein, specific binding or binding of an antibody molecule described herein refers to binding mediated by one or more CDR portions of the antibody. Not all CDRs may be required for specific binding. Specific binding to a particular described target or antigen, which shows a significant increase in binding compared to an isotype control antibody, can be demonstrated, for example, by ELISA.

[0015] As used herein, an "epitope" refers to the binding determinant of an antibody or fragment thereof described herein that is minimally required for specific binding to a target antigen. When the target antigen is a polypeptide, the epitope can be a continuous or discontinuous epitope. A continuous epitope is formed by one region of the target antigen, while a discontinuous epitope can be formed from two or more separate regions. A discontinuous epitope can be formed, for example, when the target antigen adopts a tertiary structure that brings together two amino acid sequences to form a three-dimensional structure that is bound by the antibody. When the target antigen is a polypeptide, the epitope is generally a plurality of amino acids linked in the polypeptide chain. A continuous epitope can include 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous amino acids. An epitope can include a continuous polymer of amino acids, but not all amino acids of the polymer may be contacted by the amino acid residues of the antibody. Such non-contact amino acids can be important for the structure and linkage of the contact amino acids and thus also form part of the epitope. One of ordinary skill in the art can determine, for example, by cross-blocking experiments with a reference antibody, whether any given antibody binds to the epitope of the reference antibody. In certain embodiments, described herein are antibodies that bind to the same epitope of the described antibody. In certain embodiments, described herein are antibodies that are competitively blocked by the described antibody. In certain embodiments, described herein are antibodies that compete with the described antibody for binding.

[0016] Among the antibodies provided are antibody fragments. An "antibody fragment" refers to a molecule other than an intact antibody that includes a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab’, Fab’-SH, F(ab’)2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv or sFv); and multispecific antibodies formed from antibody fragments. In certain embodiments, the antibody is a single-chain antibody fragment that includes a variable heavy chain region and / or a variable light chain region, such as scFv.

[0017] Antibody fragments can be made by a variety of techniques including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells. In some embodiments, the antibody is a recombinantly produced fragment, e.g., one that includes a non-naturally occurring configuration, e.g., two or more antibody regions or chains linked by a synthetic linker, e.g., a polypeptide linker, and / or one that is not produced by enzymatic digestion of a naturally occurring intact antibody. In some aspects, the antibody fragment is scFv.

[0018] A "humanized" antibody is an antibody in which all or substantially all of the CDR amino acid residues are derived from non-human CDRs and all or substantially all of the FR amino acid residues are derived from human FRs. A humanized antibody may, in some cases, include at least a portion of the antibody constant region derived from a human antibody. A "humanized form" of a non-human antibody refers to a variant of the non-human antibody that has been humanized such that it retains the specificity and affinity of the parental non-human antibody while typically reducing immunogenicity to humans. In some embodiments, some FR residues in the humanized antibody are replaced with the corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived) so as to, for example, restore or improve antibody specificity or affinity.

[0019] Among the antibodies provided are human antibodies. A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell, or a non-human source utilizing a human antibody repertoire or other human antibody coding sequences (including a human antibody library). The term excludes humanized forms of non-human antibodies that include non-human antigen-binding regions, e.g., those in which all or substantially all of the CDRs are non-human.

[0020] Human antibodies can be prepared by administering an immunogen to a transgenic animal that is modified to produce intact human antibodies or intact antibodies having human variable regions in response to antigen administration. Such animals typically contain all or part of the human immunoglobulin locus, which may replace the endogenous immunoglobulin locus, be present episomally, or be randomly integrated into the chromosomes of the animal. In such transgenic animals, the endogenous immunoglobulin locus is generally inactivated. Also, human antibodies can be derived from human antibody libraries, including phage display, and cell-free libraries containing antibody coding sequences derived from the human repertoire.

[0021] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. The polypeptides provided, including antibodies and antibody chains, as well as other peptides such as linkers and binding peptides, can contain amino acid residues including natural amino acid residues and / or non-natural amino acid residues. The terms also include post-expression modifications of polypeptides such as glycosylation, sialylation, acetylation, phosphorylation, etc. In some embodiments, a polypeptide can contain modifications to the native or natural sequence as long as the protein maintains the desired activity. Such modifications can be intentional, such as by site-directed mutagenesis, or accidental, such as due to mutations in the host producing the protein or errors resulting from PCR amplification. In some embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. Variants typically differ from the polypeptides specifically disclosed herein by one or more substitutions, deletions, additions, and / or insertions. Such variants can occur naturally or can be generated synthetically, for example, by modifying one or more of the previous polypeptide sequences of the invention, evaluating one or more of the biological activities of the polypeptides described herein, and / or using any of several known techniques. For example, it may be desirable to improve the binding affinity and / or other biological properties of an antibody. Amino acid sequence variants of an antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from residues within the amino acid sequence of the antibody, and / or insertions into such residues, and / or substitutions of such residues. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct has the desired properties, such as antigen binding.

[0022] The percent sequence identity to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after aligning the sequences without considering any conservative substitutions as part of sequence identity and introducing gaps, if necessary, to achieve the maximum percent sequence identity. The alignment for the purpose of determining the percent amino acid sequence identity can be achieved by various known methods using publicly available computer software such as, for example, BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. It is possible to determine appropriate parameters for aligning the sequences, including an algorithm necessary to achieve the maximum alignment over the entire length of the sequences being compared. However, for the purposes herein, the percent amino acid sequence identity value is generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and the source code has been submitted with user documentation at the U.S. Copyright Office, Washington D.C., 20559 (where it is registered under U.S. Copyright Registration No. TXU510087). The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, Calif.) or can be compiled from the source code. The ALIGN-2 program should be compiled for use in a UNIX (registered trademark) operating system including Digital UNIX (registered trademark) V4.0D. All sequence comparison parameters are set by and are invariant with the ALIGN-2 program.

[0023] In situations where ALIGN-2 is used for amino acid sequence comparison, the percent amino acid sequence identity of a given amino acid sequence A to or against, or with, a given amino acid sequence B (which may otherwise be expressed as a given amino acid sequence A having, or including, a particular percent amino acid sequence identity to or with a given amino acid sequence B) is calculated as follows: 100 × fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in the alignment of the programs for A and B, and Y is the total number of amino acid residues in B. It will be appreciated that the length of amino acid sequence A is not equal to the length of amino acid sequence B and that the percent amino acid sequence identity of A to B is not equal to the percent amino acid sequence identity of B to A. Unless otherwise specified, all percent amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0024] In some embodiments, the antibodies provided herein have a dissociation constant (K D ) of about 1 μM, 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM or less (e.g., 10 -8 M or less, e.g., 10 -8 M to 10 -13 M, e.g., 10 -9 M to 10 -13 M) with respect to the antibody target. In some embodiments, the antibodies provided herein have a dissociation constant (K D ) of about 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM, or 0.001 nM or more (e.g., 10 -8 M or less, e.g., 10 -8 M to 10 -13 M, e.g., 10 -9 M to 10 -13 M) with respect to the antibody target. K DIt can be measured by any suitable assay. In certain embodiments, KD can be measured using a surface plasmon resonance assay (e.g., using a BIACORE-2000®, BIACORE-3000®, or Octet).

[0025] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibodies provided herein to generate Fc region variants. The Fc region herein is the C-terminal region of the immunoglobulin heavy chain containing at least a portion of the constant region. Fc regions include native sequence Fc regions and variant Fc regions. Fc region variants may include human Fc region sequences (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc regions) containing amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0026] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibodies provided herein to generate Fc region variants. The Fc region herein is the C-terminal region of the immunoglobulin heavy chain containing at least a portion of the constant region. Fc regions include native sequence Fc regions and variant Fc regions. Fc region variants may include human Fc region sequences (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc regions) containing amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0027] In some examples, the Fc region of an immunoglobulin is important for many important antibody functions (e.g., effector functions) such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cell-mediated phagocytosis (ADCP), and results in the death of target cells through different mechanisms. Thus, in some embodiments, the antibodies described herein comprise the variable domains of the invention combined with constant domains comprising various Fc regions selected based on the biological activity of the antibody for the intended use. In a particular example, human IgG can be classified into, for example, four subclasses: IgG1, IgG2, IgG3, and IgG4. Each of these has an Fc region with a unique profile for binding to one or more of the Fcγ receptors (activating receptors FcγRI (CD64), FcγRIIA, FcγRIIC (CD32); FcγRIIIA and FcγRIIIB (CD16), and inhibitory receptor FcγRIIB) and for the first component of complement (C1q). Human IgG1 and IgG3 bind to all Fcγ receptors; IgG2 binds to FcγRIIA H131 and has a lower affinity for FcγRIIA R131 than for FcγRIIIA V158 ; IgG4 binds to FcγRI, FcγRIIA, FcγRIIB, FcγRIIC, and FcγRIIIA V158 ; and the inhibitory receptor FcγRIIB has a lower affinity for IgG1, IgG2, and IgG3 than for all other Fcγ receptors. Studies have shown that FcγRI does not bind to IgG2 and that FcγRIIIB does not bind to IgG2 or IgG4. Ibid. Generally, with respect to ADCC activity, human IgG1 ≥ IgG3 ≥ IgG4 ≥ IgG2.

[0028] In some embodiments, the antibodies of the disclosure are variants with reduced effector function, thereby making them desirable candidates for applications where certain effector functions (such as complement fixation and ADCC) are unnecessary or harmful. Such antibodies may have reduced complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), or antibody-dependent cellular phagocytosis (ADCP). In some embodiments, the antibodies of the disclosure are variants with increased effector function for applications where increased immunogenicity would be beneficial. Such antibodies may have increased CDC, ADCC, or ADCP, or combinations thereof. Non-limiting examples of in vitro assays for evaluating the ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362 and U.S. Patent No. 5,821,337. Additionally, non-radioactive assay methods (such as the ACTI™ and CytoTox 96® non-radioactive cytotoxicity assays) may be used. Effector cells useful in such assays include peripheral blood mononuclear cells (PBMCs), monocytes, macrophages, and natural killer (NK) cells.

[0029] Antibodies can have an extended half-life and improved binding to the neonatal Fc receptor (FcRn) (see, e.g., U.S. Patent Application Publication No. 2005 / 0014934). Such antibodies can include an Fc region having one or more substitutions that improve the binding of the Fc region to FcRn, and can have substitutions at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434 (according to the EU numbering system) (see, e.g., U.S. Patent No. 7,371,826). Other examples of Fc region variants are also contemplated (see, e.g., Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent Nos. 5,648,260 and 5,624,821; and International Publication No. 94 / 29351 pamphlet). One such mutation that confers an extended half-life is the "YTE" mutation having the M252Y / S254T / T256E mutation according to EU numbering. Another such mutation that confers an extended half-life is the "LS" mutation having the Met428Leu / Asn434Ser mutation according to EU numbering.

[0030] In some embodiments, it may be desirable to generate a cysteine-engineered antibody, such as a "thioMAb," in which one or more residues of the antibody are replaced with cysteine residues. In some embodiments, the residue substitution occurs at an accessible site of the antibody. To create an immune complex, reactive thiol groups can be positioned at sites for conjugation to other moieties, such as a drug moiety or a linker-drug moiety. In some embodiments, any one or more of the following residues: V205 of the light chain (Kabat numbering); A118 of the heavy chain (EU numbering); and S400 of the heavy chain Fc region (EU numbering) can be replaced with cysteine.

[0031] In some embodiments, the antibodies provided herein can be further modified to contain additional non - proteinaceous moieties that are known and available. Suitable moieties for derivatizing antibodies include, but are not limited to, water - soluble polymers. Non - limiting examples of water - soluble polymers include polyethylene glycol (PEG), copolymers of ethylene glycol / propropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly - 1,3 - dioxolane, poly - 1,3,6 - trioxane, ethylene / maleic anhydride copolymer, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n - vinyl pyrrolidone) polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyol (e.g., glycerol), polyvinyl alcohol, and mixtures thereof, but are not limited thereto. Polyethylene glycol propionaldehyde may have advantages in manufacture due to its stability in water. The polymer can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if two or more polymers are attached, they can be the same molecule or different molecules.

[0032] The antibodies described herein can be encoded by nucleic acids. Nucleic acids are a type of polynucleotide that contains two or more nucleotide bases. In certain embodiments, the nucleic acid is a component of a vector that can be used to transfer into a cell a polypeptide encoding the polynucleotide. As used herein, the term "vector" refers to a nucleic acid molecule that is capable of transporting another nucleic acid to which the nucleic acid molecule is linked. One type of vector is an integrative vector or "integrating vector" that can be integrated into the chromosomal DNA of a host cell. Another type of vector is an "episomal" vector, e.g., a nucleic acid capable of extrachromosomal replication. A vector capable of directing the expression of an operably linked gene is referred to herein as an "expression vector". Suitable vectors include plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, viral vectors, and the like. In an expression vector, regulatory elements such as promoters, enhancers, polyadenylation signals, etc. used for the control of transcription can be derived from mammalian, microbial, viral, or insect genes. Usually, an origin of replication that confers the ability to replicate in a host and a selectable gene for facilitating the recognition of transformants may additionally be incorporated. Vectors derived from viruses such as lentiviruses, retroviruses, adenoviruses, adeno-associated viruses, etc. can be used. Plasmid vectors can be linearized for integration into genomic regions. In certain embodiments, the expression vector is a plasmid. In certain embodiments, the expression vector is a lentivirus, an adenovirus, or an adeno-associated virus. In certain embodiments, the expression vector is an adenovirus. In certain embodiments, the expression vector is an adeno-associated virus. In certain embodiments, the expression vector is a lentivirus.

[0033] When used herein to describe an amino acid or nucleic acid sequence as compared to a reference sequence, the terms “identical,” “identity,” or “percent identity” as used herein can be determined using the equations described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990, modified as in Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such equations are incorporated into the basic local alignment search tool (BLAST) programs of Altschul et al. (J. Mol. Biol. 215:403-410, 1990). The percent identity of a sequence can be determined using the most recent version of BLAST as of the filing date of the present application.

[0034] The nucleic acids encoding the antibodies described herein can be used to infect appropriate cells, transfect the cells, transform them, or otherwise make the cells transgenic for the nucleic acid, thereby enabling the production of antibodies for commercial or therapeutic use. Standard cell lines and methods for generating antibodies from large-scale cell cultures are known in the art. See, e.g., Li et al., “Cell culture processes for monoclonal antibody production.” MAbs. 2010 Sep-Oct;2(5):466-477. In certain embodiments, the cells are eukaryotic cells. In certain embodiments, the eukaryotic cells are mammalian cells. In certain embodiments, the mammalian cells are cell lines useful for producing antibodies, including Chinese hamster ovary (CHO) cells, NS0 mouse myeloma cells, or PER.C6® cells. In certain embodiments, the nucleic acid encoding the antibody is integrated into the genomic locus of a cell useful for producing the antibody. In certain embodiments, described herein is a method of making an antibody, comprising culturing a cell comprising a nucleic acid encoding the antibody under in vitro conditions sufficient to permit production and secretion of the antibody.

[0035] In certain embodiments, described herein is: (a) a mammalian cell line comprising a nucleic acid encoding an antibody described herein, integrated at a genomic locus; and (b) a master cell bank comprising a cryoprotectant. In certain embodiments, the cryoprotectant comprises glycerol or DMSO. In certain embodiments, the master cell bank is contained within a suitable vial or container capable of withstanding freezing by liquid nitrogen.

[0036] Also described herein is a method of making an antibody described herein. Such a method includes incubating a cell or cell line comprising a nucleic acid encoding the antibody in a cell culture medium under conditions sufficient to permit expression and secretion of the antibody, and further harvesting the antibody from the cell culture medium. Harvesting may further include one or more purification steps to remove live cells, cell debris, non-antibody proteins, or polypeptides, unwanted salts, buffers, and medium components. In certain embodiments, additional purification steps include centrifugation, ultracentrifugation, protein A, protein G, protein A / G, or protein L purification, and / or ion exchange chromatography.

[0037] As used herein, "treating," "treatment," or "treat" refers to an intentional intervention in a physiological disorder that provides, for example, alleviation of the severity of a disease or symptom; shortening of the duration of the course of a symptom; amelioration or elimination of one or more symptoms associated with the disease or symptom; or provision of a beneficial effect to a subject having the disease or symptom. Treatment does not require cure of the underlying disease or condition.

[0038] A "therapeutically effective amount", "effective dosage", "effective amount", or "therapeutically effective dosage" of a drug or therapeutic agent, when used alone or in combination with another therapeutic agent, is any amount of the drug that protects a subject from the onset of a disease, or promotes regression of the disease as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of periods without disease symptoms, or prevention of a disorder or physical impairment resulting from the disease. The ability of a therapeutic agent to promote regression of a disease can be evaluated, for example, in human subjects during a clinical trial, in an animal model system that predicts efficacy in humans, using various methods known to those of skill in the art, or by assessing the activity of the agent in an in vitro assay.

[0039] As used herein, "pharmaceutically acceptable" in relation to a "carrier", "excipient", or "diluent" includes any physiologically compatible solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. In some embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, intraspinal, or topical administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., the antibody, may be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0040] The pharmaceutical compounds described herein may include one or more pharmaceutically acceptable salts. "Pharmaceutically acceptable salts" refer to salts that retain the desired biological activity of the parent compound and do not impart any undesirable toxicological effects (see, for example, Berge, S.M., et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous, etc., and those derived from non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, etc., and those derived from non-toxic organic amines such as Ν,Ν’-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, etc.

[0041] Described herein, in one aspect, is an antibody or antigen-binding fragment thereof that binds to the insulin-like growth factor 1 receptor (IGF1R), the antibody or antigen-binding fragment thereof comprising: an immunoglobulin heavy chain CDR1 (HCDR1) comprising any one of the amino acid sequences of SEQ ID NOs: 30-33; an immunoglobulin heavy chain CDR2 (HCDR2) comprising any one of the amino acid sequences of SEQ ID NOs: 34-40; an immunoglobulin heavy chain CDR3 (HCDR3) comprising any one of the amino acid sequences of SEQ ID NOs: 41 or 42; an immunoglobulin light chain CDR1 (LCDR1) comprising any one of the amino acid sequences of SEQ ID NOs: 43-45; an immunoglobulin light chain CDR2 (LCDR2) comprising the amino acid sequence of SEQ ID NO: 46; and / or an immunoglobulin light chain CDR3 (LCDR3) comprising any one of the amino acid sequences of SEQ ID NOs: 47-49; the antibody or antigen-binding fragment thereof does not include an immunoglobulin heavy chain variable region identical to SEQ ID NO: 1 and / or an immunoglobulin light chain variable region identical to SEQ ID NO: 2.

[0042] Described herein, in one aspect, is an antibody or an antigen-binding fragment thereof that binds to insulin-like growth factor 1 receptor (IGF1R), and the antibody or the antigen-binding fragment thereof comprises the following: (a) an immunoglobulin heavy chain CDR1 (HCDR1) comprising the amino acid sequence SX1GMH, wherein X1 is H, Y, A, or T; (b) an immunoglobulin heavy chain CDR2 (HCDR2) comprising the amino acid sequence X1IX2X3DX4SX5TYYADSVRG, wherein X1 is I, T, or Y, X2 is W, N, or A, X3 is F, H, A, or G, X4 is G or A, and X5 is S or T; (c) an immunoglobulin heavy chain CDR3 (HCDR3) comprising the amino acid sequence ELX1RRYFDL, wherein X1 is G or N; (d) an immunoglobulin light chain CDR1 (LCDR1) comprising the amino acid sequence RASQSVSSX1LA, wherein X1 is Y, A, or T; (e) an immunoglobulin light chain CDR2 (LCDR2) comprising the amino acid sequence DASKRAT; and / or an immunoglobulin light chain CDR3 (LCDR3) comprising the amino acid sequence QQRX1KX2PPWT, wherein X1 is S or G and X2 is Y or W); the antibody or the antigen-binding fragment thereof does not comprise an immunoglobulin heavy chain variable region identical to SEQ ID NO: 1 and / or an immunoglobulin light chain variable region identical to SEQ ID NO: 2). In certain embodiments, the antibody or the antigen-binding fragment thereof comprises an immunoglobulin heavy chain and an immunoglobulin light chain, the immunoglobulin heavy chain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 3; the immunoglobulin light chain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 4. In certain embodiments, the antibody or the antigen-binding fragment thereof comprises an immunoglobulin heavy chain and an immunoglobulin light chain, the immunoglobulin heavy chain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 5; the immunoglobulin light chain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 6.In certain embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 7; and the immunoglobulin light chain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 8. In certain embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 9; and the immunoglobulin light chain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 10. In certain embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 11; and the immunoglobulin light chain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 12. In certain embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 13; and the immunoglobulin light chain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 14. In certain embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 15; and the immunoglobulin light chain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 16.In certain embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 17, and the immunoglobulin light chain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 18. In certain embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 19, and the immunoglobulin light chain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 20. In certain embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 21, and the immunoglobulin light chain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 22. In certain embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 51, and the immunoglobulin light chain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 52. In certain embodiments, the antibody is an IgG antibody. In certain embodiments, the antibody or antigen-binding fragment is a Fab, F(ab)2, or single-chain variable fragment (scFv). In certain embodiments, the antibody or antigen-binding fragment thereof is chimeric or humanized. In certain embodiments, the antibody inhibits signal transduction via IGF1R. In certain embodiments, the antibody has a K of less than 5×10. -9 K less than M D In certain embodiments, the antibody has a K of less than 1×10 -9 K less than M DIt has. In certain embodiments, the antibody has a K of less than 5×10 -10 M D It has. In certain embodiments, the antibody has a half-life of 14 days or more in humans. In certain embodiments, the antibody has a half-life of 21 days or more in humans. In certain embodiments, the antibody comprises the M252Y / S254T / T256E substitution according to EU numbering in one or both heavy chain constant regions.

[0043] Described herein is, in one aspect, an antibody or antigen-binding fragment thereof that binds to insulin-like growth factor 1 receptor (IGF1R), the antibody or antigen-binding fragment thereof comprising: (a) HCDR1 comprising the amino acid sequence SHGMH; (b) HCDR2 comprising the amino acid sequence YIWFDGSSTYYADSVRG; (c) HCDR3 comprising the amino acid sequence ELGRRYFDL; (d) LCDR1 comprising the amino acid sequence RASQSVSSALA; (e) LCDR2 comprising the amino acid sequence DASKRAT; and / or (f) LCDR3 comprising the amino acid sequence QQRSKYPPWT. In certain embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain and an immunoglobulin light chain, the immunoglobulin heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 17, and the immunoglobulin light chain comprising an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 18. In certain embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain and an immunoglobulin light chain, the immunoglobulin heavy chain comprising the same amino acid sequence as that shown in SEQ ID NO: 17, and the immunoglobulin light chain comprising the same amino acid sequence as that shown in SEQ ID NO: 18. In certain embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain and an immunoglobulin light chain, the immunoglobulin heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 51, and the immunoglobulin light chain comprising an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 52. In certain embodiments, the antibody or antigen-binding fragment thereof is an IgG antibody. In certain embodiments, the antibody or antigen-binding fragment thereof is a Fab, F(ab)2, or single-chain variable fragment (scFv). In certain embodiments, the antibody or antigen-binding fragment thereof is chimeric or humanized. In certain embodiments, the antibody inhibits signal transduction via IGF1R. In certain embodiments, the antibody has a K of less than 5×10 -9 M D . In certain embodiments, the antibody has a K of 1×10 -9Less than K of M D has. In certain embodiments, the antibody is 5×10 -10 Less than K of M D has. In certain embodiments, the antibody has a half-life of 14 days or more in humans. In certain embodiments, the antibody has a half-life of 21 days or more in humans. In certain embodiments, the antibody comprises the M252Y / S254T / T256E substitution according to EU numbering in one or both heavy chain constant regions.

[0044] Described herein, in one aspect, is an antibody or an antigen-binding fragment thereof that binds to the insulin-like growth factor 1 receptor (IGF1R), and the antibody or the antigen-binding fragment thereof comprises the following: (a) HCDR1 comprising the amino acid sequence SYGMH; (b) HCDR2 comprising the amino acid sequence IIWFDGSSTYYADSVRG; (c) HCDR3 comprising the amino acid sequence ELGRRYFDL; (d) LCDR1 comprising the amino acid sequence RASQSVSSYLA; (e) LCDR2 comprising the amino acid sequence DASKRAT; and / or (f) LCDR3 comprising the amino acid sequence QQRSKYPPWT. In certain embodiments, the antibody or the antigen-binding fragment thereof is an IgG antibody. In certain embodiments, the antibody or the antigen-binding fragment thereof comprises an immunoglobulin heavy chain and an immunoglobulin light chain, the immunoglobulin heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 7, and the immunoglobulin light chain comprising an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 8. In certain embodiments, the antibody or the antigen-binding fragment thereof comprises an immunoglobulin heavy chain and an immunoglobulin light chain, the immunoglobulin heavy chain comprising the same amino acid sequence as that shown in SEQ ID NO: 7, and the immunoglobulin light chain comprising the same amino acid sequence as that shown in SEQ ID NO: 8. In certain embodiments, the antibody or the antigen-binding fragment thereof is a Fab, F(ab)2, or single-chain variable fragment (scFv). In certain embodiments, the antibody or the antigen-binding fragment thereof is chimeric or humanized. In certain embodiments, the antibody inhibits signal transduction via IGF1R. In certain embodiments, the antibody has a K of less than 5×10 -9 M D . In certain embodiments, the antibody has a K of less than 1×10 -9 M D . In certain embodiments, the antibody has a K of less than 5×10 -10 M DIt has. In certain embodiments, the antibody has a half-life of 14 days or more in humans. In certain embodiments, the antibody has a half-life of 21 days or more in humans. In certain embodiments, the antibody comprises an M252Y / S254T / T256E substitution according to EU numbering in one or both heavy chain constant regions.

[0045] Described herein is, in one aspect, an antibody or antigen-binding fragment thereof that binds to insulin-like growth factor 1 receptor (IGF1R), wherein the antibody or antigen-binding fragment thereof comprises: (a) HCDR1 comprising the amino acid sequence SHGMH; (b) HCDR2 comprising the amino acid sequence IIAGDASTTYYADSVRG; (c) HCDR3 comprising the amino acid sequence ELGRRYFDL; (d) LCDR1 comprising the amino acid sequence RASQSVSSYLA; (e) LCDR2 comprising the amino acid sequence DASKRAT; and / or (f) LCDR3 comprising the amino acid sequence QQRSKYPPWT. In certain embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 5, and the immunoglobulin light chain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to that shown in SEQ ID NO: 6. In certain embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain comprises the same amino acid sequence as that shown in SEQ ID NO: 5, and the immunoglobulin light chain comprises the same amino acid sequence as that shown in SEQ ID NO: 6. In certain embodiments, the antibody or antigen-binding fragment thereof is an IgG antibody. In certain embodiments, the antibody or antigen-binding fragment thereof is a Fab, F(ab)2, or single-chain variable fragment (scFv). In certain embodiments, the antibody or antigen-binding fragment thereof is chimeric or humanized. In certain embodiments, the antibody inhibits signal transduction via IGF1R. In certain embodiments, the antibody has a K -9 less than 5×10 D In certain embodiments, the antibody has a K -9Less than K of M D has. In certain embodiments, the antibody is 5×10 -10 Less than K of M D has. In certain embodiments, the antibody has a half-life of 14 days or more in humans. In certain embodiments, the antibody has a half-life of 21 days or more in humans. In certain embodiments, the antibody has a half-life of 25 days or more in humans. In certain embodiments, the antibody has a half-life of 30 days or more in humans. In certain embodiments, the antibody comprises the M252Y / S254T / T256E substitution according to EU numbering in one or both heavy chain constant regions.

[0046] In one aspect, described herein is a teplizumab derivative with increased affinity, the teplizumab derivative comprising a substitution of the tryptophan at position 94 of SEQ ID NO: 2 with tyrosine.

[0047] IGF1R signaling is disrupted (e.g., increased) in several human-related diseases. The antibodies described herein are potentially useful for the treatment of such diseases associated with this abnormal signaling. The antibodies described herein can be used to effectively treat individuals with IGF1R disorders by inhibiting IGF1R signaling. IGF1R signaling occurs mainly via the PI3K pathway and the RAS pathway. IGF1R signaling or inhibition of IGF1R signaling can be determined by phosphorylation of IGF1R. In certain embodiments, the antibodies described herein exhibit inhibition with an EC50 of 10 ng / mL or less. In certain embodiments, the antibodies described herein exhibit inhibition with an EC50 of 9 ng / mL or less. In certain embodiments, the antibodies described herein exhibit inhibition with an EC50 of 8 ng / mL or less. In certain embodiments, the antibodies described herein exhibit inhibition with an EC50 of 7 ng / mL or less. In certain embodiments, the antibodies described herein exhibit inhibition with an EC50 of 6 ng / mL or less. In certain embodiments, the antibodies described herein exhibit inhibition with an EC50 of 5 ng / mL or less. Such assays for determining EC50 are described herein and can be performed with 200 ng / mL of recombinant human IGF-1 using wells in a flat-bottom 96-well plate with 4 x 10^4 NCI-H322 cells / well.

[0048] In certain embodiments, the antibody can be administered to a subject in need thereof (e.g., a subject suffering from a disease associated with IGF1R signaling disorder or abnormal IGF1R signaling) by any route suitable for administration of an antibody-containing pharmaceutical composition, such as subcutaneously, intraperitoneally, intravenously, intramuscularly, or intratumorally. In certain embodiments, the antibody is administered intravenously. In certain embodiments, the antibody is administered subcutaneously. In certain embodiments, the antibody is administered intratumorally. In certain embodiments, the antibody is administered based on a suitable dosing schedule, such as once a week, twice a week, once a month, twice a month, once every two weeks, once every three weeks, or once a month. In certain embodiments, the antibody is administered once every three weeks. The antibody can be administered in any therapeutically effective amount. In certain embodiments, the therapeutically acceptable amount is from about 0.1 mg / kg to about 50 mg / kg. In certain embodiments, the therapeutically acceptable amount is from about 1 mg / kg to about 40 mg / kg. In certain embodiments, the therapeutically acceptable amount is from about 1 mg / kg to about 20 mg / kg. In certain embodiments, the therapeutically acceptable amount is from about 1 mg / kg to about 10 mg / kg. In certain embodiments, the therapeutically acceptable amount is from about 5 mg / kg to about 30 mg / kg. In certain embodiments, the therapeutically acceptable amount is from about 5 mg / kg to about 20 mg / kg.

[0049] Pharmaceutically acceptable excipients, carriers, and diluents In certain embodiments, the anti-IGF1R antibodies of the present disclosure are included in a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, carriers, and diluents. Pharmaceutically acceptable excipients, carriers, and diluents can be included to increase the shelf life, stability, or administrability of the antibody. Such compounds include salts, pH buffers, detergents, anticoagulants, and preservatives. In certain embodiments, the antibodies of the present disclosure are administered by suspending them in a sterile solution. In certain embodiments, the solution contains about 0.9% NaCl. In certain embodiments, the solution contains about 5.0% dextrose. In certain embodiments, the solution further contains one or more of the following: buffers such as acetate, citrate, histidine, succinate, phosphate, bicarbonate, and hydroxymethylaminomethane (Tris); surfactants such as polysorbate 80 (Tween® 80), polysorbate 20 (Tween® 20), and poloxamer 188; polyols / disaccharides / polysaccharides such as glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40; amino acids such as glycine or arginine; antioxidants such as ascorbic acid, methionine; or chelating agents such as EDTA or EGTA.

[0050] Antibodies in formulations for subcutaneous administration generally exist in a high-concentration form containing more than 50 mg / ml, 100 mg / ml, 200 mg / ml, or 300 mg / ml. Many excipients useful for subcutaneous formulations are known. See, for example, Wang et al., Antibody Therapeutics, 2021, Vol. 4, No. 4 262-273.

[0051] In certain embodiments, the antibodies of the present disclosure can be lyophilized for transport / storage and reconstituted prior to administration. In certain embodiments, the lyophilized antibody formulation includes a bulking agent such as mannitol, sorbitol, sucrose, trehalose, dextran 40, or combinations thereof. The lyophilized formulation can be contained within a vial composed of glass or other suitable non-reactive material. The antibody, when formulated, can be buffered at a specific pH, generally less than 7.0, regardless of whether it is reconstituted. In certain embodiments, the pH can be 4.5 - 7.0, 4.5 - 6.5, 4.5 - 6.0, 4.5 - 5.5, 4.5 - 5.0, or 5.0 - 6.0.

[0052] Also described herein is a kit comprising one or more of the antibodies described herein and one or more additional components selected from the following, in a suitable container: instructions for use; diluents, excipients, carriers, and devices for administration (e.g., syringe / needle or other injector).

[0053] In certain embodiments, described herein is a method for preparing a composition for inhibiting IGF1R signaling in an individual, the method comprising mixing one or more pharmaceutically acceptable excipients, carriers, or diluents with an antibody of the present disclosure. In certain embodiments, described herein is a method for preparing a cancer treatment for storage or transport, the method comprising lyophilizing one or more antibodies of the present disclosure.

Examples

[0054] The following illustrative examples are representative of embodiments of the compositions and methods described herein and are not intended to be limiting in any way.

[0055] Example 1 - Determination of Antibody Affinity To develop antibodies with higher affinity than required for subcutaneous formulations, multiple variants of a reference antibody with the teprotumumab variable region (including the variable region containing SEQ ID NO: 1 and SEQ ID NO: 2) were tested to determine binding affinity. The antibodies described herein were tested by Octet for binding affinity to IGF1R. The results are shown in Table 1 below. Sequence alignments of all teprotumumab variants are shown in FIGS. 1A and 1B. Surprisingly, a mutation of tryptophan at position 94 of the light chain to tyrosine was present in all antibodies. This was surprising because generally, light chain CDRs are not as important for binding affinity as heavy chain CDRs.

[0056]

Table 1

[0057] Affinity measurement of teprotumumab variants by biolayer interferometry Human IGF-I R (Glu 31-Asn 932 with a polyhistidine tag at the C-terminus) expressed from human 293 cells (HEK293) was purchased from ACROBiosystems (Newark, DE). A Dip and Read Ni-NTA (NTA) biosensor from Sartorius (Bohemia, NY), pre-immobilized with nickel-charged tris-NTA, enabled the evaluation of the kinetic properties of novel anti-human IGF-I R antibodies. Briefly, antibody variants were digested into purified Fab fragments (to enable 1:1 binding and global fitting). Monovalent binding Fab domains of each antibody variant were generated using the FabALACTICA Fab kit from GENOVIS (Cambridge, MA). Subsequently, the Fab fragments were separated from Fc using a CaptureSelect™ Fc column. Binding of variant Fabs to NTA-captured human IGF-I R (His-tagged) was monitored by Octet RED96e (Sartorius) using biolayer interferometry (BLI). After charging the NTA biosensor with 10 mM NiCl2, approximately 5 μg / mL of His-tagged human IGF-I R was loaded up to an average loading response of 0.67 nm shift. Variant Fabs were assayed from 0 to 100 nM in 10× kinetics buffer from Sartorius (1× PBS, 0.1% BSA, 0.02% Tween®-20, and Kathon as a preservative). Binding kinetics and affinity were determined by applying 1:1 global fitting to double-reference subtracted data using Octet Analysis Studio Software (Sartorius).

[0058] Example 2 - Biological Activity of Teprotumumab Variants The biological activity of teprotumumab variants was determined by assessing the inhibition of IGF-1R phosphorylation and the binding of ligands IGF-1 and 2 to the receptor. The results are shown in Tables 2 to 4. The clones generally showed biological activity equivalent to or better than that of the reference teprotumumab, indicating that the improved affinity seen in Example 2 also extends to biological activity.

[0059]

Table 2

[0060]

Table 3

[0061]

Table 4

[0062] Inhibition of IGF-1R phosphorylation 4×10^4 H322 cells / well were seeded in a flat-bottom 96-well plate and incubated overnight at 37°C in a 5% incubator. The cells were treated with different concentrations of anti-IGF1R for 1 hour and stimulated with 200 ng / mL of recombinant human IGF-1 protein for 30 minutes. Phosphorylated IGF-1 in the cell lysates was then measured according to the manufacturing protocol using the Insulin Signaling Panel whole cell lysate kit (MSD).

[0063] IGF-1 binding inhibition assay Maxisorp plates were coated with 1.5 μg / mL of IGF-1R overnight at 4°C. The plates were washed and blocked with 1% BSA. Subsequently, serially diluted anti-IGF1R antibody was incubated at room temperature for 30 minutes and then incubated with IGF-1 biotin at a final concentration of 20 ng / mL. The plates were washed and streptavidin-horseradish peroxidase was added. After the final wash, 3,3’,5,5’-tetramethylbenzidine was added for 8 minutes. The reaction was stopped with HCL-based stop solution. The plates were measured for absorbance at 450 nM and the data were analyzed using Softmax Pro 7.1 software.

[0064] IGF-2 Binding Inhibition Assay Maxisorp plates were coated with 1.5 μg / mL of IGF-1R overnight at 4°C. The plates were washed and blocked with 1% BSA. Subsequently, serially diluted anti-IGF1R antibody was incubated at room temperature for 30 minutes and then incubated with IGF-2 biotin at a final concentration of 200 ng / mL. The plates were washed and streptavidin-horseradish peroxidase was added. After the final wash, 3,3’,5,5’-tetramethylbenzidine was added for 8 minutes. The reaction was stopped with HCL-based stop solution. The plates were measured for absorbance at 450 nM and the data were analyzed using Softmax Pro 7.1 software.

[0065] Clone D03 with the YTE mutation in Example 3 exhibits ADCC activity equivalent to that of teprotumumab. The higher the affinity of an antibody, the greater the antibody-dependent cell-mediated cytotoxicity (ADCC) it can promote when administered to an individual. For use in treating ophthalmic symptoms and other symptoms associated with autoimmune or inflammatory symptoms, an increase in ADCC would be undesirable. Clone D03 was selected to further test its ADCC activity using different heavy chain constant region mutations (the "YTE" mutation with the M252Y / S254T / T256E mutation according to EU numbering; and the "LS" mutation of Met428Leu / Asn434Ser according to EU numbering). As shown in Figure 2, clone D03 formatted with the LS mutation ("D03-LS") surprisingly showed an increase in ADCC compared to clone D03 with the YTE mutation ("D03-YTE") and compared to teprotumumab (inverted triangle). D03-YTE showed ADCC equivalent to that of teprotumumab.

[0066] ADCC assay The antibody was incubated with DU145 cells at a specified concentration for 4 hours. At that time, effector cells with a luminescent ADCC reporter were added and incubated overnight, and BioGlo detection reagent was added for 10 minutes, and the results were read with a luminometer.

[0067] Clone D03 with the YTE mutation showed a 2- to 3-fold extension of the half-life compared to teprotumumab. The half-life of clone D03 was tested in cynomolgus monkeys. Cynomolgus monkeys were dosed with D03-YTE at 150 mg / kg IV, 150 mg / kg subcutaneous, or 75 mg / kg subcutaneous. Serum samples were collected at 2, 6, 24, 96, 168, 336, 504, 672, 1008, 1344, 1680, and 2016 hours. Serum samples were analyzed for D03-YTE by ELISA. As shown in Table 5 below, D03 has an extended serum half-life compared to teprotumumab.

[0068]

Table 5

[0069] Clone D03 with the YTE mutation shows an increased inhibition of IGF-1R phosphorylation compared to teprotumumab. D03-YTE was tested for its ability to inhibit IGF-1R phosphorylation. The experiment described in Example 2 was performed with the following modifications: The dilution of the anti-IGF-1R antibody was started at 100 μg / mL and serially diluted 1 / 8-fold, allowing 100% inhibition by teprotumumab. The experiment in Figure 3 shows that in this experimental setting, teprotumumab (circles) showed an IC50 of 397.4 ng / mL, compared to 20.51 ng / mL for D03-YTE (squares).

[0070] Example 5 - Teprotumumab variants retain the manufacturability of the parental clone. One limiting factor in developing an antibody for subcutaneous injection is that, in addition to binding with high affinity and / or having a longer in vivo half-life, the antibody must have favorable biophysical properties, such as low hydrophobicity, a tendency not to form aggregates, and the ability to be present in a formulation with low viscosity. Any mutation made to the antibody has the potential to adversely affect these properties. However, as shown in this example, despite the increased affinity and biological potency, there were no detrimental changes with respect to the major manufacturability criteria. As shown in Table 6, D03 with the YTE mutation did not show an increase in degradation when subjected to forced degradation at 40°C for 28 days compared to teprotumumab with the YTE mutation (buffer composition: 20 mM histidine / histidine-HCl, 40 mM L-methionine, 210 mM trehalose, pH 5.5, 0.2% PX188).

[0071] [Table 6]

[0072] The anti-IGF-1R variants were evaluated to ensure that Fc and variable domain mutations did not significantly increase viscosity. See Table 7. An increase in viscosity is undesirable as it can limit the options for liquid formulations for subcutaneous administration. The viscosity of the anti-IGF-1R variants was first evaluated at approximately 130 mg / ml. One variant (B09-YTE) showed an increase in viscosity compared to other variants and the control mAb. This variant was not evaluated at higher concentrations. Since there was little differentiation at approximately 130 mg / ml, the viscosity was evaluated at a higher concentration (approximately 170 mg / ml). At 170 mg, all D03 and E01 variants had viscosity measurements of less than 20 cP and were similar to the teprotumumab control.

[0073] The antibodies were formulated in the following buffer: 20 mM histidine / histidine-HCl, 40 mM L-methionine, 210 mM trehalose, pH 5.5, 0.2% PX188. The antibodies were evaluated at concentrations of 130 mg / ml and 170 mg / ml and at 20 °C and 25 °C. The antibody concentration was determined by SoloVPE (in triplicate). Viscosity was evaluated by RheoSense m-VROC applying a shear sweep rate of 400 - 2700 / sec at 20 °C and 25 °C collecting 8 - 12 segments.

[0074]

Table 7

[0075] Anti-IGF-1R variants were evaluated by hydrophobic interaction chromatography (HIC) analysis (Table 8). HIC is an emerging assessment of the hydrophobic properties of antibodies and other proteins. Excessively hydrophobic properties are detrimental to overall stability and can lead to increased non-specific binding and self-interaction. All anti-IGF-1R variants had elution profiles similar to that of the teprotumumab control. No increase in hydrophobic properties was observed. Teprotumumab and its variants eluted earlier than the positive control (NISTmAb) and significantly earlier than CNTO607 (a mAb known to have hydrophobic properties that cause developmental problems). These data suggest that the mutations introduced to increase either IGF-1R affinity or half-life extension did not affect the overall hydrophobic properties of the variants.

[0076] HIC was performed using an Agilent 1260 Infinity II HPLC equipped with a ProPac HIC-10, 5 μm, 4.6 × 100 mm column (ThermoFisher PN: 063655). Method parameters were obtained directly from the MabPAC-10 product insert. The following buffers were used: Buffer A: 2 M ammonium sulfate, 0.1 M sodium phosphate, 2-propanol (93:7 v / v), pH 7.0; Buffer B: 0.1 M sodium phosphate, 2-propanol (93:7 v / v), pH 7.0. After equilibration in 95% Buffer A and 5% Buffer B for 5 minutes, a 25-minute gradient ending with 100% Buffer B was used. The temperature was 30 °C, and protein elution was visualized using UV detection at 214 nM. Also, NISTmAb (positive control human IgG1) and CNTO607 (a mAb with known hydrophobic properties) were evaluated as comparators.

[0077]

Table 8

[0078] Preferred embodiments of the present invention have been shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Here, those skilled in the art will envision various changes and substitutions without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein can be used in the practice of the present invention.

[0079] Publications, patent applications, issued patents, and other documents cited herein are hereby incorporated by reference into this specification as if each individual publication, patent application, issued patent, or other document were specifically and individually indicated as being incorporated by reference in its entirety. Definitions contained in the text incorporated by reference are excluded to the extent they are inconsistent with the definitions in this disclosure.

[0080] The sequences described herein [Chemical formula] [Chemical formula] [Chemical formula]

[0081] [Table 9]

Claims

1. An antibody or antigen-binding fragment thereof that binds to the insulin-like growth factor 1 receptor (IGF1R), wherein the antibody or antigen-binding fragment comprises an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, and the immunoglobulin heavy chain variable region is: a. Amino acid sequence SX １ A GMH-containing immunoglobulin heavy chain CDR1 (HCDR1), wherein X １ is H, Y, A, or T, with HCDR1; b. Amino acid sequence X １ IX ２ X ３ DX ４ SX ５ An immunoglobulin heavy chain CDR2 (HCDR2) containing TYYADSVRG, wherein X １ is I, T, or Y, and X ２ is W, N, or A, and X ３ is F, H, A, or G, and X ４ is G or A, and X ５ is S or T, and the HCDR2; c. Amino acid sequence ELX １ An immunoglobulin heavy chain CDR3 (HCDR3) containing RRYFDL, wherein X １ HCDR3 is G or N The immunoglobulin light chain variable region includes: d. Amino acid sequence RASQSVSX １ An immunoglobulin light chain CDR1 (LCDR1) containing LA, wherein X １ is Y, A, or T, with LCDR1; e. Immunoglobulin light chain CDR2 (LCDR2) containing the amino acid sequence DASKRAT; f. Amino acid sequence QQRX １ KX ２ An immunoglobulin light chain CDR3 (LCDR3) containing PPWT, wherein X １ is S or G, X ２ Y is LCDR3; An antibody or its antigen-binding fragment, including the above.

2. a. The HCDR1 comprises the amino acid sequence (SHGMH) of SEQ ID NO: 30; b. The HCDR2 comprises the amino acid sequence of SEQ ID NO: 35 (YIWFDGSSTYYADSVRG); c. The HCDR3 comprises the amino acid sequence of SEQ ID NO: 41 (ELGRRYFDL); d. The LCDR1 comprises the amino acid sequence of SEQ ID NO: 43 (RASQSVSSALA); e. The LCDR2 comprises the amino acid sequence of SEQ ID NO: 46 (DASKRAT); f. The antibody or antigen-binding fragment thereof according to claim 1, wherein the LCDR3 comprises the amino acid sequence of SEQ ID NO: 48 (QQRSKYPPWT).

3. The antibody or antigen-binding fragment according to claim 1, wherein the immunoglobulin heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 17, and the immunoglobulin light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

18.

4. a. The HCDR1 comprises the amino acid sequence (SYGMH) of SEQ ID NO: 33; b. The HCDR2 comprises the amino acid sequence of SEQ ID NO: 38 (IIWFDGSSTYYADSVRG); c. The HCDR3 comprises the amino acid sequence of SEQ ID NO: 41 (ELGRRYFDL); d. The LCDR1 comprises the amino acid sequence of SEQ ID NO: 45 (RASQSVSSYLA); e. The LCDR2 comprises the amino acid sequence of SEQ ID NO: 46 (DASKRAT); f. The antibody or antigen-binding fragment thereof according to claim 1, wherein the LCDR3 comprises the amino acid sequence of SEQ ID NO: 48 (QQRSKYPPWT).

5. a. The HCDR1 comprises the amino acid sequence (SHGMH) of SEQ ID NO: 30; b. The HCDR2 comprises the amino acid sequence of SEQ ID NO: 34 (IIAGDASTTYYADSVRG); c. The HCDR3 comprises the amino acid sequence of SEQ ID NO: 41 (ELGRRYFDL); d. The LCDR1 comprises the amino acid sequence of SEQ ID NO: 45 (RASQSVSSYLA); e. The LCDR2 comprises the amino acid sequence of SEQ ID NO: 46 (DASKRAT); f. The antibody or antigen-binding fragment thereof according to claim 1, wherein the LCDR3 comprises the amino acid sequence of SEQ ID NO: 48 (QQRSKYPPWT).

6. The antibody or antigen-binding fragment according to claim 1, wherein the immunoglobulin heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 3, and the immunoglobulin light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

4.

7. The antibody or antigen-binding fragment according to claim 1, wherein the immunoglobulin heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 5, and the immunoglobulin light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

6.

8. The antibody or antigen-binding fragment according to claim 1, wherein the immunoglobulin heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7, and the immunoglobulin light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

8.

9. The antibody or antigen-binding fragment according to claim 1, wherein the immunoglobulin heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9, and the immunoglobulin light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

10.

10. The antibody or antigen-binding fragment thereof according to claim 1, wherein the immunoglobulin heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 11, and the immunoglobulin light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

12.

11. The antibody or antigen-binding fragment according to claim 1, wherein the immunoglobulin heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 13, and the immunoglobulin light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

14.

12. The antibody or antigen-binding fragment thereof according to claim 1, wherein the immunoglobulin heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15, and the immunoglobulin light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

16.

13. The antibody or antigen-binding fragment according to claim 1, wherein the immunoglobulin heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 19, and the immunoglobulin light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

20.

14. The antibody or antigen-binding fragment thereof according to claim 1, wherein the immunoglobulin heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 21, and the immunoglobulin light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

22.

15. The antibody or antigen-binding fragment according to claim 1, wherein the antibody is an IgG antibody or an antigen-binding fragment thereof.

16. The antigen-binding fragment is Fab, F(ab) ２ The antibody or antigen-binding fragment according to claim 1, which is a single-chain variable fragment (scFv).

17. The antibody or antigen-binding fragment thereof according to claim 1, comprising an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain includes M252Y, S254T, and T256E substitutions according to EU numbering.

18. The antibody or antigen-binding fragment thereof according to claim 17, characterized by a half-life of 25 days or more in humans.

19. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof inhibits IGF1R-mediated signal transduction.

20. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody binds to human IGF1R with a KD of less than 5 × 10⁻⁹ M.

21. A nucleic acid encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 20.

22. A cell line comprising the nucleic acid described in claim 21.

23. The cell line according to claim 22, which is a Chinese hamster ovary cell line.

24. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 20, and a pharmaceutically acceptable excipient, carrier, or diluent.

25. The pharmaceutical composition according to claim 24, formulated for intravenous administration.

26. The pharmaceutical composition according to claim 24, formulated for subcutaneous administration.

27. ​​A monoclonal antibody that binds to IGF1R, comprising an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain is: HCDR1 containing the amino acid sequence (SHGMH) of SEQ ID NO: 30; HCDR2 containing the amino acid sequence of SEQ ID NO: 35 (YIWFDGSTYYADSVRG); HCDR3 containing the amino acid sequence of SEQ ID NO: 41 (ELGRRYFDL) The immunoglobulin light chain comprises a variable region of the immunoglobulin heavy chain, the immunoglobulin light chain being: LCDR1 containing the amino acid sequence of SEQ ID NO: 43 (RASQSVSALA); LCDR2 containing the amino acid sequence of SEQ ID NO: 46 (DASKRAT); LCDR3 containing the amino acid sequence of SEQ ID NO: 48 (QQRSKYPPWT) A monoclonal antibody containing the immunoglobulin light chain variable region.

28. The monoclonal antibody according to claim 27, wherein the immunoglobulin heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 17, and the immunoglobulin light chain variable region comprises the amino acid sequence of SEQ ID NO:

18.

29. The monoclonal antibody according to claim 27, wherein the immunoglobulin heavy chain comprises a human IgG1 constant region having M252Y, S254T, and T256E substitutions according to EU numbering.

30. The monoclonal antibody according to claim 27, wherein the immunoglobulin heavy chain comprises the amino acid sequence of SEQ ID NO: 51, and the immunoglobulin light chain comprises the amino acid sequence of SEQ ID NO:

52.

31. A pharmaceutical composition comprising a monoclonal antibody according to any one of claims 27 to 30 and a pharmaceutically acceptable excipient, carrier, or diluent.

32. A nucleic acid encoding a monoclonal antibody according to any one of claims 27 to 30.

33. A cell line comprising the nucleic acid described in Claim 32.

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