Igf1r antibodies

Abstract

Described herein are antibodies that bind to Insulin-like Growth Factor Receptor 1 and inhibit signaling via Insulin-like Growth Factor Receptor 1.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 500,168, filed on May 4, 2023, and U.S. Provisional Application No. 63 / 351,077, filed on June 10, 2022, both of which are incorporated herein by reference in their entirety. Summary of the invention

[0003] Subcutaneous administration of antibody therapy to patients is a more convenient and cost-effective delivery route than intravenous administration, because such treatment can be applied at home, and does not require the presence of a trained medical practitioner. However, an obstacle to subcutaneous administration of antibodies is that the volume of the antibody formulations to be used in prefilled syringes, onbody infusors, autoinjectors, etc. is significantly reduced. In order to solve this problem, high-concentration antibody formulations are required, but due to viscosity problems, aggregation reactions and other unfavorable factors for formulating macromolecules, this may be impractical for subcutaneous delivery. A solution is to use a novel antibody that binds to a target with higher affinity or exerts higher biological activity compared to known antibodies. In addition, antibodies comprising certain Fc region mutations have a longer half-life in vivo, which can further reduce the amount of antibodies contained in the subcutaneous formulation.

[0004] IGF1R antibodies with high binding affinity and high bioinhibitory activity are described herein. In addition, IGF1R antibodies with extended half-life and low-level ADCC activity are described herein. Such antibodies can be effectively included in formulations for subcutaneous administration to improve the ease of administration to patients. In one aspect, an antibody or antigen-binding fragment thereof is described herein that binds to insulin-like growth factor 1 receptor (IGF1R), wherein the antibody or antigen-binding fragment thereof comprises: (a) an immunoglobulin heavy chain CDR1 (HCDR1) comprising the amino acid sequence SX 1 GMH, where X 1 is H, Y, A or T; (b) an immunoglobulin heavy chain CDR2 (HCDR2) comprising the amino acid sequence X 1 IX 2 X 3 DX 4 SX 5 TYYADSVRG, where X 1 is I, T or Y, X 2 W, N or A, X 3 is F, H, A or G, X 4 It is G or A, X 5is S or T; (c) an immunoglobulin heavy chain CDR3 (HCDR3) comprising the amino acid sequence ELX 1 RRYFDL, where X 1 is G or N; (d) an immunoglobulin light chain CDR1 (LCDR1) comprising the amino acid sequence RASQSVSSX 1 LA, where X 1 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 QQRX 1 KX 2 PPWT, where X 1 It is S or G, X 2 is Y or W; wherein the antibody or 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 one embodiment, the antibody or antigen-binding fragment thereof binds to insulin-like growth factor 1 receptor (IGF1R), wherein the antibody or antigen-binding fragment thereof comprises: (a) an immunoglobulin heavy chain CDR1 (HCDR1) comprising the amino acid sequence SX 1 GMH, where X 1 is H, Y, A or T; (b) an immunoglobulin heavy chain CDR2 (HCDR2) comprising the amino acid sequence X 1 IX 2 X 3 DX 4 SX 5 TYYADSVRG, where X 1 is I, T or Y, X 2 W, N or A, X 3 is F, H, A or G, X 4 It is G or A, X 5 is S or T; (c) an immunoglobulin heavy chain CDR3 (HCDR3) comprising the amino acid sequence ELX 1 RRYFDL, where X 1 is G or N; (d) an immunoglobulin light chain CDR1 (LCDR1) comprising the amino acid sequence RASQSVSSX 1 LA, where X 1 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 QQRX 1 KX 2 PPWT, where X 1 It is S or G, X2is Y or W; wherein the antibody or antigen-binding fragment thereof does not comprise 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 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), wherein: (a) the HCDR1 comprises the amino acid sequence SHGMH; (b) the HCDR2 comprises the amino acid sequence YIWFDGSSTYYADSVRG; (c) the HCDR3 comprises the amino acid sequence ELGRRYFDL; (d) the LCDR1 comprises the amino acid sequence RASQSVSSALA; (e) the LCDR2 comprises the amino acid sequence DASKRAT; and / or (f) the LCDR3 comprises the amino acid sequence QQRSKYPPWT. In certain embodiments, the antibody or 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), wherein: (a) the HCDR1 comprises the amino acid sequence SYGMH; (b) the HCDR2 comprises the amino acid sequence IIWFDGSSTYYADSVRG; (c) the HCDR3 comprises the amino acid sequence ELGRRYFDL; (d) the LCDR1 comprises the amino acid sequence RASQSVSSYLA; (e) the LCDR2 comprises the amino acid sequence DASKRAT; and / or (f) the LCDR3 comprises the amino acid sequence QQRSKYPPWT. In certain embodiments, the antibody or 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), wherein: (a) the HCDR1 comprises the amino acid sequence SHGMH; (b) the HCDR2 comprises the amino acid sequence IIAGDASTTYYADSVRG; (c) the HCDR3 comprises the amino acid sequence ELGRRYFDL; (d) the LCDR1 comprises the amino acid sequence RASQSVSSYLA; (e) the LCDR2 comprises the amino acid sequence DASKRAT; and / or (f) the LCDR3 comprises the amino acid sequence QQRSKYPPWT.In certain embodiments, the CDRs are defined according to Kabat. In certain embodiments, the CDRs are defined according to Chothia. In certain embodiments, the CDRs are defined according to IMGT. 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 having at least about 90%, 95%, 97%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 3; and wherein the immunoglobulin light chain variable region comprises an amino acid sequence having at least about 90%, 95%, 97%, 99% or 100% identity with the amino acid sequence 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 having at least about 90%, 95%, 97%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 5; and wherein the immunoglobulin light chain variable region comprises an amino acid sequence having at least about 90%, 95%, 97%, 99% or 100% identity with the amino acid sequence 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 having at least about 90%, 95%, 97%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 7; and wherein the immunoglobulin light chain variable region comprises an amino acid sequence having at least about 90%, 95%, 97%, 99% or 100% identity with the amino acid sequence 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 the amino acid sequence shown in SEQ ID NO:9; and wherein the immunoglobulin light chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99% or 100% identical to the amino acid sequence 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 the amino acid sequence shown in SEQ ID NO:11; and wherein the immunoglobulin light chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99% or 100% identical to the amino acid sequence 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 the amino acid sequence shown in SEQ ID NO:13; and wherein the immunoglobulin light chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99% or 100% identical to the amino acid sequence 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 the amino acid sequence shown in SEQ ID NO:15; and wherein the immunoglobulin light chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99% or 100% identical to the amino acid sequence 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 the amino acid sequence shown in SEQ ID NO:17; and wherein the immunoglobulin light chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99% or 100% identical to the amino acid sequence 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 the amino acid sequence shown in SEQ ID NO:19; and wherein the immunoglobulin light chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99% or 100% identical to the amino acid sequence 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 having at least about 90%, 95%, 97%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 21; and wherein the immunoglobulin light chain variable region comprises an amino acid sequence having at least about 90%, 95%, 97%, 99% or 100% identity with the amino acid sequence 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 thereof is Fab, F(ab). 2Or a single chain variable fragment (scFv). In certain embodiments, the antibody or its antigen-binding fragment is chimeric or humanized. In certain embodiments, the antibody or its antigen-binding fragment comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain comprises an amino acid sequence having at least about 90%, 95%, 97%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 51; and wherein the immunoglobulin light chain comprises an amino acid sequence having at least about 90%, 95%, 97%, 99% or 100% identity with the amino acid sequence 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 a M252Y / S254T / T256E substitution according to EU numbering in one or both heavy chain constant regions. In certain embodiments, the antibody inhibits signaling through IGF1R. In certain embodiments, the antibody has less than 5x10 -9 M of K D In certain embodiments, the antibody has a -9 M of K D In certain embodiments, the antibody has a -10 M of K D . Also described herein are nucleic acids encoding the antibodies or antigen binding fragments. Also described herein are cell lines comprising nucleic acids encoding the antibodies or antigen binding fragments. In certain embodiments, the cell line is a Chinese hamster ovary cell line. Also described herein are pharmaceutical compositions comprising the antibodies or antigen binding fragments thereof and a pharmaceutically acceptable excipient, carrier, or diluent. In certain embodiments, the pharmaceutical compositions are formulated for intravenous administration. In certain embodiments, the pharmaceutical compositions are formulated for subcutaneous administration. In certain embodiments, the antibodies or antigen binding fragments thereof or pharmaceutical compositions are used in methods of inhibiting IGF1R signaling in an individual. BRIEF DESCRIPTION OF THE DRAWINGS

[0005] The novel features described herein are particularly set forth in the appended claims. A better understanding of the features and advantages of the features described herein will be obtained by referring to the following detailed description and the accompanying drawings, which set forth illustrative examples in which the principles of the features described herein are utilized, and the accompanying drawings, in which:

[0006] Figure 1A A multiple sequence alignment of the heavy chain variable regions described herein is shown.

[0007] Figure 1B A multiple sequence alignment of the light chain variable regions described herein is shown.

[0008] Figure 2 Antibody-dependent cellular cytotoxicity (ADCC) assays performed with clone D03 with different heavy chain constant region formats ("YTE" mutation with mutations at M252Y / S254T / T256E according to EU numbering; and "LS" mutation Met428Leu / Asn434Ser according to EU numbering) are shown. RLU = relative light unit.

[0009] Figure 3 Inhibition of IGF-1R signaling by teixumab and D03-YTE was demonstrated. DETAILED DESCRIPTION

[0010] In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments. However, those skilled in the art will appreciate that the embodiments provided can be practiced without these details. Unless the context otherwise requires, throughout this specification and the claims that follow, the words "comprise" and its variants (such as "comprise / comprising") should be considered open, inclusive meanings, i.e., "including but not limited to". Unless the content clearly indicates otherwise, as used in this specification and the appended claims, the singular forms "a / an" and "the" include plural referents. It should also be noted that the term "or" is generally used in its meaning that includes "and / or", unless the context clearly indicates otherwise. In addition, the titles provided herein are for convenience only and do not explain the scope or meaning of the claimed embodiments.

[0011] As used herein, the term "about" refers to an amount that is approximately 10% or less of the stated amount.

[0012] As used herein, the terms "individual," "patient," or "subject" refer to an individual diagnosed with, suspected of having, or at risk of developing at least one disease for which the described compositions and methods can be used to treat. 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.

[0013] The antibodies provided include monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies and multireactive antibodies) and antibody fragments. Antibodies include antibody conjugates and molecules comprising antibodies, such as chimeric molecules. Therefore, antibodies include, but are not limited to, full-length and native antibodies, as well as fragments and portions thereof that retain their binding specificity, such as any specific binding portion thereof, including those having any number of immunoglobulin classes and / or isotypes (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE, and IgM); and biologically relevant (antigen binding) fragments or specific binding portions thereof, including, but not limited to, Fab, F(ab') and F(ab'). 2 , Fv and scFv (single chain or related entities). A monoclonal antibody is generally one of a substantially homogeneous antibody composition; thus, any individual antibodies contained within a monoclonal antibody composition are identical except for possible naturally occurring mutations that may be present in minor amounts. A monoclonal antibody may comprise a human IgG1 constant region. A monoclonal antibody may comprise a human IgG4 constant region.

[0014] The term "antibody" as used herein is used in the broadest sense and includes monoclonal antibodies, and includes whole antibodies and functional (antigen-binding) antibody fragments thereof, including the following fragments: antigen-binding (Fab) fragments, F(ab') fragments, 2 The term "antibody" refers to a fragment of a single-chain antibody, a fragment of a Fab' fragment, a Fv fragment, a recombinant IgG (rIgG) fragment, a single-chain antibody fragment (including a single-chain variable fragment (sFv or scFv)) and a single-domain antibody (e.g., sdAb, sdFv, nanobody) fragment. The term covers genetically engineered and / or otherwise modified forms of immunoglobulins, such as intracellular antibodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies and heteroconjugate antibodies, multispecific (e.g., bispecific) antibodies, double antibodies, three antibodies and four antibodies, tandem two-scFv, tandem three-scFv. Unless otherwise indicated, the term "antibody" should be understood to cover its functional antibody fragments. The term also covers complete or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA and IgD. The antibody may include a human IgG1 constant region. The antibody may include a human IgG4 constant region.

[0015] The terms "complementarity determining region" and "CDR" (which are synonymous with "hypervariable region" or "HVR") are known in the art to refer to non-contiguous amino acid sequences within an antibody variable region that confer antigen specificity and / or binding affinity. Typically, there are three CDRs (CDR-H1, CDR-H2, CDR-H3) in each heavy chain variable region, and three CDRs (CDR-L1, CDR-L2, CDR-L3) in each light chain variable region. "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. Typically, there are four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) in each full-length heavy chain variable region, and four FRs (FR-L1, FR-L2, FR-L3, and FR-L4) in each full-length light chain variable region. The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 ("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.(“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 (“IMGT” numbering scheme); Honegger A and Plückthun 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, (“Aho” numbering scheme); and Whitelegg NR and Rees AR, “WAM: an improved algorithm for modelling antibodies on the WEB [WAM: An improved algorithm for modeling antibodies on the WEB]," Protein Eng. 2000 Dec; 13(12):819-24 ("AbM" numbering scheme). In certain embodiments, the CDRs of the antibodies described herein may be defined by a method selected from the group consisting of Kabat, Chothia, IMGT, Aho, AbM, or a combination thereof.

[0016] The boundary of a given CDR or FR may differ according to the scheme used for identification. For example, the Kabat scheme is based on structural alignment, and the Chothia scheme is based on structural information. The numbering of the Kabat and Chothia schemes is based on the most common antibody region sequence length, with the insertion provided by inserting letters (e.g., "30a") and the disappearance occurring in some antibodies. These two schemes place certain insertions and deletions ("indels") in different positions, thereby producing difference numbers. The Contact scheme is based on the analysis of complex crystal structures, and is similar to the Chothia numbering scheme in many respects.

[0017] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of a native antibody (V H and V L ) generally have a similar structure, wherein each domain comprises four conserved framework regions (FRs) and three CDRs (see, e.g., Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007)). A single V H or V L A domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a specific antigen may use a V H or V L The domains are separated from antibodies that bind to the antigen to screen for complementary V L or V H Libraries of domains (see, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

[0018] The specific binding or binding of antibody molecules described herein refers to the binding mediated by one or more CDR portions of the antibody. Not all CDRs are necessary for specific binding. Specific binding can be demonstrated, for example, by ELISA for a specific target or antigen listed, which shows a significant increase in binding compared to an isotype control antibody.

[0019] As described herein, "epitope" refers to a binding determinant of an antibody or fragment described herein, which is minimally necessary for the specific binding of the antibody or its fragment to the target antigen. When the target antigen is a polypeptide, the epitope will 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 by two or more independent regions. For example, a discontinuous epitope may be formed when the target antigen adopts a tertiary structure that combines two amino acid sequences and forms a three-dimensional structure that binds to the antibody. When the target antigen is a polypeptide, the epitope is usually connected to multiple amino acids 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 continuous amino acids. Although the epitope can include a continuous amino acid polymer, not every amino acid of the polymer can contact the amino acid residues of the antibody. Such non-contact amino acids will still include part of the epitope because they can be important for the structure and bond of the contacted amino acids. One skilled in the art can determine whether any given antibody binds to an epitope of a reference antibody, for example, by cross-blocking experiments with the reference antibody. In certain embodiments, antibodies that bind to the same epitope of the antibody are described herein. In certain embodiments, antibodies that are competitively blocked by the antibody are described herein. In certain embodiments, antibodies that compete for binding with the antibody are described herein.

[0020] Among the antibodies provided are antibody fragments. An "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an 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 by antibody fragments. In a particular embodiment, the antibody is a single-chain antibody fragment (such as, scFv) comprising a variable heavy chain region and / or a variable light chain region.

[0021] Antibody fragments can be prepared by a variety of techniques, including but not limited to proteolytic digestion of intact antibodies and production by recombinant host cells. In some embodiments, antibodies are recombinantly produced fragments, such as fragments comprising non-naturally occurring rearrangements, such as those having two or more antibody regions or chains joined by synthetic linkers (e.g., polypeptide linkers), and / or those not produced by enzymatic digestion of naturally occurring intact antibodies. In some aspects, antibody fragments are scFv.

[0022] "Humanized" antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDR and all or substantially all FR amino acid residues are derived from human FR. Humanized antibodies optionally may include at least a portion of an antibody constant region derived from a human antibody. "Humanized form" of a non-human antibody refers to a variant of a non-human antibody that has undergone humanization, typically to reduce immunogenicity to people, while retaining the specificity and affinity of the parent non-human antibody. In certain embodiments, some FR residues in a humanized antibody are replaced by corresponding residues from a non-human antibody (e.g., an antibody derived from CDR residues), such as to restore or improve antibody specificity or affinity.

[0023] Among the antibodies provided are human antibodies. A "human antibody" is an antibody whose amino acid sequence corresponds to that of an antibody produced by a human or human cell or a non-human source utilizing a human antibody library or other human antibody encoding sequences (including human antibody libraries). The term excludes humanized forms of non-human antibodies comprising non-human antigen binding regions, such as those in which all or substantially all CDRs are non-human.

[0024] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce complete human antibodies or complete antibodies with human variable regions in response to antigenic stimulation. Such animals typically contain all or part of a human immunoglobulin locus that replaces an endogenous immunoglobulin locus, or it exists outside the chromosome or is randomly integrated into the chromosome of the animal. In such transgenic animals, the endogenous immunoglobulin locus is usually inactivated. Human antibodies can also be derived from human antibody libraries, including phage display and cell-free libraries, which contain antibody coding sequences derived from human libraries.

[0025] The terms "polypeptide" and "protein" are used interchangeably and refer to polymers of amino acid residues, and are not limited to a minimum length. Polypeptides (including antibodies and antibody chains provided and other peptides, such as linkers and binding peptides) may contain amino acid residues, including natural and / or non-natural amino acid residues. These terms also include post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, phosphorylation, etc. In some aspects, these polypeptides may contain modifications relative to natural or natural sequences, as long as the protein maintains the desired activity. These modifications may be deliberate (such as by site-directed mutagenesis), or may be accidental (such as by mutations of the host producing the protein or due to errors caused by PCR amplification). In some embodiments, it is envisioned that amino acid sequence variants of antibodies provided herein. Variants are typically different from the polypeptides specifically disclosed herein in terms of one or more substitutions, deletions, additions and / or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more of the above polypeptide sequences of the present invention and evaluating one or more biological activities of the polypeptides as described herein and / or using any of a variety of known techniques. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. The amino acid sequence variants of the 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 and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions and substitutions can be performed to obtain the final construct, provided that the final construct has the desired characteristics, such as antigen binding.

[0026] The percentage of sequence identity (%) relative to a reference polypeptide sequence is to achieve the maximum percentage of sequence identity and not to consider any conservative substitution as part of sequence identity and to align the sequences and introduce a gap (if necessary), the percentage of the amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence. The comparison for determining the percentage of amino acid sequence identity can be achieved in a known variety of ways, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. It is possible to determine the appropriate parameters for aligning sequences, including the algorithm required for achieving maximum alignment on the full length of the sequence being compared. However, for the purposes of this article, the amino acid sequence identity % value is generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program is created by Genentech, Inc., and the source code has been submitted to the U.S. Copyright Office (USCopyright Office) (Washington DC 20559) together with user documentation, and registered in the U.S. Copyright Office with U.S. Copyright Registration Number TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.

[0027] Where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with or relative to a given amino acid sequence B (which may alternatively be phrased as a given amino acid sequence A having or comprising a certain % amino acid sequence identity to, with or relative to a given amino acid sequence B) is calculated as follows: the fraction X / Y multiplied by 100, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in this program's alignment of A and B, and where Y is the total number of amino acid residues in B. It should be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. Unless expressly stated otherwise, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0028] In some embodiments, the antibodies provided herein have an affinity for the antibody target 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, for example, 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 The dissociation constant (K D In some embodiments, the antibodies provided herein have a specific affinity for the antibody target 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 greater (e.g., 10 -8 M or less, for example, 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 The dissociation constant (K D K can be measured by any suitable assay. D In certain embodiments, surface plasmon resonance can be used to measure or Octet) to measure KD.

[0029] In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of the antibody provided herein, thereby generating Fc region variants. The Fc region herein is the C-terminal region of at least a portion of the constant region containing the heavy chain of an immunoglobulin. The Fc region includes a native sequence Fc region and a variant Fc region. The Fc region variant can be included in a human Fc region sequence (e.g., human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0030] In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of the antibody provided herein, thereby generating Fc region variants. The Fc region herein is the C-terminal region of at least a portion of the constant region containing the heavy chain of an immunoglobulin. The Fc region includes a native sequence Fc region and a variant Fc region. The Fc region variant can be included in a human Fc region sequence (e.g., human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0031] In some cases, the Fc region of an immunoglobulin is important for many important antibody functions (e.g., effector functions), such as antigen-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cell-mediated phagocytosis (ADCP), which, despite different mechanisms, lead to the killing of target cells. Therefore, in some embodiments, the antibodies described herein comprise variable domains of the present invention in combination with constant domains comprising different Fc regions, which are selected based on the biological activity of the antibodies for intended use. In some cases, human IgG, for example, can be classified into four subclasses, IgG1, IgG2, IgG3, and IgG4, and each of these subclasses comprises an Fc region having a unique property of binding to one or more Fcγ receptors (activating receptors FcγRI (CD64), FcγRIIA, FcγRIIC (CD32); FcγRIIIA and FcγRIIIB (CD16), and inhibiting receptors FcγRIIB) and the first component of complement (C1q). Human IgG1 and IgG3 bind to all Fcγ receptors; IgG2 binds to FcγRIIA H131 Binds to FcγRIIA R131 , FcγRIIIA V158 IgG4 has a lower affinity for FcγRI, FcγRIIA, FcγRIIB, FcγRIIC, and FcγRIIIA V158 Binds; and the inhibitory receptor FcγRIIB has a lower affinity for IgG1, IgG2, and IgG3 than all other Fcγ receptors. Studies have shown that FcγRI does not bind to IgG2, and FcγRIIIB does not bind to IgG2 or IgG4. Same as above. In general, with respect to ADCC activity, human IgG1≥IgG3>>IgG4≥IgG2.

[0032] In some embodiments, the antibody of the present disclosure is a variant with reduced effector function, which makes it an ideal candidate for applications in which certain effector functions (such as complement fixation and ADCC) are unnecessary or harmful. Such an antibody can have reduced complement dependent cytotoxicity (CDC), antibody dependent cellular toxicity (ADCC) or antibody dependent cellular phagocytosis (ADCP). In some embodiments, the antibody of the present disclosure is a variant with increased effector function, which will be a useful application for increased immunogenicity. Such an antibody can have increased CDC, ADCC or ADCP or a combination thereof. Non-limiting examples of in vitro assays for evaluating the ADCC activity of target molecules are described in U.S. Patent Nos. 5,500,362 and 5,821,337. Alternatively, non-radioactive assays (e.g., ACTI TM and CytoTox Non-radioactive cytotoxicity assays). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs), monocytes, macrophages, and natural killer (NK) cells.

[0033] The antibody may have increased half-life and improved binding to the neonatal Fc receptor (FcRn) (see, e.g., US 2005 / 0014934). Such an antibody may comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn, and including those having substitutions at one or more of the following Fc region residues, these Fc region residues are 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 and Winter, Nature 322:738-40 (1988); U.S. Pat. Nos. 5,648,260 and 5,624,821; and WO94 / 29351). One such mutation that confers increased half-life is the "YTE" mutation, which has mutations at M252Y / S254T / T256E according to EU numbering. Another such mutation that confers increased half-life is the "LS" mutation, which has mutations at Met428Leu / Asn434Ser according to EU numbering.

[0034] In some embodiments, it may be desirable to generate cysteine ​​engineered antibodies, such as "thioMAbs," in which one or more residues of an antibody are substituted with cysteine ​​residues. In some embodiments, the substituted residues occur at accessible sites of the antibody. Reactive thiol groups may be located at sites for conjugation with other moieties (e.g., drug moieties or linker drug moieties) to produce immunoconjugates. In some embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region.

[0035] In certain embodiments, the antibodies provided herein can be further modified to contain known and available other non-protein parts. Suitable parts for antibody derivatization include but are not limited to water-soluble polymers. Non-limiting examples of water-soluble polymers include but are not limited to polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers) and dextran or poly (n vinyl pyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylene polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde can have advantages in manufacturing due to its stability in water. Polymer can be any molecular weight, and can be branched or non-branched. The number of polymers attached to the antibody can vary, and if two or more polymers are attached, they can be identical or different molecules.

[0036] The antibodies described herein can be encoded by nucleic acids. Nucleic acid is a polynucleotide comprising two or more nucleotide bases. In certain embodiments, nucleic acid is a component of a vector that can be used to transfer a polypeptide encoding a polynucleotide into a cell. As used herein, the term "vector" refers to a nucleic acid molecule that can transport another nucleic acid connected thereto. One type of vector is a genomic integration vector or "integration vector", which can be integrated into the chromosomal DNA of a host cell. Another type of vector is an "additional" vector, such as a nucleic acid capable of extrachromosomal replication. A vector capable of directing the expression of a gene operably connected thereto is referred to herein as an "expression vector". Suitable vectors include plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, viral vectors, etc. In an expression vector, regulatory elements (such as promoters, enhancers, polyadenylation signals) for controlling transcription can be derived from mammals, microorganisms, viruses, or insect genes. The ability to replicate in a host and selection genes that promote transformant recognition, which are usually conferred by a replication origin, can be additionally incorporated. Vectors derived from viruses (such as slow viruses, retroviruses, adenoviruses, adeno-associated viruses, etc.) can be used. Plasmid vectors can be linearized to be integrated 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.

[0037] As used herein, the terms "homologous," "homology," or "percent homology" when used herein to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence can be determined using the formula described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990, as modified in Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such a formula is incorporated into the Basic Local Alignment Search Tool (BLAST) program of Altschul et al. (J. Mol. Biol. 215:403-410, 1990). The percent homology of a sequence can be determined using the most recent version of BLAST as of the filing date of this application.

[0038] Nucleic acids encoding the antibodies described herein can be used to infect, transfect, transform or otherwise genetically modify suitable cells to produce antibodies for commercial or therapeutic use. Standard cell lines and methods for producing antibodies from large-scale cell cultures are known in the art. See, for example, Li et al., "Cell culture processes for monoclonal antibody production," Mabs. 2010 Sep-Oct; 2(5):466–477. In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the eukaryotic cell is a mammalian cell. In certain embodiments, the mammalian cell is a cell line used to produce antibodies, which is a Chinese hamster ovary cell (CHO) cell, an NS0 mouse myeloma cell, or Cells. In certain embodiments, nucleic acids encoding antibodies are integrated into a cellular genomic locus that can be used to produce antibodies. In certain embodiments, methods for preparing antibodies are described herein, comprising culturing cells containing nucleic acids encoding antibodies under in vitro conditions sufficient to allow production and secretion of the antibody.

[0039] In certain embodiments, described herein is a master cell bank comprising: (a) a mammalian cell line comprising a nucleic acid encoding an antibody described herein integrated at a genomic location; and (b) a cryoprotectant. In certain embodiments, the cryoprotectant comprises glycerol or DMSO. In certain embodiments, the master cell bank is contained in a suitable vial or container capable of withstanding liquid nitrogen freezing.

[0040] Also described herein is a method for preparing antibodies as herein described. Such methods include hatching cells or cell lines comprising nucleic acids encoding antibodies in a cell culture medium under conditions sufficient to allow antibody expression and secretion, and further harvesting the antibodies from the cell culture medium. Harvesting may further include one or more purification steps to remove living cells, cell debris, non-antibody proteins or polypeptides, unwanted salts, buffers, and culture medium components. In certain embodiments, one or more additional purification steps include centrifugation, ultracentrifugation, protein A, protein G, protein A / G, or protein L purification and / or ion exchange chromatography.

[0041] As used herein, "treat," "treatment," or "treating" refers to an intentional intervention in a physiological disease state, such as to reduce the severity of the disease or condition; reduce the duration of the course of the condition; improve or eliminate one or more symptoms associated with the disease or condition; or provide a beneficial effect to a subject suffering from the disease or condition. Treatment does not require a cure for the underlying disease or condition.

[0042] A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dose" of a drug or therapeutic agent is any amount of the drug, when used alone or in combination with another therapeutic agent, that protects a subject from the onset of a disease or promotes regression of a disease as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or the prevention of impairment or disability due to the disease distress. The ability of a therapeutic agent to promote regression of a disease can be evaluated using a variety of methods known to skilled practitioners, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by measuring the activity of the agent in in vitro assays.

[0043] As used herein, "pharmaceutically acceptable carriers, excipients or diluents" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible. In some aspects, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound (i.e., antibody) can be coated in a material to protect the compound from the action of acids and other natural conditions that can inactivate the compound.

[0044] The pharmaceutical compounds described herein may contain one or more pharmaceutically acceptable salts. "Pharmaceutically acceptable salts" refers to salts that retain the desired biological activity of the parent compound and do not impart any undesirable toxicological effects (see, for example, Berge, SM, et al. (1977) J. Pharm. Sci. [Pharmacy Journal] 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 acid, etc., and 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, and the like, and nontoxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.

[0045] In one aspect, an antibody or antigen-binding fragment thereof is described herein that binds to insulin-like growth factor 1 receptor (IGF1R), wherein the antibody or antigen-binding fragment thereof comprises: an immunoglobulin heavy chain CDR1 (HCDR1) comprising an amino acid sequence of any one of SEQ ID NOs: 30 to 33; an immunoglobulin heavy chain CDR2 (HCDR2) comprising an amino acid sequence of any one of SEQ ID NOs: 34 to 40; an immunoglobulin heavy chain CDR3 (HCDR3) comprising an amino acid sequence of any one of SEQ ID NOs: 41 or 42; an immunoglobulin light chain CDR1 (LCDR1) comprising an amino acid sequence of any one of SEQ ID NOs: 43 to 45; an immunoglobulin light chain CDR2 (LCDR2) comprising an amino acid sequence of SEQ ID NO: 46; and / or an immunoglobulin light chain CDR3 (LCDR3) comprising an amino acid sequence of any one of SEQ ID NOs: 47 to 49; wherein the antibody or antigen-binding fragment thereof does not comprise an amino acid sequence identical to SEQ ID NOs: 48 to 49. The immunoglobulin heavy chain variable region is identical to SEQ ID NO:1 and / or the immunoglobulin light chain variable region is identical to SEQ ID NO:2.

[0046] In one aspect, described herein is 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) an immunoglobulin heavy chain CDR1 (HCDR1) comprising the amino acid sequence SX 1 GMH, where X 1 is H, Y, A or T; (b) an immunoglobulin heavy chain CDR2 (HCDR2) comprising the amino acid sequence X 1 IX 2 X 3DX 4 SX 5 TYYADSVRG, where X 1 is I, T or Y, X 2 W, N or A, X 3 is F, H, A or G, X 4 It is G or A, X 5 is S or T; (c) an immunoglobulin heavy chain CDR3 (HCDR3) comprising the amino acid sequence ELX 1 RRYFDL, where X 1 is G or N; (d) an immunoglobulin light chain CDR1 (LCDR1) comprising the amino acid sequence RASQSVSSX 1 LA, where X 1 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 QQRX 1 KX 2 PPWT, where X 1 It is S or G, X 2is Y or W; wherein the antibody or 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 antigen-binding fragment thereof comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain comprises an amino acid sequence having at least about 90%, 95%, 97%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 3; and wherein the immunoglobulin light chain comprises an amino acid sequence having at least about 90%, 95%, 97%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 4. 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 having at least about 90%, 95%, 97%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 5; and wherein the immunoglobulin light chain comprises an amino acid sequence having at least about 90%, 95%, 97%, 99% or 100% identity with the amino acid sequence 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 having at least about 90%, 95%, 97%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 7; and wherein the immunoglobulin light chain comprises an amino acid sequence having at least about 90%, 95%, 97%, 99% or 100% identity with the amino acid sequence 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 having at least about 90%, 95%, 97%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 9; and wherein the immunoglobulin light chain comprises an amino acid sequence having at least about 90%, 95%, 97%, 99% or 100% identity with the amino acid sequence 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 having at least about 90%, 95%, 97%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 11; and wherein the immunoglobulin light chain comprises an amino acid sequence having at least about 90%, 95%, 97%, 99% or 100% identity with the amino acid sequence 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 having at least about 90%, 95%, 97%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 13; and wherein the immunoglobulin light chain comprises an amino acid sequence having at least about 90%, 95%, 97%, 99% or 100% identity with the amino acid sequence 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 having at least about 90%, 95%, 97%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 15; and wherein the immunoglobulin light chain comprises an amino acid sequence having at least about 90%, 95%, 97%, 99% or 100% identity with the amino acid sequence 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 having at least about 90%, 95%, 97%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 17; and wherein the immunoglobulin light chain comprises an amino acid sequence having at least about 90%, 95%, 97%, 99% or 100% identity to the amino acid sequence 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 having at least about 90%, 95%, 97%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 19; and wherein the immunoglobulin light chain comprises an amino acid sequence having at least about 90%, 95%, 97%, 99% or 100% identity to the amino acid sequence 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 the amino acid sequence shown in SEQ ID NO:21; and wherein the immunoglobulin light chain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99% or 100% identical to the amino acid sequence 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 having at least about 90%, 95%, 97%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 51; and wherein the immunoglobulin light chain comprises an amino acid sequence having at least about 90%, 95%, 97%, 99% or 100% identity with the amino acid sequence 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 a single chain variable fragment (scFv). In certain embodiments, the antibody or its antigen-binding fragment is chimeric or humanized. In certain embodiments, the antibody inhibits signaling via IGF1R. In certain embodiments, the antibody has less than 5x10 -9 M of K D In certain embodiments, the antibody has a -9 M of K D In certain embodiments, the antibody has a -10 M of K D In certain embodiments, the antibody has a half-life of 14 days or longer in humans. In certain embodiments, the antibody has a half-life of 21 days or longer in humans. In certain embodiments, the antibody comprises a M252Y / S254T / T256E substitution according to EU numbering in one or both heavy chain constant regions.

[0047] In one aspect, the present invention describes an antibody or an antigen-binding fragment thereof that binds to the 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 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, wherein the immunoglobulin heavy chain comprises an amino acid sequence having at least about 90%, 95%, 97%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 17; and wherein the immunoglobulin light chain comprises an amino acid sequence having at least about 90%, 95%, 97%, 99% or 100% identity to the amino acid sequence 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 identical to the amino acid sequence shown in SEQ ID NO: 17; and wherein the immunoglobulin light chain comprises an amino acid sequence identical to the amino acid sequence 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 having at least about 90%, 95%, 97%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO:51; and wherein the immunoglobulin light chain comprises an amino acid sequence having at least about 90%, 95%, 97%, 99% or 100% identity to the amino acid sequence 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) or 2 or a single chain variable fragment (scFv). In certain embodiments, the antibody or its antigen-binding fragment is chimeric or humanized. In certain embodiments, the antibody inhibits signaling via IGF1R. In certain embodiments, the antibody has less than 5x10 -9 M of K D In certain embodiments, the antibody has a -9 M of K D In certain embodiments, the antibody has a -10 M of K DIn certain embodiments, the antibody has a half-life of 14 days or longer in humans. In certain embodiments, the antibody has a half-life of 21 days or longer in humans. In certain embodiments, the antibody comprises a M252Y / S254T / T256E substitution according to EU numbering in one or both heavy chain constant regions.

[0048] In one aspect, an antibody or antigen-binding fragment thereof is described herein 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 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 antigen-binding fragment thereof is an IgG antibody. 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 having at least about 90%, 95%, 97%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO:7; and wherein the immunoglobulin light chain comprises an amino acid sequence having at least about 90%, 95%, 97%, 99% or 100% identity to the amino acid sequence 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 identical to the amino acid sequence shown in SEQ ID NO:7; and wherein the immunoglobulin light chain comprises an amino acid sequence identical to the amino acid sequence shown in SEQ ID NO:8. In certain embodiments, the antibody or antigen-binding fragment thereof is a Fab, F(ab) or Fab fragment thereof. 2 or a single chain variable fragment (scFv). In certain embodiments, the antibody or its antigen-binding fragment is chimeric or humanized. In certain embodiments, the antibody inhibits signaling via IGF1R. In certain embodiments, the antibody has less than 5x10 -9 M of K D In certain embodiments, the antibody has a -9 M of K D In certain embodiments, the antibody has a -10 M of K DIn certain embodiments, the antibody has a half-life of 14 days or longer in humans. In certain embodiments, the antibody has a half-life of 21 days or longer in humans. In certain embodiments, the antibody comprises a M252Y / S254T / T256E substitution according to EU numbering in one or both heavy chain constant regions.

[0049] In one aspect, the present invention describes an antibody or an antigen-binding fragment thereof that binds to the 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 having at least about 90%, 95%, 97%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO:5; and wherein the immunoglobulin light chain comprises an amino acid sequence having at least about 90%, 95%, 97%, 99% or 100% identity with the amino acid sequence 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 identical to the amino acid sequence shown in SEQ ID NO:5; and wherein the immunoglobulin light chain comprises an amino acid sequence identical to the amino acid sequence 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) or Fab fragment thereof. 2 or a single chain variable fragment (scFv). In certain embodiments, the antibody or its antigen-binding fragment is chimeric or humanized. In certain embodiments, the antibody inhibits signaling via IGF1R. In certain embodiments, the antibody has less than 5x10 -9 M of K D In certain embodiments, the antibody has a -9 M of K D In certain embodiments, the antibody has a -10 M of K D. In certain embodiments, the antibody has a half-life of 14 days or longer in humans. In certain embodiments, the antibody has a half-life of 21 days or longer in humans. In certain embodiments, the antibody has a half-life of 25 days or longer in humans. In certain embodiments, the antibody has a half-life of 30 days or longer in humans. In certain embodiments, the antibody comprises a M252Y / S254T / T256E substitution according to EU numbering in one or both heavy chain constant regions.

[0050] In one aspect, described herein are tefiximab derivatives with increased affinity, wherein the tefiximab derivative comprises a substitution of tryptophan at position 94 of SEQ ID NO: 2 with tyrosine.

[0051] IGF1R signaling is disturbed (e.g., increased) in several diseases associated with humans, and the antibodies described herein may be used to treat 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 primarily through PI3K and RAS pathways. IGF1R signaling or inhibition of IGF1R signaling can be determined by phosphorylation of IGF1R. In certain embodiments, the antibodies described herein exhibit inhibition, wherein the EC50 is 10 ng / mL or less. In certain embodiments, the antibodies described herein exhibit inhibition, wherein the EC50 is 9 ng / mL or less. In certain embodiments, the antibodies described herein exhibit inhibition, wherein the EC50 is 8 ng / mL or less. In certain embodiments, the antibodies described herein exhibit inhibition, wherein the EC50 is 7 ng / mL or less. In certain embodiments, the antibodies described herein exhibit inhibition, wherein the EC50 is 6 ng / mL or less. In certain embodiments, the antibodies described herein exhibit inhibition, wherein the EC50 is 5 ng / mL or less. Such an assay for determining the EC50 is described herein and can be performed in a flat-bottom 96-well plate using 4x10^4 NCI-H322 cells / well with 200 ng / mL of recombinant human IGF-1.

[0052] In certain embodiments, the antibody can be administered to a subject in need (e.g., suffering from an IGF1R signaling disorder or disease associated with abnormal IGF1R signaling) by any route suitable for administering a pharmaceutical composition containing the antibody (e.g., such as subcutaneous, intraperitoneal, intravenous, intramuscular, or intratumoral). 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 in a suitable dosage schedule (e.g., once a week, twice a week, once a month, twice a month, once every two weeks, once every three weeks, or once a month, etc.). 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 about 0.1 mg / kg to about 50 mg / kg. In certain embodiments, the therapeutically acceptable amount is about 1 mg / kg to about 40 mg / kg. In certain embodiments, the therapeutically acceptable amount is about 1 mg / kg to about 20 mg / kg. In certain embodiments, the therapeutically acceptable amount is about 1 mg / kg to about 10 mg / kg. In certain embodiments, the therapeutically acceptable amount is about 5 mg / kg to about 30 mg / kg. In certain embodiments, the therapeutically acceptable amount is about 5 mg / kg to about 20 mg / kg.

[0053] Pharmaceutically acceptable excipients, carriers and diluents

[0054] In certain embodiments, the anti-IGF1R antibodies of the present disclosure are contained in a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, carriers, and diluents. Pharmaceutically acceptable excipients, carriers, and diluents may be included to increase the shelf life, stability, or applicability of the antibody. These compounds include salts, pH buffers, detergents, anticoagulants, and preservatives. In certain embodiments, the antibodies of the present disclosure are suspended in a sterile solution for administration. In certain embodiments, the solution comprises about 0.9% NaCl. In certain embodiments, the solution comprises about 5.0% dextrose. In certain embodiments, the solution further comprises one or more of the following: a buffer, e.g., acetate, citrate, histidine, succinate, phosphate, bicarbonate, and hydroxymethylaminomethane (Tris); a surfactant, e.g., polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and poloxamer 188; a polyol / disaccharide / polysaccharide, e.g., glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40; an amino acid, e.g., glycine or arginine; an antioxidant, e.g., ascorbic acid, methionine; or a chelating agent, e.g., EDTA or EGTA.

[0055] Antibodies in formulations for subcutaneous administration are typically present in high concentrations comprising greater than 50 mg / ml, 100 mg / ml, 200 mg / ml, or 300 mg / ml. Many excipients that can be used for subcutaneous formulations are known. See, for example, Wang et al., Antibody Therapeutics, 2021, Vol. 4, No. 4, 262–273.

[0056] In certain embodiments, the antibodies disclosed herein can be freeze-dried for transportation / storage and reconstructed before administration. In certain embodiments, freeze-dried antibody formulations include swelling agents, for example, mannitol, sorbitol, sucrose, trehalose, dextran 40 or a combination thereof. Freeze-dried formulations may be contained in vials consisting of glass or other suitable non-reactive materials. Antibodies may be buffered at a certain pH (generally less than 7.0) when formulated (regardless of whether they are reconstructed). In certain embodiments, pH may be between 4.5 and 7.0, 4.5 and 6.5, 4.5 and 6.0, 4.5 and 5.5, 4.5 and 5.0 or 5.0 and 6.0.

[0057] Also described herein are kits comprising one or more antibodies described herein in a suitable container and one or more additional components selected from the group consisting of: instructions for use; diluents, excipients, carriers, and administration devices (e.g., syringe / needle or other injector).

[0058] In certain embodiments, described herein are methods of 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 disclosure. In certain embodiments, described herein are methods of preparing a cancer treatment for storage or transport, the method comprising lyophilizing one or more antibodies of the disclosure.

[0059] Examples

[0060] The following illustrative examples represent embodiments of the compositions and methods described herein and are not meant to be limiting in any way.

[0061] Example 1 - Determination of Antibody Affinity

[0062] In order to develop antibodies with higher affinity required for subcutaneous formulations, multiple mutants of a reference antibody having a tebuconazole variable region (comprising a variable region containing SEQ ID NO: 1 and SEQ ID NO 2) were tested to determine binding affinity. The binding affinity of the antibodies described herein to IGF1R was tested by Octet. The results are shown in Table 1 below. The sequence alignment of all tebuconazole mutants is shown in Figure 1A and Figure 1BSurprisingly, the light chains of all antibodies had a tryptophan to tyrosine mutation at position 94. This is surprising because light chain CDRs are generally less important for binding affinity than heavy chain CDRs.

[0063]

[0064] Affinity measurements of tefixime variants by biolayer interferometry

[0065] Human IGF-IR (Glu 31-Asn 932, with a polyhistidine tag at the C-terminus) expressed from human 293 cells (HEK293) was purchased from ACROBiosystems (Newark, DE). Immersion and read Ni-NTA (NTA) biosensors from Sartorius (Bohemia, NY) pre-fixed with Tris-NTA with nickel were used for kinetic characterization of novel anti-human IGF-IR antibodies. Briefly, antibody variants were digested into purified Fab fragments (to achieve 1:1 binding and global fit). The FabALACTICA Fab kit from GENOVIS (Cambridge, MA) was used to generate monovalent binding Fab domains for each antibody variant, followed by the CaptureSelect TM The Fc column separates the Fab fragment from the Fc.By biolayer interferometry (BLI) on Octet RED96e (Sartorius), the combination of variant Fab and human IGF-IR (His-labeled) captured by NTA is monitored.NTA biosensor is equipped with 10mM NiCl2, then the average loading response of human IGF-IR to 0.67nm displacement with about 5ug / mL loaded with His-labeled is measured from 0-100nM in 10X kinetic buffer (1XPBS, 0.1% BSA, 0.02% Tween-20 plus Kathon as preservative) from Sartorius.Use Octet Analysis Studio software (Sartorius) to determine binding kinetics and affinity by subtracting data to carry out 1:1 global fitting to double reference.

[0066] Example 2 - Biological activity of tetumumab mutants

[0067] The biological activity of the telotumumab mutants was determined by evaluating the inhibition of IGF-1R phosphorylation and the binding of the ligands IGF-1 and IGF-2 to the receptor. The results are shown in Tables 2 to 4. Overall, the clones showed comparable or better biological activity than the reference telotumumab, indicating that the improvement in affinity seen in Example 2 also extends to biological activity.

[0068]

[0069]

[0070]

[0071]

[0072]

[0073] Inhibition of IGF-1R phosphorylation

[0074] 4x10^4 NCI-H322 cells / well were seeded in a flat-bottom 96-well plate and incubated overnight in a 37°C, 5% incubator. Cells were treated with different concentrations of anti-IGF1R for 1 hour and stimulated with 200ng / mL recombinant human IGF-1 protein for 30 minutes. Phosphorylated IGF-1 in cell lysates was then measured by the Insulin Signaling Panel Whole Cell Lysate Kit (MSD) according to the manufacturer's protocol.

[0075] IGF-1 inhibition of binding assay

[0076] Maxisorp plates were coated with 1.5ug / mL of IGF-1R overnight at 4°C. The plates were washed and blocked with 1% BSA. Subsequently, serially diluted anti-IGF1R antibodies were incubated at room temperature for 30min and then incubated with IGF-1 biotin at a final concentration of 20ng / 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 an HCL-based stop solution. The absorbance of the plates was measured at 450nM and the data was analyzed using Softmax Pro 7.1 software.

[0077] IGF-2 inhibition of binding assay

[0078] Maxisorp plates were coated with 1.5ug / mL of IGF-1R overnight at 4°C. The plates were washed and blocked with 1% BSA. Subsequently, serially diluted anti-IGF1R antibodies were incubated at room temperature for 30min and then incubated with IGF-2 biotin at a final concentration of 200ng / 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 an HCL-based stop solution. The absorbance of the plates was measured at 450nM and the data was analyzed using Softmax Pro 7.1 software.

[0079] Example 3 - Clone D03 with YTE mutation shows ADCC activity comparable to tebuconazole

[0080] When administered to an individual, antibodies with higher affinity can promote greater antibody-dependent cellular cytotoxicity (ADCC). For use in the treatment of ophthalmic conditions and other conditions associated with autoimmune or inflammatory conditions, an increase in ADCC would be undesirable. Clone D03 was selected for further testing of its ADCC activity using different heavy chain constant region mutations (a "YTE" mutation with a mutation at M252Y / S254T / T256E according to EU numbering; and a "LS" mutation Met428Leu / Asn434Ser according to EU numbering). Figure 2 As shown, clone D03 formatted with the LS ("D03-LS") mutation surprisingly showed increased ADCC compared to clone D03 with the YTE mutation ("D03-YTE") compared to tebuconazole (inverted triangles). D03-YTE showed ADCC comparable to tebuconazole.

[0081] ADCC assay

[0082] Antibodies were incubated with DU145 cells at the indicated concentrations for 4 hours, at which time effector cells with a luminescent ADCC reporter were added for overnight incubation, BioGlo detection reagent was added for 10 minutes, and the results were read on a luminometer.

[0083] Example 3 - Clone D03 with the YTE mutation exhibits a 2-3 fold increased half-life compared to tebuconazole

[0084] The half-life of clone D03-YTE was tested in cynomolgus monkeys. D03-YTE (150 mg / kg IV, 150 mg / kg subcutaneous or 75 mg / kg subcutaneous) was given to cynomolgus monkeys. 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 increased serum half-life compared to tilimumab.

[0085]

[0086]

[0087] Example 4 - Clone D03 with the YTE mutation exhibits increased inhibition of IGF-1R phosphorylation compared to tebuconazole

[0088] The ability of D03-YTE to inhibit IGF-1R phosphorylation was tested. The experiment was performed as described in Example 2 with the following modifications: dilution of anti-IGF-1R antibody started at 100 ug / mL and was diluted 1 / 8 to allow for 100% inhibition of tetumumab. Figure 3 The experiment in shows that in this experimental setting, tefixime (circles) exhibited an IC50 of 397.4 ng / mL, while D03-YTE (squares) had an IC50 of 20.51 ng / mL.

[0089] Example 5 - Tetumumab mutants retain the manufacturability of the parental clone

[0090] When developing antibodies for subcutaneous injection, a limiting factor 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 to form aggregates, and the ability to exist in a formulation with low viscosity. Any mutation to the antibody is likely to negatively affect these features, however, as shown in this example, despite the increase in affinity and biological efficacy, there are no deleterious changes in key manufacturability criteria. As shown in Table 6, when compared to the teitutumumab with YTE mutation (buffer composition: 20mM histidine / histidine-HCl, 40mM L-methionine, 210mM ​​trehalose (pH 5.5), 0.2% PX188), D03 with YTE mutation did not show increased degradation at 40°C when forced degradation for 28 days.

[0091]

[0092]

[0093] Anti-IGF-1R variants were evaluated to ensure that Fc and variable domain mutations would not significantly increase viscosity. See Table 7. Increased viscosity is undesirable because it may limit the selection of liquid formulations for subcutaneous administration. The viscosity of anti-IGF-1R variants was initially evaluated to be about 130 mg / ml. Compared with other variants and control mAbs, a variant (B09-YTE) exhibited increased viscosity, and the variant was not evaluated at higher concentrations. Since there was almost no differentiation at about 130 mg / ml, viscosity was evaluated at higher concentrations (about 170 mg / ml). At 170 mg / ml, the viscosity measurements of all D03 and E01 variants were all below 20 cP, and were similar to the control for tilimumab.

[0094] 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 130 mg / ml and 170 mg / ml concentrations and at 20°C and 25°C. Antibody concentrations were determined by SoloVPE (triplicate). Viscosity was assessed by RheoSense m-VROC collecting 8-12 segments at 20°C and 25°C with an applied shear scan rate of 400-2700 / s.

[0095]

[0096]

[0097] The anti-IGF-1R variants were evaluated by hydrophobic interaction chromatography (HIC) analysis (Table 8). HIC is an emerging assessment of the hydrophobic characteristics of antibodies and other proteins. Too much hydrophobic characteristics may be detrimental to overall stability and lead to an increase in nonspecific binding and self-interactions. All anti-IGF-1R variants had elution profiles similar to the tebuconazole control. No increase in hydrophobic characteristics was observed. Tebuconazole and its variants eluted earlier than the positive control (NISTmAb) and significantly earlier than CNTO607 (a mAb with known hydrophobic characteristics that cause developability issues). These data indicate that mutations introduced to increase IGF-1R affinity or extend half-life do not affect the overall hydrophobic characteristics of the variants.

[0098] HIC was performed using an Agilent 1260 Infinity II HPLC with a ProPac HIC-10, 5 μm, 4.6×100 mm column (ThermoFisher PN: 063655). The method parameters were taken directly from the MabPAC-10 product manual. 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 for 5 minutes in 95% buffer A and 5% buffer B, a 25-minute gradient was used, ending with 100% buffer B. The temperature was 30°C, and UV detection at 214 nM was used to visualize protein elution. NISTmAb (positive control human IgG1) and CNTO607 (mAb with known hydrophobic characteristics) were also evaluated as comparators.

[0099]

[0100]

[0101] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided only as examples. Numerous variations, changes, and substitutions will now be contemplated by those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be used to practice the present invention.

[0102] All publications, patent applications, issued patents, and other documents mentioned in this specification are incorporated herein by reference as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions contained in text incorporated by reference are excluded to the extent they conflict with definitions in this disclosure.

[0103] Sequences described herein

[0104] SEQ ID NO 1

[0105] >Tetaxel_VH

[0106] QVELVESGGGVVQPGRSQRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAIIWFDGSSTYYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCARELGRRYFDLWGRGTLVSVSS

[0107] SEQ ID NO 2

[0108] >Tiotumumab_VL

[0109] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASKRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSKWPPWTFGQGTKVESK

[0110] SEQ ID NO 3

[0111] >B02_VH

[0112] QVELVESGGGVVQPGRSQRLSCAASGFTFSSTGMHWVRQAPGKGLEWVAYIWFDGSSTYYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCARELNRRYFDLWGRGTLVSVSS

[0113] SEQ ID NO 4

[0114] >B02_VL

[0115] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASKRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSKYPPWTFGQGTKVESK

[0116] SEQ ID NO 5

[0117] >B09_VH

[0118] QVELVESGGGVVQPGRSQRLSCAASGFTFSSHGMHWVRQAPGKGLEWVAIIAGDASTTYYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCARELGRRYFDLWGRGTLVSVSS

[0119] SEQ ID NO 6

[0120] >B09_VL

[0121] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASKRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSKYPPWTFGQGTKVESK

[0122] SEQ ID NO 7

[0123] >E01_VH

[0124] QVELVESGGGVVQPGRSQRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAIIWFDGSSTYYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCARELGRRYFDLWGRGTLVSVSS

[0125] SEQ ID NO 8

[0126] >E01_VL

[0127] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASKRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSKYPPWTFGQGTKVESK

[0128] SEQ ID NO 9

[0129] >B10_VH

[0130] QVELVESGGGVVQPGRSQRLSCAASGFTFSSYGMHWVRQAPGKGLEWVATIWFDGSSTYYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCARELGRRYFDLWGRGTLVSVSS

[0131] SEQ ID NO 10

[0132] >B10_VL

[0133] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASKRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSKYPPWTFGQGTKVESK

[0134] SEQ ID NO 11

[0135] >D10_VH

[0136] QVELVESGGGVVQPGRSQRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAIINFDGSSTYYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCARELGRRYFDLWGRGTLVSVSS

[0137] SEQ ID NO 12

[0138] >D10_VL

[0139] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASKRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSKYPPWTFGQGTKVESK

[0140] SEQ ID NO 13

[0141] >C10_VH

[0142] QVELVESGGGVVQPGRSQRLSCAASGFTFSSAGMHWVRQAPGKGLEWVAIIWADGSSTYYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCARELGRRYFDLWGRGTLVSVSS

[0143] SEQ ID NO 14

[0144] >C10_VL

[0145] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASKRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSKYPPWTFGQGTKVESK

[0146] SEQ ID NO 15

[0147] >A11_VH

[0148] QVELVESGGGVVQPGRSQRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAYIWFDGSSTYYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCARELGRRYFDLWGRGTLVSVSS

[0149] SEQ ID NO 16

[0150] >A11_VL

[0151] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASKRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSKYPPWTFGQGTKVESK

[0152] SEQ ID NO 17

[0153] >D03_VH

[0154] QVELVESGGGVVQPGRSQRLSCAASGFTFSSHGMHWVRQAPGKGLEWVAYIWFDGSSTYYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCARELGRRYFDLWGRGTLVSVSS

[0155] SEQ ID NO 18

[0156] >D03_VL

[0157] EIVLTQSPATLSLSPGERATLSCRASQSVSSALAWYQQKPGQAPRLLIYDASKRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSKYPPWTFGQGTKVESK

[0158] SEQ ID NO 19

[0159] >F02_VH

[0160] QVELVESGGGVVQPGRSQRLSCAASGFTFSSHGMHWVRQAPGKGLEWVATIWFDGSSTYYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCARELGRRYFDLWGRGTLVSVSS

[0161] SEQ ID NO 20

[0162] >F02_VL

[0163] EIVLTQSPATLSLSPGERATLSCRASQSVSSTLAWYQQKPGQAPRLLIYDASKRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSKYPPWTFGQGTKVESK

[0164] SEQ ID NO 21

[0165] >H11_VH

[0166] QVELVESGGGVVQPGRSQRLSCAASGFTFSSYGMHWVRQAPGKGLEWVATIWHDGSSTYYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCARELGRRYFDLWGRGTLVSVSS

[0167] SEQ ID NO 22

[0168] >H11_VL

[0169] EIVLTQSPATLSLSPGERATLSCRASQSVSSALAWYQQKPGQAPRLLIYDASKRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRGKYPPWTFGQGTKVESK

[0170]

[0171]

Claims

1. An antibody or an antigen-binding fragment thereof that binds to insulin-like growth factor 1 receptor (IGF1R), wherein the antibody or the antigen-binding fragment thereof comprises: 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 f. 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 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.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the amino acid residue corresponding to X2 of LCDR3 is tyrosine.

3. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, wherein: a. the HCDR1 comprises the amino acid sequence of SEQ ID NO:30 (SHGMH); 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); and f. The LCDR3 comprises the amino acid sequence of SEQ ID NO:48 (QQRSKYPPWT).

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein: a. the HCDR1 comprises the amino acid sequence of SEQ ID NO:33 (SYGMH); 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); and f. The LCDR3 comprises the amino acid sequence of SEQ ID NO:48 (QQRSKYPPWT).

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein: a. the HCDR1 comprises the amino acid sequence of SEQ ID NO:30 (SHGMH); 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); and f. The LCDR3 comprises the amino acid sequence of SEQ ID NO:48 (QQRSKYPPWT).

6. The antibody or antigen-binding fragment thereof of claim 1 or 2, comprising 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 the amino acid sequence shown in SEQ ID NO:3; and wherein the immunoglobulin light chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:

4.

7. The antibody or antigen-binding fragment thereof of claim 1 or 2, comprising 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 the amino acid sequence shown in SEQ ID NO:5; and wherein the immunoglobulin light chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:

6.

8. The antibody or antigen-binding fragment thereof of claim 1 or 2, comprising 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 the amino acid sequence shown in SEQ ID NO:7; and wherein the immunoglobulin light chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:

8.

9. The antibody or antigen-binding fragment thereof of claim 1 or 2, comprising 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 the amino acid sequence shown in SEQ ID NO:9; and wherein the immunoglobulin light chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:

10.

10. The antibody or antigen-binding fragment thereof of claim 1 or 2, comprising 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 the amino acid sequence shown in SEQ ID NO: 11; and wherein the immunoglobulin light chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:

12.

11. The antibody or antigen-binding fragment thereof of claim 1 or 2, comprising 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 the amino acid sequence shown in SEQ ID NO: 13; and wherein the immunoglobulin light chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:

14.

12. The antibody or antigen-binding fragment thereof of claim 1 or 2, comprising 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 the amino acid sequence shown in SEQ ID NO: 15; and wherein the immunoglobulin light chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:

16.

13. The antibody or antigen-binding fragment thereof of claim 1 or 2, comprising 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 the amino acid sequence shown in SEQ ID NO: 17; and wherein the immunoglobulin light chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:

18.

14. The antibody or antigen-binding fragment thereof of claim 1 or 2, comprising 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 the amino acid sequence shown in SEQ ID NO: 19; and wherein the immunoglobulin light chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:

20.

15. The antibody or antigen-binding fragment thereof of claim 1 or 2, comprising 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 the amino acid sequence shown in SEQ ID NO:21; and wherein the immunoglobulin light chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:

22.

16. The antibody or antigen-binding fragment thereof of claim 1 or 2, comprising 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 the amino acid sequence shown in SEQ ID NO:51; and wherein the immunoglobulin light chain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:

52.

17. The antibody or antigen-binding fragment thereof of any one of claims 1 to 16, wherein the antibody or antigen-binding fragment thereof is an IgG antibody.

18. The antibody or antigen-binding fragment thereof of any one of claims 1 to 16, wherein the antibody or antigen-binding fragment thereof comprises Fab, F(ab)2, or a single-chain variable fragment (scFv).

19. The antibody or antigen-binding fragment thereof of any one of claims 1 to 18, wherein the antibody or antigen-binding fragment thereof is chimeric or humanized.

20. The antibody or antigen-binding fragment thereof of any one of claims 1 to 19, comprising a M252Y / S254T / T256E substitution according to EU numbering in one or both heavy chain constant regions.

21. The antibody or antigen-binding fragment thereof of any one of claims 1 to 20, wherein the antibody or antigen-binding fragment thereof is characterized by a half-life in humans of 25 days or more.

22. The antibody or antigen-binding fragment thereof of any one of claims 1 to 20, wherein the antibody or antigen-binding fragment thereof is characterized by a half-life in humans of 30 days or more.

23. The antibody or antigen-binding fragment thereof of any one of claims 1 to 22, wherein the antibody inhibits signaling through IGF1R.

24. The antibody or antigen-binding fragment thereof of any one of claims 1 to 23, wherein the antibody inhibits phosphorylation of IGF1R with an EC50 of 10 ng / mL or less.

25. The antibody or antigen-binding fragment thereof of any one of claims 1 to 23, wherein the antibody inhibits phosphorylation of IGF1R with an EC50 of 9 ng / mL or less.

26. The antibody or antigen-binding fragment thereof of any one of claims 1 to 23, wherein the antibody inhibits phosphorylation of IGF1R with an EC50 of 7 ng / mL or less.

27. The antibody or antigen-binding fragment thereof of any one of claims 1 to 26, wherein the antibody has a -9 M of K D .

28. The antibody or antigen-binding fragment thereof of any one of claims 1 to 26, wherein the antibody has a -9 M of K D .

29. The antibody or antigen-binding fragment thereof of any one of claims 1 to 26, wherein the antibody has a -10 M of K D .

30. A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 29.

31. A cell line comprising the nucleic acid of claim 30.

32. The cell line of claim 31, wherein the cell line is a Chinese hamster ovary cell line.

33. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 29, and a pharmaceutically acceptable excipient, carrier or diluent.

34. The pharmaceutical composition of claim 33, formulated for intravenous administration.

35. The pharmaceutical composition of claim 34, formulated for subcutaneous administration.

36. The antibody or antigen-binding fragment thereof of any one of claims 1 to 29 or the pharmaceutical composition of any one of claims 33 to 35 for use in a method of inhibiting IGF1R signaling in a subject.

37. A method of inhibiting IGF1R signaling in a subject, the method comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1 to 29 or the pharmaceutical composition of any one of claims 33 to 35.

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