Anti-IGF1R antibody and its use

Abstract

Anti-IGF1R (insulin-like growth factor 1 receptor) antibodies, antigen-binding fragments, and their uses are provided.

Description

[Technical Field]

[0001] Claim of priority This application claims priority to PCT / CN2023 / 086082, filed on 4 April 2023, and PCT / CN2023 / 115246, filed on 28 August 2023. The entirety of the aforementioned applications is incorporated herein by reference.

[0002] Technical field This disclosure relates to an anti-IGF1R (insulin-like growth factor 1 receptor) antibody and its use. [Background technology]

[0003] Cancer is currently one of the leading causes of death in humans. According to World Health Organization statistics, in 2012, there were 14 million new cancer cases and 8.2 million cancer deaths worldwide. In China, there were 3.07 million newly diagnosed cancer cases and 2.2 million deaths. Recent clinical and commercial successes of anti-cancer antibodies have drawn considerable interest to antibody-based therapies.

[0004] Furthermore, thyroid eye disorders (TED), such as thyroid-associated eye disease (TED), which are frequently associated with Graves' disease, remain undertreated. Current medical treatments, primarily consisting of glucocorticoids, have limited efficacy and raise safety concerns.

[0005] There is a need to develop antibodies for use in various antibody-based therapies to treat cancer, autoimmune diseases, or eye diseases such as thyroid-related eye disease. [Overview of the project]

[0006] This disclosure relates to an anti-IGF1R antibody, its antigen-binding fragment, and its use.

[0007] In one aspect, the present disclosure relates to a heavy chain variable region (VH) comprising complementarity determining regions (CDR) 1, 2, and 3, wherein in some embodiments, the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VH CDR3 amino acid sequence, a light chain variable region (VL) comprising CDR1, 2, and 3, wherein in some embodiments, the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VL CDR3 amino acid sequence, and an antibody or an antigen-binding fragment thereof that binds to insulin-like growth factor 1 receptor (IGF1R). In some embodiments, the selected VH CDR1, 2, and 3 amino acid sequences, and the selected VL CDR1, 2, and 3 amino acid sequences are one of the following. (1) The amino acid sequences of the selected VH CDR1, 2, 3 are shown in SEQ ID NO: 1, 2, 3 respectively, and the amino acid sequences of the selected VL CDR1, 2, 3 are shown in SEQ ID NO: 4, 5, 6 respectively. (2) The amino acid sequences of the selected VH CDR1, 2, 3 are shown in SEQ ID NO: 25, 26, 27 respectively, and the amino acid sequences of the selected VL CDR1, 2, 3 are shown in SEQ ID NO: 4, 5, 6 respectively. (3) The amino acid sequences of the selected VH CDR1, 2, 3 are shown in SEQ ID NO: 7, 8, 9 respectively, and the amino acid sequences of the selected VL CDR1, 2, 3 are shown in SEQ ID NO: 10, 11, 12 respectively. (4) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 28, 29, and 30, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 10, 11, and 12, respectively. (5) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 13, 14, and 15, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 16, 17, and 18, respectively. (6) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 31, 32, and 33, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 16, 17, and 18, respectively. (7) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 19, 20, and 21, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 22, 23, and 24, respectively. (8) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 34, 35, and 36, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 22, 23, and 24, respectively.

[0008] In some embodiments, based on Kabat's definition, the VH includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, and the VL includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 4, 5, and 6, respectively. In some embodiments, based on Kabat's definition, the VH includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 7, 8, and 9, respectively, and the VL includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 10, 11, and 12, respectively. In some embodiments, based on Kabat's definition, the VH includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 13, 14, and 15, respectively, and the VL includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 16, 17, and 18, respectively. In some embodiments, based on Kabat's definition, the VH includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 19, 20, and 21, respectively, and the VL includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 22, 23, and 24, respectively. In some embodiments, based on Chothia's definition, the VH includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 25, 26, and 27, respectively, and the VL includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 4, 5, and 6, respectively. In some embodiments, based on Chothia's definition, the VH includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 28, 29, and 30, respectively, and the VL includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 10, 11, and 12, respectively. In some embodiments, based on Chothia's definition, the VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 31, 32, and 33, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 16, 17, and 18, respectively.In some embodiments, based on Chothia's definition, the VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 34, 35, and 36, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 22, 23, and 24, respectively.

[0009] In some embodiments, the antibody or antigen-binding fragment specifically binds to human IGF1R or monkey IGF1R. In some embodiments, the antibody or antigen-binding fragment is a human antibody or its antigen-binding fragment, or a humanized antibody or its antigen-binding fragment (e.g., human IgG1 antibody or IgG4 antibody). In some embodiments, the antibody or antigen-binding fragment is a single-chain variable fragment (scFv) or a multispecific antibody (e.g., a bispecific antibody).

[0010] In one embodiment, the disclosure relates to nucleic acids comprising polynucleotides that encode a polypeptide including the following: (1) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which contain the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3, wherein the VH binds to IGF1R when paired with a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO: 38. (2) An immunoglobulin light chain or fragment thereof comprising a VL containing CDR1, 2, and 3, respectively, which contain the amino acid sequences shown in SEQ ID NOs: 4, 5, and 6, wherein the VL binds to IGF1R when paired with a VH containing the amino acid sequence shown in SEQ ID NO: 37, (3) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which contain the amino acid sequences shown in SEQ ID NOs. 25, 26, and 27, wherein the VH binds to IGF1R when paired with a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO. 38. (4) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which contain the amino acid sequences shown in SEQ ID NOs: 7, 8, and 9, wherein the VH binds to IGF1R when paired with a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO: 40, (5) An immunoglobulin light chain or fragment thereof comprising a VL containing CDR1, 2, and 3, respectively, which contain the amino acid sequences shown in SEQ ID NOs. 10, 11, and 12, wherein the VL binds to IGF1R when paired with a VH containing the amino acid sequence shown in SEQ ID NO. 39. (6) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which include the amino acid sequences shown in SEQ ID NOs. 28, 29, and 30, wherein the VH binds to IGF1R when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 40. (7) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which include the amino acid sequences shown in SEQ ID NOs. 13, 14, and 15, wherein the VH binds to IGF1R when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 42. (8) An immunoglobulin light chain or fragment thereof comprising a VL containing CDR1, 2, and 3, respectively, which contain the amino acid sequences shown in SEQ ID NOs: 16, 17, and 18, wherein the VL binds to IGF1R when paired with a VH containing the amino acid sequence shown in SEQ ID NO: 41, (9) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which include the amino acid sequences shown in SEQ ID NOs. 31, 32, and 33, wherein the VH binds to IGF1R when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 42. (10) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which include the amino acid sequences shown in SEQ ID NOs: 19, 20, and 21, wherein the VH binds to IGF1R when paired with a light chain variable region (VL) which includes the amino acid sequence shown in SEQ ID NO: 44. (11) An immunoglobulin light chain or fragment thereof comprising a VL containing CDR1, 2, and 3, respectively, which contain the amino acid sequences shown in SEQ ID NOs. 22, 23, and 24, wherein the VL binds to IGF1R when paired with a VH containing the amino acid sequence shown in SEQ ID NO. 43, or (12) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which include the amino acid sequences shown in SEQ ID NOs: 34, 35, and 36, wherein the VH binds to IGF1R when paired with a light chain variable region (VL) which includes the amino acid sequence shown in SEQ ID NO: 44.

[0011] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide, comprising an immunoglobulin heavy chain or fragment thereof, comprising VH containing CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs. 1, 2, and 3, or SEQ ID NOs. 25, 26, and 27, respectively. In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide, comprising an immunoglobulin light chain or fragment thereof, comprising VL containing CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs. 4, 5, and 6, respectively.

[0012] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide, comprising an immunoglobulin heavy chain or fragment thereof, comprising VH containing CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs. 7, 8, and 9, or SEQ ID NOs. 28, 29, and 30, respectively. In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide, comprising an immunoglobulin light chain or fragment thereof, comprising VL containing CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs. 10, 11, and 12, respectively.

[0013] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide, comprising an immunoglobulin heavy chain or fragment thereof, comprising a VH containing CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs. 13, 14, and 15, or SEQ ID NOs. 31, 32, and 33, respectively. In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide, comprising an immunoglobulin light chain or fragment thereof, comprising a VL containing CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs. 16, 17, and 18, respectively.

[0014] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide, comprising an immunoglobulin heavy chain or fragment thereof, comprising VH containing CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs. 19, 20, and 21, or SEQ ID NOs. 34, 35, and 36, respectively. In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide, comprising an immunoglobulin light chain or fragment thereof, comprising VL containing CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs. 22, 23, and 24, respectively.

[0015] In some embodiments, the VH specifically binds to human IGF1R or monkey IGF1R when paired with the VL, or the VL specifically binds to human IGF1R or monkey IGF1R when paired with the VH. In some embodiments, the immunoglobulin heavy chain or fragment thereof is a human immunoglobulin heavy chain or fragment thereof or a humanized immunoglobulin heavy chain or fragment thereof (e.g., a human IgG1 heavy chain or fragment thereof), and the immunoglobulin light chain or fragment thereof is a human immunoglobulin light chain or fragment thereof or a humanized immunoglobulin light chain or fragment thereof. In some embodiments, the nucleic acid encodes a single-stranded variable fragment (scFv), a multispecific antibody (e.g., a bispecific antibody), or a chimeric antigen receptor (CAR). In some embodiments, the nucleic acid is cDNA.

[0016] In one embodiment, the disclosure relates to a vector comprising one or more nucleic acids described herein. In one embodiment, the disclosure relates to a vector comprising two nucleic acids described herein, wherein in some embodiments, the vector encodes a VH region and a VL region that bind together to IGF1R. In one embodiment, the disclosure relates to a pair of vectors, wherein in some embodiments, each vector comprises one of the nucleic acids described herein, and in some embodiments, the pair of vectors encode a pair of VH and VL regions that bind together to IGF1R.

[0017] In one embodiment, the disclosure relates to a cell comprising a vector or a pair of vectors described herein. In some embodiments, the cell is a CHO cell. In one embodiment, the disclosure relates to a cell comprising one or more nucleic acids described herein. In one embodiment, the disclosure relates to a cell comprising two nucleic acids described herein. In some embodiments, the two nucleic acids encode a pair of VH and VL regions that bind together to IGF1R.

[0018] In one embodiment, the present disclosure relates to a method for producing an antibody or an antigen-binding fragment thereof, the method comprising (a) culturing cells described herein under conditions sufficient to enable the cells to produce the antibody or the antigen-binding fragment, and (b) recovering the antibody or the antigen-binding fragment produced by the cells.

[0019] In one embodiment, the present disclosure relates to an antibody or antigen-binding fragment thereof that binds to IGF1R, comprising a heavy chain variable region (VH) comprising an amino acid sequence that is at least 80% identical to a selected VH sequence, and a light chain variable region (VL) comprising an amino acid sequence that is at least 80% identical to a selected VL sequence, wherein in some embodiments, the selected VH sequence and the selected VL sequence are one of the following: (1) the selected VH sequence is SEQ ID NO: 37 and the selected VL sequence is SEQ ID NO: 38, (2) the selected VH sequence is SEQ ID NO: 39 and the selected VL sequence is SEQ ID NO: 40, (3) the selected VH sequence is SEQ ID NO: 41 and the selected VL sequence is SEQ ID NO: 42, and (4) the selected VH sequence is SEQ ID NO: 43 and the selected VL sequence is SEQ ID NO: 44.

[0020] In some embodiments, VH includes the sequence of sequence number 37, and VL includes the sequence of sequence number 38. In some embodiments, VH includes the sequence of sequence number 39, and VL includes the sequence of sequence number 40. In some embodiments, VH includes the sequence of sequence number 41, and VL includes the sequence of sequence number 42. In some embodiments, VH includes the sequence of sequence number 43, and VL includes the sequence of sequence number 44.

[0021] In some embodiments, the antibody or antigen-binding fragment specifically binds to human IGF1R or monkey IGF1R. In some embodiments, the antibody or antigen-binding fragment is a human antibody or its antigen-binding fragment, or a humanized antibody or its antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment is a single-chain variable fragment (scFv) or a multispecific antibody (e.g., a bispecific antibody).

[0022] In one embodiment, this disclosure relates to an antibody or an antigen-binding fragment that cross-competes with an antibody or an antigen-binding fragment described herein.

[0023] In one embodiment, the present disclosure relates to an antibody or antigen-binding fragment thereof that binds to IGF1R, comprising a heavy chain variable region (VH) including VH CDR1, VH CDR2, and VH CDR3 which are identical to VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence, and a light chain variable region (VL) including VL CDR1, VL CDR2, and VL CDR3 which are identical to VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein in some embodiments, the selected VH sequence and the selected VL sequence are one of the following. (1) The selected VH sequence is sequence number 37 and the selected VL sequence is sequence number 38, (2) The selected VH sequence is sequence number 39 and the selected VL sequence is sequence number 40, (3) The selected VH sequence is sequence number 41 and the selected VL sequence is sequence number 42, and (4) The selected VH sequence is sequence number 43 and the selected VL sequence is sequence number 44.

[0024] In one embodiment, the present disclosure relates to an antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof, covalently bound to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic agent or a cell proliferation inhibitor.

[0025] In one embodiment, the present disclosure relates to a method for treating a subject having cancer, the method comprising administering to the subject a therapeutically effective dose of a composition comprising an antibody or antigen-binding fragment thereof as described herein, or an antibody-drug conjugate. In some embodiments, the subject has solid tumors, brain tumors, lung cancer, melanoma, prostate cancer, esophageal squamous cell carcinoma, leukemia, lymphoma, ovarian cancer, colorectal cancer, head and neck cancer, bladder cancer, kidney cancer, and breast cancer. In some embodiments, the method described herein further comprises administering to the subject a therapeutically effective dose of anti-OX40 antibody, anti-PD1 antibody, anti-PDL1 antibody, anti-PDL2 antibody, anti-LAG-3 antibody, anti-TIGIT antibody, anti-CTLA-4 antibody, anti-GITR antibody, anti-TIM-3 antibody, anti-4-1BB antibody, and / or anti-CD40 antibody.

[0026] In one embodiment, the present disclosure relates to a method for reducing the rate of tumor growth, the method comprising contacting tumor cells with an effective amount of an antibody or antigen-binding fragment thereof as described herein, or a composition comprising an antibody-drug conjugate.

[0027] In one embodiment, the present disclosure relates to a method for killing tumor cells, the method comprising contacting the tumor cells with an effective amount of an antibody or antigen-binding fragment thereof as described herein, or a composition comprising an antibody-drug conjugate.

[0028] In one embodiment, the present disclosure relates to a method for treating a subject having an eye disease or eye disorder, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof as described herein, or an antibody-drug conjugate.

[0029] In some embodiments, the subject has a thyroid eye disease (TED). In some embodiments, the thyroid eye disease is a thyroid-associated eye disease.

[0030] In one embodiment, the present disclosure relates to a method for treating a subject having a bone disease or bone disorder, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof as described herein, or an antibody-drug conjugate.

[0031] In one embodiment, this disclosure relates to a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described herein and a pharmaceutically acceptable carrier. In one embodiment, this disclosure relates to a pharmaceutical composition comprising an antibody-drug conjugate as described herein and a pharmaceutically acceptable carrier.

[0032] In some embodiments, the antibody concentration is approximately 50 to 150 mg / mL (for example, approximately 100 mg / mL).

[0033] In one embodiment, this disclosure relates to a chimeric antigen receptor (CAR) comprising an antibody or an antigen-binding fragment thereof as described herein.

[0034] As used herein, “thyroid eye disease (TED)” refers to a condition in which the muscles of the eye, eyelids, lacrimal glands, and fatty tissue behind the eye become inflamed. Symptoms of thyroid eye disease include, but are not limited to, redness and swelling of the eye and eyelids, and “staring” or “protruding” of the eye.

[0035] As used herein, the term “antibody” means any antigen-binding molecule that contains at least one (e.g., 1, 2, 3, 4, 5, or 6) complementarity-determining regions (CDRs) (e.g., any of three CDRs derived from an immunoglobulin light chain or any of three CDRs derived from an immunoglobulin heavy chain) and is capable of specifically binding to an epitope. Non-limiting examples of antibodies include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, antibodies may contain the Fc region of a human antibody. The term antibody also includes derivatives, such as bispecific antibodies, single-chain antibodies, diabodies, linear antibodies, and multispecific antibodies formed from antibody fragments.

[0036] As used herein, the term “antigen-binding fragment” means a portion of a full-length antibody, the portion of which is specifically capable of binding to an antigen. In some embodiments, the antigen-binding fragment contains at least one variable domain (e.g., a variable domain in the heavy chain or a variable domain in the light chain). Non-limiting examples of antibody fragments include, for example, Fab, Fab', F(ab')2, and Fv fragments.

[0037] As used herein, the term “human antibody” means an antibody encoded by endogenous nucleic acids of human origin (e.g., rearranged human immunoglobulin heavy or light chain loci). In some embodiments, human antibodies are recovered from humans or produced in human cell culture media (e.g., in human hybridoma cells). In some embodiments, human antibodies are produced in non-human cells (e.g., mouse or hamster cell lines). In some embodiments, human antibodies are produced in bacterial or yeast cells. In some embodiments, human antibodies are produced in transgenic non-human animals (e.g., cattle) containing unarranged or rearranged human immunoglobulin loci (e.g., heavy or light chain human immunoglobulin loci).

[0038] As used herein, the term “chimeric antibody” means an antibody containing sequences present in at least two different species (e.g., an antibody derived from two different mammalian species, such as a human and a mouse antibody). Non-limiting examples of chimeric antibodies include antibodies containing a variable domain sequence (e.g., all or part of the light chain and / or heavy chain variable domain sequence) of a non-human (e.g., mouse) antibody, as well as a constant domain of a human antibody. Further examples of chimeric antibodies are described herein and are well known in the art.

[0039] As used herein, the term “humanized antibody” means a non-human antibody that contains minimal sequences derived from non-human (e.g., mouse) immunoglobulin and sequences derived from human immunoglobulin. In non-limiting examples, a humanized antibody is a human antibody (recipient antibody) in which residues in the hypervariable (e.g., CDR) region of the recipient antibody are replaced by residues in the hypervariable (e.g., CDR) region of a non-human antibody (e.g., donor antibody), such as a mouse, rat, or rabbit antibody having desired specificity, affinity, and capability. In some embodiments, Fv framework residues of human immunoglobulin are replaced by corresponding non-human (e.g., mouse) immunoglobulin residues. In some embodiments, the humanized antibody may contain residues not found in the recipient antibody or donor antibody. These modifications can further refine the performance of the antibody. In some embodiments, the humanized antibody contains at least one and typically two variable domains substantially all of which, all or substantially all of the hypervariable loop (CDR) corresponds to the hypervariable loop of a non-human (e.g., mouse) immunoglobulin, and all or substantially all of the framework region is a human immunoglobulin sequence. The humanized antibody may also contain an immunoglobulin constant region (Fc), typically at least a portion of the constant region of a human immunoglobulin. The humanized antibody can be produced using molecular biological methods well known in the art. Non-limiting examples of methods for producing the humanized antibody are described herein.

[0040] As used herein, the term “single-chain antibody” means a single polypeptide containing at least two immunoglobulin variable domains (e.g., variable domains of mammalian immunoglobulin heavy or light chains) that are specifically capable of binding to an antigen. Non-limiting examples of single-chain antibodies are described herein.

[0041] Where used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and refer to an animal, human, or non-human being to be treated according to the methods of the present invention. Veterinary and non-veterinary uses are conceived in this disclosure. Human patients may be adult humans or young humans (e.g., humans under 18 years of age). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. Examples include non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), rabbits, pigs (e.g., pigs, miniature pigs), equids, canids, felines, bovines, and other domestic, livestock, and zoo animals.

[0042] As used herein, when referring to an antibody, the terms "specifically binding" and "specifically binds" mean that the antibody interacts more with its target molecule (e.g., IGF1R) than with other molecules, because the interaction depends on the presence of a specific structure (i.e., an antigenic determinant or epitope) on the target molecule; in other words, the reagent recognizes and binds to a molecule containing a specific structure, rather than to all general molecules. An antibody that specifically binds to a target molecule may also be called a target-specific antibody. For example, an antibody that specifically binds to the IGF1R molecule may be called an IGF1R-specific antibody or an anti-IGF1R antibody.

[0043] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably and mean polymers of at least two amino acids of any length.

[0044] As used herein, the terms “polynucleotide,” “nucleic acid molecule,” and “nucleic acid sequence” are used interchangeably and mean, but are not limited to, polymers of nucleotides of any length, of at least two nucleotides, including, but not limited to, DNA, RNA, DNA / RNA hybrids, and modifications thereof.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. Methods and materials for use in the present invention are described herein, but other suitable methods and materials well known in the art may also be used. Materials, methods, and examples are illustrative and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references referenced herein are incorporated by reference in their entirety. In case of any conflict, this specification, including definitions, shall prevail.

[0046] Other features and advantages of the present invention will become apparent from the following detailed description and drawings, as well as from the claims. [Brief explanation of the drawing]

[0047] [Figure 1] List the Kabat CDR sequences of anti-IGF1R antibodies. [Figure 2] List the Chothia CDR sequences of anti-IGF1R antibodies. [Figure 3-1] The sequences of the heavy chain variable region and light chain variable region of the anti-IGF1R antibody, as well as the amino acid sequences discussed in this disclosure, are listed below. [Figure 3-2] Same as above [Figure 3-3] Same as above [Figure 3-4]Same as above [Figure 4] This study demonstrates the internalization of anti-IGF1R antibodies (12A4, 12D1, 10D1, and 10G1). Teprotumumab analogs were used as controls. [Figure 5] Figures A and B show the inhibition of IGF1R autophosphorylation by anti-IGF1R antibodies (12A4, 12D1, 10D1, and 10G1). Teprotumumab analogs were used as controls. [Figure 6] This study demonstrates inhibition of IGF-1-induced OPN secretion by anti-IGF1R antibodies (12A4, 12D1, 10D1, and 10G1). Teprotumumab analogs were used as controls. [Figure 7] This shows the pharmacokinetic characteristics of the anti-IGF1R antibody. [Figure 8] The study demonstrated ADCC cytotoxicity of anti-IGF1R antibodies (12A4-YTE and 12D1-QL). Teprotumumab analogs were used as positive controls. [Figure 9] This shows the release levels of cytokine INF-γ treated with anti-IGF1R antibodies (12A4-YTE and 12D1-QL). Teprotumumab analogs were used as controls. [Figure 10] This shows the release levels of hyaluronic acid (HA) treated with anti-IGF1R antibodies (12A4-YTE and 12D1-QL). Teprotumumab analogs were used as a control. [Figure 11] This shows the release levels of hyaluronic acid (HA) treated with the anti-IGF1R antibody 12A4-YTE. Teprotumumab analogs were used as a control. [Modes for carrying out the invention]

[0048] The insulin-like growth factor 1 receptor (IGF1R) is a ubiquitously expressed membrane-bound tyrosine kinase receptor that recognizes its two major ligands, IGF1 and IGF2, and regulates several important cellular functions. IGF activity is highly associated with several chronic lung diseases, including lung cancer. Targeting IGF signaling is an attractive therapeutic strategy because IGF1R signaling is significantly involved as a crucial contributor to cancer cell proliferation, survival, migration, and resistance to anticancer treatments. IGF1R has also been shown to be associated with thyroid eye disease (TED) and bone diseases such as osteoporosis and osteosarcoma. Therefore, anti-IGF1R antibodies have the potential to be used as a treatment for cancer as well as other diseases such as eye and bone diseases.

[0049] This disclosure provides examples of antibodies that bind to IGF1R (insulin-like growth factor 1 receptor) and their antigen-binding fragments.

[0050] IGF1R IGF1R (insulin-like growth factor 1 receptor, accession number P08069, UniProt protein database entry IGF1R_HUMAN) is a receptor tyrosine kinase that mediates the action of insulin-like growth factor 1 (IGF-1). It binds to IGF-1 with high affinity and to IGF-2 and insulin (INS) with lower affinity. Activated IGF1R is involved in regulating cell proliferation and survival. IGF1R is crucial for tumor transformation and malignant cell survival. Ligand binding activates the receptor kinase, leading to receptor autophosphorylation and tyrosine phosphorylation of multiple substrates that function as signaling adapter proteins, including insulin receptor substrates (IRS1 / 2), Shc, and 14-3-3 protein. Phosphorylation of IRS proteins leads to activation of two major signaling pathways: the PI3K-AKT / PKB pathway and the Ras-MAPK pathway. Activation of the MAPK pathway increases cell proliferation, while activation of the PI3K pathway suppresses apoptosis and stimulates protein synthesis. Phosphorylated IRS1 can activate the 85kDa regulatory subunit of PI3K (PIK3R1), leading to the activation of several downstream substrates, including the protein AKT / PKB. Phosphorylation of AKT enhances protein synthesis through mTOR activation and triggers the anti-apoptotic effect of IGF1R through BAD phosphorylation and inactivation. In parallel with PI3K-driven signaling, recruitment of Grb2 / SOS by phosphorylated IRS1 or Shc leads to Ras recruitment and activation of the ras-MAPK pathway. In addition to these two main signaling pathways, IGF1R also signals through the Janus kinase / signaling and transcriptional activator pathway (JAK / STAT). Phosphorylation of JAK proteins may lead to phosphorylation / activation of signaling and transcriptional activator (STAT) proteins. In particular, activation of STAT3 may be essential for the transformative activity of IGF1R. The JAK / STAT pathway activates gene transcription and may be involved in transformative activity. JNK kinase can also be activated by IGF1R.IGF1 exerts inhibitory activity against JNK activation through phosphorylation and inhibition of MAP3K5 / ASK1, which can directly bind to IGF1R.

[0051] IGF1R is currently being evaluated as a pharmacological target in clinical trials for tumor patients, including those with non-small cell lung cancer (NSCLC). The role of IGF1R in the implantation and progression of lung metastases in TME will be studied using the Lewis lung cancer (LLC) model as a reproducible syngeneic model of NSCLC. The involvement of IGF1R as a cancer promoter in TME will be studied by analyzing tumor samples from NSCLC patients and generating LLC models by performing ectopic transplantation or lung metastases under the condition of IGF1R deficiency.

[0052] Thyroid-associated ophthalmopathy, a symptom frequently associated with Graves' disease, remains undertreated. Current medical treatments, primarily consisting of glucocorticoids, have limited efficacy and raise safety concerns. Inhibition of insulin-like growth factor I receptor (IGF-IR) may offer a novel therapeutic strategy to weaken the underlying autoimmune etiology of ophthalmopathy.

[0053] IGF1R has also been found to synergistically interact with TSHR to increase osteopontin (OPN) secretion in bone disease. Thyroid-stimulating hormone (TSH), also known as thyrotropin, is a hormone that activates the TSH receptor (TSHR) to stimulate thyroid development in the uterus and on thyroid cells, stimulating the production of thyroxine (T4) and triiodothyronine (T3), which are thyroid hormones in adults. Thyroid hormones are essential for skeletal development and healthy bone metabolism. Clinical studies have demonstrated that normal thyroid function in adults is important for bone homeostasis. Hyperthyroidism leads to increased bone resorption, which causes bone loss and bone mineralization.

[0054] Further information on IGF1R can be found, for example, in the following references, which are incorporated herein by reference in their entirety: Boutin et al., Front. Endocrinol., 20 May 2020 Sec. Thyroid Endocrinology; Girnita et al., Cell Mol Life Sci. 2014, 71:2403-27; Wang et al., Biomed Res Int. 2018, 2018:6057589; and Griffiths et al., Nature. 2020, 583:615-9; Ullrich et al., EMBO J. 5:2503-2512 (1986); and Kato et al., J. Biol. Chem. 268:2655-2661 (1993); Kellar et al., Biomed Res These can be found in Int.2015, 2015:621324, Alfaro-Arnedo et al., Oncogene volume 41, pages 3625-3639, 2022, Smith et al., N Engl J Med. 2017 May 4, 376(18):1748-1761, and Barbesino et al., The Journal of Clinical Endocrinology & Metabolism, Volume 107, Issue Supplement_1, September 2022, Pages S47-S56, the entirety of each of these is incorporated herein by reference.

[0055] Anti-IGF1R antibody and antigen-binding fragment This disclosure provides antibodies and antigen-binding fragments that specifically bind to IGF1R (e.g., human IGF1R). The antibodies and antigen-binding fragments described herein are capable of binding to IGF1R. In some embodiments, these antibodies block the binding of human IGF1R to IGF1R ligands (e.g., human insulin-like growth factor 1, i.e., IGF-1, or human insulin-like growth factor 2, i.e., IGF-2). In some embodiments, these antibodies inhibit human IGF-1-induced cell proliferation. In some embodiments, these antibodies block the IGF-1 / IGF1R or IGF-2 / IGF1R signaling pathway. In some embodiments, these antibodies can initiate complement-dependent cell-mediated cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, these antibodies bind to cells expressing IGF1R.

[0056] This disclosure provides, for example, anti-IGF1R antibodies 12A4, 12D1, 10D1, 10G1, and modified antibodies thereof (including, for example, chimeric antibodies, humanized antibodies, and human antibodies).

[0057] CDR sequences for 12A4 and antibodies derived from 12A4 (e.g., humanized antibodies) include the CDRs of the heavy chain variable domain, SEQ ID NOs: 1, 2, 3, and the CDRs of the light chain variable domain, SEQ ID NOs: 4, 5, 6, as defined by Kabat. CDRs can also be defined by the Chothia system. Under the Chothia definition, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NOs: 25, 26, 27, and the CDR sequences of the light chain variable domain are shown in SEQ ID NOs: 4, 5, 6.

[0058] The CDR sequences for 12D1 and antibodies derived from 12D1 include the heavy chain variable domain CDRs, SEQ ID NOs. 7, 8, and 9, and the light chain variable domain CDRs, SEQ ID NOs. 10, 11, and 12, as defined by Kabat. Under Chothia's definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOs. 28, 29, and 30, and the light chain variable domain CDRs are shown in SEQ ID NOs. 10, 11, and 12.

[0059] The CDR sequences for 10D1 and antibodies derived from 10D1 include the heavy chain variable domain CDRs, SEQ ID NOs. 13, 14, and 15, and the light chain variable domain CDRs, SEQ ID NOs. 16, 17, and 18, as defined by Kabat. Under Chothia's definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOs. 31, 32, and 33, and the light chain variable domain CDRs are shown in SEQ ID NOs. 16, 17, and 18.

[0060] The CDR sequences for 10G1 and antibodies derived from 10G1 include the heavy chain variable domain CDRs, SEQ ID NOs: 19, 20, and 21, and the light chain variable domain CDRs, SEQ ID NOs: 22, 23, and 24, as defined by Kabat. Under Chothia's definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOs: 34, 35, and 36, and the light chain variable domain CDRs are shown in SEQ ID NOs: 22, 23, and 24.

[0061] The amino acid sequence for the heavy chain variable region of the 12A4 antibody is shown in SEQ ID NO: 37. The amino acid sequence for the light chain variable region of the 12A4 antibody is shown in SEQ ID NO: 38.

[0062] The amino acid sequence for the heavy chain variable region of the 12D1 antibody is shown in SEQ ID NO: 39. The amino acid sequence for the light chain variable region of the 12D1 antibody is shown in SEQ ID NO: 40.

[0063] The amino acid sequence for the heavy chain variable region of the 10D1 antibody is shown in SEQ ID NO: 41. The amino acid sequence for the light chain variable region of the 10D1 antibody is shown in SEQ ID NO: 42.

[0064] The amino acid sequence for the heavy chain variable region of the 10G1 antibody is shown in SEQ ID NO: 43. The amino acid sequence for the light chain variable region of the 10G1 antibody is shown in SEQ ID NO: 44.

[0065] The amino acid sequences for the heavy chain variable region and light chain variable region of the modified antibody are also provided. In some embodiments, the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs. 38, 40, 42, or 44 is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs. 38, 40, 42, or 44. The heavy chain variable region sequence can be paired with a corresponding light chain variable region sequence, which together bind to IGF1R.

[0066] In some embodiments, the variable region is entirely human, derived from, for example, human heavy chain immunoglobulin loci (e.g., a combination of human IGHV, IGHD, and IGHJ genes) and / or human kappa chain immunoglobulin loci (e.g., a combination of human IGKV and IGKJ genes).

[0067] Furthermore, in some embodiments, the antibodies or antigen-binding fragments described herein may also contain one, two, or three heavy chain variable region CDRs selected from the group consisting of SEQ ID NOs: 1-3, SEQ ID NOs: 7-9, SEQ ID NOs: 13-15, SEQ ID NOs: 19-21, SEQ ID NOs: 25-27, SEQ ID NOs: 28-30, SEQ ID NOs: 31-33, and SEQ ID NOs: 34-36, and / or one, two, or three light chain variable region CDRs selected from the group consisting of SEQ ID NOs: 4-6, SEQ ID NOs: 10-12, SEQ ID NOs: 16-18, and SEQ ID NOs: 22-24.

[0068] In some embodiments, the antibody may have a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region contains or comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of the selected VH CDR1; the CDR2 region contains or comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of the selected VH CDR2; and the CDR3 region contains or comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of the selected VH CDR3. In some embodiments, the antibody may have a light chain variable region (VL) comprising CDR1, 2, and 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of the selected VL CDR1; the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of the selected VL CDR2; and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of the selected VL CDR3. The amino acid sequences of the selected VH CDR1, 2, and 3 and the selected VL CDR1, 2, and 3 are shown in Figure 1 (CDR according to Kabat's definition) and Figure 2 (CDR according to Chothia's definition).

[0069] In some embodiments, the antibody or antigen-binding fragment described herein may contain one, two, or three heavy chain variable domains of the CDRs of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3

[0070] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9

[0071] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15

[0072] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 19 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, SEQ ID NO: 20 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, or SEQ ID NO: 21 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0073] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0074] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30, all of which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0075] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 33, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0076] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0077] In some embodiments, the antibody or antigen-binding fragments described herein may contain one, two, or three light chain variable domains of the CDRs of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0078] In some embodiments, the antibody or antigen-binding fragments described herein may contain one, two, or three light chain variable domains of the CDRs of SEQ ID NO: 10 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, SEQ ID NO: 11 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, or SEQ ID NO: 12 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0079] In some embodiments, the antibody or antigen-binding fragments described herein may contain one, two, or three light chain variable domains of the CDRs of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 17, and SEQ ID NO: 18, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0080] In some embodiments, the antibody or antigen-binding fragments described herein may contain one, two, or three light chain variable domains of the CDRs of SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0081] Insertions, deletions, and substitutions can be located within the CDR sequence or at one or both ends of the CDR sequence. In some embodiments, the CDR is determined based on a Kabat-defined scheme. In some embodiments, the CDR is determined based on a Chothia-defined scheme. In some embodiments, the CDR is determined based on a combination of the Kabat and Chothia-defined schemes. In some embodiments, the CDR is determined based on the IMGT definition. In some embodiments, the CDR is determined based on the contact definition.

[0082] This disclosure also provides an antibody or antigen-binding fragment thereof that binds to IGF1R. The antibody or antigen-binding fragment thereof contains a heavy chain variable region (VH) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH sequence, and a light chain variable region (VL) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 37 and the selected VL sequence is SEQ ID NO: 38. In some embodiments, the selected VH sequence is SEQ ID NO: 39 and the selected VL sequence is SEQ ID NO: 40. In some embodiments, the selected VH sequence is SEQ ID NO: 41 and the selected VL sequence is SEQ ID NO: 42. In some embodiments, the selected VH sequence is SEQ ID NO: 43 and the selected VL sequence is SEQ ID NO: 44.

[0083] To measure the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (for example, gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences to ensure optimal alignment for comparison, and non-homologous sequences may be ignored). The length of the reference sequence aligned for comparison is at least 80% of the reference sequence length, and in some embodiments, at least 90%, 95%, or 100%. Subsequently, amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at that position. The percentage of identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced to optimally align the two sequences and the length of each gap. For example, sequence comparison and measurement of identity percentage between two sequences can be performed using a Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0084] This disclosure also provides nucleic acids comprising polynucleotides encoding polypeptides containing an immunoglobulin heavy chain or an immunoglobulin light chain. The immunoglobulin heavy chain or immunoglobulin light chain contains a CDR as shown in Figure 1 or Figure 2, or has the sequence shown in Figure 3. When a polypeptide forms a pair with a corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds to IGF1R.

[0085] Anti-IGF1R antibodies and antigen-binding fragments may also be antibody variants (including derivatives and conjugates) of antibodies or antibody fragments, as well as multispecific (e.g., bispecific) antibodies or antibody fragments. Additional antibodies provided herein include polyclonal, monoclonal, multimer, multispecific (e.g., bispecific), human antibodies, chimeric antibodies (e.g., human-mouse chimeric), single-chain antibodies, intracellularly produced antibodies (i.e., intrabodies), and their antigen-binding fragments. Antibodies or their antigen-binding fragments may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In some embodiments, the antibody or its antigen-binding fragment is an IgG antibody or its antigen-binding fragment.

[0086] Antibody fragments are suitable for use in the provided manner, insofar as they retain the desired affinity and specificity of the full-length antibody. Therefore, an antibody fragment that binds to IGF1R retains its ability to bind to IGF1R. An Fv fragment is an antibody fragment containing a complete antigen recognition and binding site. This region consists of a dimer in which one heavy chain variable domain and one light chain variable domain are closely associated, which can essentially be covalent, for example, in scFv. In this configuration, the three CDRs of each variable domain interact to define the antigen-binding site on the surface of the VH-VL dimer. Together, the six CDRs, or subsets thereof, confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) can have the ability to recognize and bind to an antigen, though usually with lower affinity than the entire binding site. A single-stranded Fv or (scFv) antibody fragment contains the VH and VL domains (or regions) of the antibody, and these domains are located within a single polypeptide chain. Generally, scFv polypeptides further contain a polypeptide linker between the VH and VL domains, which allows the scFv to form a desirable structure for antigen binding.

[0087] This disclosure also provides an antibody or an antigen-binding fragment thereof that cross-competes with any antibody or antigen-binding fragment described herein. Cross-competition assays are well known in the art and are described, for example, in Moore et al., “Antibody cross-competition analysis of the human immunodeficiency virus type 1 gp120 exterior envelope glycoprotein.” Journal of Virology 70.3(1996):1863-1872, which in whole is incorporated herein by reference. In one embodiment, this disclosure also provides an antibody or an antigen-binding fragment thereof that binds to the same epitope or region as any antibody or antigen-binding fragment described herein. Epitope-binding assays are well known in the art and are described, for example, in Estep et al., “High throughput solution-based measurement of antibody-antigen affinity and epitope binning.” MAbs. Vol.5. No.2. Taylor & Francis, 2013, which in whole is incorporated herein by reference.

[0088] Antibody and antigen-binding fragments This disclosure provides various antibodies and their antigen-binding fragments derived from the anti-IGF1R antibody described herein. Generally, an antibody (also called an immunoglobulin) consists of two classes of polypeptide chains: a light chain and a heavy chain. An unspecified example of an antibody in this disclosure may be an intact four-immunoglobulin chain antibody containing two heavy chains and two light chains. The heavy chain of the antibody may be any isotype including IgM, IgG, IgE, IgA, or IgD, or a subisotype including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chain may be a κ light chain or a λ light chain. The antibody may contain two identical copies of the light chain and two identical copies of the heavy chain, each containing one variable domain (or variable region, VH The heavy chain, which contains a variable domain (or variable region), binds to each other via disulfide bonds within its constant domains, forming the "stem" of the antibody. Each of these contains one variable domain (or variable region, V L Each light chain containing a constant domain (or constant region) is bound to a heavy chain via a disulfide bond. The variable region of each light chain is aligned with the variable region of the heavy chain to which it is bound. The variable regions of both the light and heavy chains contain three hypervariable regions sandwiched between more conserved framework regions (FRs).

[0089] The hypervariable regions, known as complementarity-determining regions (CDRs), form loops containing the antigen-binding surface of the antibody. The four framework regions are largely adapted to the β-sheet structure, and the CDRs form loops connecting the β-sheet structure, and in some cases, form part of the β-sheet structure. The CDRs of each chain are held in close proximity by the framework regions and, together with the CDRs of other chains, contribute to the formation of the antigen-binding region.

[0090] Methods for identifying the CDR region of an antibody by analyzing its amino acid sequence are well-known, and several definitions of CDRs are commonly used. Kabat's definition is based on sequence variability, while Chothia's definition is based on the location of the structural loop region. These methods and definitions are, for example, found in Martin, “Protein sequence and structure analysis of antibody variable domains,” Antibody Engineering, Springer Berlin Heidelberg, 2001. 422-439, Abhinandan, et al. “Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains,” Molecular Immunology 45.14(2008):3832-3839, Wu, T. and Kabat, EA(1970) J. Exp. Med. 132:211-250, Martin et al., Methods Enzymol. 203:121-53(1991), Morea et al., Biophys Chem. 68(1-3):9-16(Oct. 1997), Morea et al., J Mol Biol. 275(2):269-94(Jan. 1998), Chothia This is described in et al., Nature 342(6252):877-83 (Dec. 1989), and Ponomarenko and Bourne, BMC Structural Biology 7:64 (2007), the entirety of each of these works is incorporated herein by reference.

[0091] CDRs are important for recognizing the epitopes of antigens. As used herein, an “epitope” is the smallest portion of a target molecule that can be specifically bound by the antigen-binding domain of an antibody. The minimum size of an epitope can be about 3, 4, 5, 6, or 7 amino acids, but these amino acids do not need to be in a continuous linear sequence of the primary structure of the antigen, as epitopes can depend on the three-dimensional structure of the antigen based on the secondary and tertiary structures of the antigen.

[0092] In some embodiments, the antibody is an intact immunoglobulin molecule (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). The IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved, differing in their constant regions, particularly the hinge and upper CH2 domain. The sequences and differences of IgG subclasses are well known in the art, for example, in Vidarsson, et al., “IgG subclasses and allotypes: from structure to effector functions.” Frontiers in immunology 5 (2014); Irani, et al., “Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases.” Molecular immunology 67.2 (2015):171-182; and Shakib, Farouk, ed. The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016, each of which is incorporated herein by reference.

[0093] Antibodies can also be immunoglobulin molecules derived from any species (e.g., humans, rodents, mice, camelids). Antibodies disclosed herein include, but are not limited to, polyclonal, monoclonal, monospecific, multispecific antibodies, and chimeric antibodies containing an immunoglobulin-binding domain fused to another polypeptide. The terms “antigen-binding domain” or “antigen-binding fragment” refer to any portion of an antibody that retains the specific binding activity of an intact antibody, i.e., any portion of an antibody that is specifically capable of binding to an epitope on the target molecule of an intact antibody. This includes, for example, Fab, Fab', F(ab')2, and variants of these fragments. Thus, in some embodiments, the antibody or its antigen-binding fragment may be any polypeptide containing, for example, scFv, Fv, Fd, dAb, bispecific antibodies, bispecific scFv, diabodies, linear antibodies, single-chain antibody molecules, multispecific antibodies formed from antibody fragments, and a binding domain that is an antibody-binding domain, or a binding domain homologous thereto. Non-limiting examples of antigen-binding domains include, for example, the heavy and / or light chain CDRs of an intact antibody, the heavy and / or light chain variable regions of an intact antibody, the full-length heavy or light chain of an intact antibody, or individual CDRs derived from either the heavy or light chain of an intact antibody.

[0094] Antibody fragments suitable for use in the methods described herein are also provided. Fab fragments contain a variable domain and a constant domain of the light chain, as well as a variable domain and a first constant domain (CH1) of the heavy chain. F(ab')2 antibody fragments generally contain a pair of Fab fragments, commonly linked near the carboxyl terminus by a hinge cysteine ​​between them. Other chemical linkages of antibody fragments are well known in the art.

[0095] A diabody is a small antibody fragment containing two antigen-binding sites, and this fragment contains VH (VH and VL) attached to VL within the same polypeptide chain. By using a linker that is too short to allow pairing between two domains on the same chain, the domains can be paired with complementary domains on another chain, thereby generating two antigen-binding sites.

[0096] Linear antibodies contain a pair of tandem Fd segments (VH-CH1-VH-CH1) that, together with a complementary light chain polypeptide, form a pair of antigen-binding regions. Linear antibodies may be bispecific or monospecific.

[0097] The antibodies and antibody fragments of this disclosure can be modified within the Fc region to impart a desired effector function or serum half-life.

[0098] Antibody multimerization can be achieved by the spontaneous aggregation of antibodies or by chemical or recombinant conjugation techniques known in the art. For example, a certain proportion of purified antibody preparations (e.g., one purified IgG molecule) spontaneously form protein aggregates containing antibody homodimers and other higher-order antibody multimers.

[0099] Alternatively, antibody homodimers can be formed by chemical bonding techniques well known in the art. For example, antibody polymers can be formed using heterobifunctional crosslinking agents, including but not limited to SMCC (4-(maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl) and SATA (S-acetylthioacetate N-succinimidyl). Exemplary procedures for forming antibody homodimers are described in Ghetie et al. (Proc. Natl. Acad. Sci. USA 94:7509-7514, 1997). Antibody homodimers can be converted to Fab'2 homodimers by pepsin digestion. Another method for forming antibody homodimers is the method using the autophilic T15 peptide, described in Zhao et al. (J. Immunol. 25:396-404, 2002).

[0100] In some embodiments, multispecific antibodies are bispecific antibodies. Bispecific antibodies can be produced by recombining the interface between a pair of antibody molecules to maximize the proportion of heterodimers recovered from recombinant cell culture. For example, the interface may contain at least a portion of the CH3 domain of the antibody's constant domain. In this method, one or more smaller amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the larger amino acid side chains with smaller ones (e.g., alanine or threonine), a compensatory "cavity" of the same or similar size as the larger side chain is created at the interface of the second antibody molecule. This provides a mechanism to increase the yield of heterodimers compared to other unwanted end products such as homodimers. This method is described, for example, in WO 96 / 27011, which is incorporated in its entirety by reference.

[0101] Examples of bispecific antibodies include crosslinked or "heterocomplex" antibodies. For example, one antibody in a heterocomplex can be coupled to avidin and the other to biotin. Heterocomplex antibodies can also be produced using any convenient crosslinking method. Suitable crosslinking agents and techniques are well known in the art and are disclosed in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety.

[0102] Any antibody or antigen-binding fragment described herein can be conjugated with a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or its antigen-binding fragment in a subject or in solution). Non-limiting examples of stabilizing molecules include polymers (e.g., polyethylene glycol) or proteins (e.g., serum albumin such as human serum albumin). Conjugation with a stabilizing molecule can increase the half-life of the antibody or antigen-binding fragment or extend its biological activity, either in vitro (e.g., in tissue culture medium or when stored as a pharmaceutical composition) or in vivo (e.g., in humans).

[0103] In some embodiments, the antibodies or antigen-binding fragments described herein can be conjugated with a therapeutic agent. The antibody-drug conjugate, comprising the antibody or its antigen-binding fragment, can be covalently or noncovalently bound to the therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic agent or a cell proliferation inhibitor.

[0104] In some embodiments, antigen-binding fragments can form part of a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor is a fusion of single-stranded variable fragments (scFv) described herein, fused to the CD3ζ transmembrane and endodomains. In some embodiments, the chimeric antigen receptor also includes intracellular signaling domains derived from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS). In some embodiments, the chimeric antigen receptor includes multiple signaling domains, e.g., CD3z-CD28-41BB, or CD3z-CD28-OX40, for increased potency. Thus, in one aspect, the disclosure further provides cells (e.g., T cells) expressing the chimeric antigen receptor described herein.

[0105] In some embodiments, the scFv has one heavy chain variable domain and one light chain variable domain. In some embodiments, the scFv has two heavy chain variable domains and two light chain variable domains.

[0106] In some embodiments, the sequences of antibodies or their antigen-binding fragments described herein (e.g., CDR or VH / VL sequences) can be used to generate bispecific antibodies targeting IGF1R and additional antigens (e.g., CD3, PD-1, PD-L1, HER2, or EGFR).

[0107] Characteristics of antibodies In some embodiments, the antibodies or antigen-binding fragments thereof described herein block the binding between IGF1R and an IGF1R ligand (e.g., IGF-1, IGF-2, or insulin (INS)). Thus, the antibody inhibits the IGF1R signaling pathway by binding to IGF1R. In some embodiments, the antibody can reduce tumor volume in animals. In some embodiments, the antibody can inhibit IGF-1-induced cell proliferation. In some embodiments, the antibody can inhibit IGF1R autophosphorylation. In some embodiments, the antibody can inhibit IGF-1-induced OPN secretion.

[0108] In some embodiments, the antibody (or antigen-binding fragment thereof) binds specifically to IGF1R (e.g., human IGF1R or monkey IGF1R) with a dissociation rate (k -1 ) of less than 0.1 s -1 , less than 0.01 s -1 , less than 0.001 s -1 , less than 0.0001 s -1 , or less than 0.00001 s off . In some embodiments, the dissociation rate (k off ) is greater than 0.01 s -1 , greater than 0.001 s -1 , greater than 0.0001 s -1 , greater than 0.00001 s -1 , or greater than 0.000001 s -1 .

[0109] In some embodiments, the association rate (k on ) is greater than 1×10 2 / Ms, greater than 1×10 3 / Ms, greater than 1×10 4 / Ms, greater than 1×10 5 / Ms, or greater than 1×10 6 / Ms. In some embodiments, the association rate (k on ) is less than 1×10 5 / Ms, less than 1×10 6 / Ms, or less than 1×10 7 / Ms.

[0110] Affinity is expressed as the velocity constant (KD=k off / k on It can be estimated from the quotient of ). In some embodiments, KD is 1 × 10 -6 Less than M, 1 x 10 -7 Less than M, 1 x 10 -8 Less than M, 1 x 10 -9 Less than M, or 1 × 10 -10 It is less than M. In some embodiments, KD is 50nM, 30nM, 20nM, 15nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, or less than 1nM. In some embodiments, KD is 1 × 10⁻¹⁶ -7 Super M, 1×10 -8 Super M, 1×10 -9 Super M, 1×10 -10 Super M, 1×10 -11 M or 1 × 10 -12 It is greater than M.

[0111] Common techniques for measuring the affinity of an antibody to an antigen include, for example, ELISA, RIA, and surface plasmon resonance (SPR). In some embodiments, the antibody binds to human IGF1R (SEQ ID NO: 45) and / or monkey IGF1R (e.g., SEQ ID NO: 46). In some embodiments, the antibody does not bind to human IGF1R and / or monkey IGF1R. In some embodiments, the antibody does not bind to the human insulin receptor INSR (e.g., SEQ ID NO: 47).

[0112] In some embodiments, the antibodies or antigen-binding fragments described herein have a tumor growth inhibition rate (TGI) of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or more than 200%. TVThe TGI% has a tumor growth inhibition rate of less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. The TGI% can be measured, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after the start of treatment, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the start of treatment. As used herein, the tumor growth inhibition rate (TGI%) is calculated using the following formula: TGI(%)=[1-(T i -T0) / (V i -V0)] × 100% T i V is the mean tumor volume in the treatment group on day i. T0 is the mean tumor volume in the treatment group on day 0. i V0 is the mean tumor volume in the control group on day i. V0 is the mean tumor volume in the control group on day 0.

[0113] In some embodiments, the antibodies or antigen-binding fragments described herein can inhibit IGF-1-induced cell proliferation. In some embodiments, IGF-1-induced cell proliferation is inhibited by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0114] In some embodiments, antibodies can inhibit IGF1R autophosphorylation. In some embodiments, IGF1R autophosphorylation is inhibited by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0115] In some embodiments, the antibody can inhibit IGF-1-induced OPN secretion. In some embodiments, IGF-1-induced OPN secretion is inhibited by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0116] In some embodiments, the antibody or antigen-binding fragment described herein is an IGF1R antagonist. In some embodiments, the antibody or antigen-binding fragment described herein is an IGF1R agonist.

[0117] In some embodiments, the antibodies or antigen-binding fragments described herein are non-toxic. In some embodiments, for example, at 3 mg / kg, no significant difference in body weight can be observed between the treatment group and the control group.

[0118] In some embodiments, antibodies or antigen-binding fragments can induce complement-dependent cell-mediated cytotoxicity (CDC) and / or antibody-dependent cell-mediated cytotoxicity (ADCC) and kill tumor cells.

[0119] In some embodiments, the antibody or antigen-binding fragment has a functional Fc region. In some embodiments, the effector function of the functional Fc region is antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the effector function of the functional Fc region is phagocytosis. In some embodiments, the effector functions of the functional Fc region are ADCC and phagocytosis.

[0120] In some embodiments, antibodies or antigen-binding fragments can induce complement-dependent cell-mediated cytotoxicity (CDC).

[0121] In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4. In some embodiments, the antibody is a human IgG1 antibody.

[0122] In some embodiments, the antibody or antigen-binding fragment does not have a functional Fc region. For example, the antibody or antigen-binding fragment is a Fab, Fab', F(ab')2, and Fv fragment. In some embodiments, the Fc region has an LALA mutation (L234A and L235A mutations in EU numbering) or an LALA-PG mutation (L234A, L235A, and P329G mutations in EU numbering).

[0123] In some embodiments, Fc has SI mutations (S239D and I332E mutations in EU numbering).

[0124] In some embodiments, Fc has one or more of the following mutations M252Y / S254T / T256E according to EU numbering.

[0125] In some embodiments, Fc has one or more of the following mutations T250Q / M428L according to EU numbering.

[0126] In some embodiments, the antibody or antigen-binding fragments described herein are stable under high-concentration and / or pressurized storage conditions.

[0127] In some embodiments, the stability of the antibody or antigen-binding fragments described herein is measured by the relative amounts of potential molecular variants, such as high molecular weight variants and / or low molecular weight variants. In some embodiments, the relative amounts of potential molecular variants are measured using size exclusion high-performance liquid chromatography (SEC-HPLC). Other methods for measuring antibody stability are also well known in the art.

[0128] In some embodiments, potential molecular variants account for approximately 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, and 1% or less of the total sample measured. In some embodiments, potential molecular variants are undetectable in the measured sample. In some embodiments, the main peak representing a stable antibody accounts for approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% or more of the total sample measured.

[0129] In some embodiments, the measurement is performed approximately 1, 2, 3, 4, 5, 6, or 7 days after storage. In some embodiments, the measurement is performed approximately 1, 2, 3, 4, 5, 6, or 7 weeks after storage. In some embodiments, the measurement is performed approximately 4 weeks after storage.

[0130] In some embodiments, the antibody or antigen-binding fragments described herein are stored at approximately 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, and 55°C or higher.

[0131] In some embodiments, the antibody or antigen-binding fragments described herein are stored at concentrations of at least or about 50 mg / mL, at least or about 70 mg / mL, at least or about 100 mg / mL, at least or about 120 mg / mL, or at least about 150 mg / mL or higher. In some embodiments, the antibody or antigen-binding fragments described herein are stored at concentrations of about 50 mg / mL to about 150 mg / mL, about 70 mg / mL to about 150 mg / mL, about 80 mg / mL to about 150 mg / mL, about 90 mg / mL to about 150 mg / mL, about 80 mg / mL to about 120 mg / mL, or about 90 mg / mL to about 110 mg / mL.

[0132] Method for producing anti-IGF1R antibodies Isolated fragments of human IGF1R (e.g., extracellular regions) can be used as immunogens to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. Polyclonal antibodies can be produced in animals by multiple injections (e.g., subcutaneous or intraperitoneal injection) of the antigen peptide or protein. In some embodiments, the antigen peptide or protein is injected with at least one adjuvant. In some embodiments, the antigen peptide or protein can be conjugated with an immunogenic agent in the immunized species. Animals may be injected with the antigen peptide or protein two or more times (e.g., two, three, or four times).

[0133] Full-length polypeptides or proteins can be used, or selectively, antigen peptide fragments can be used as immunogens. The protein antigen peptide contains at least 8 (e.g., at least 10, 15, 20, or 30) amino acid residues of the IGF1R amino acid sequence and includes an epitope of the protein such that the antibody produced against the peptide forms a specific immune complex with the protein. As previously mentioned, the full-length sequence of human IGF1R is well known in the art (SEQ ID NO: 45). The full-length sequence of monkey IGF1R is also well known in the art (SEQ ID NO: 46). In some embodiments, His-tagged human IGF1R protein (ACRO Biosystems Inc., catalog number: IGR-H5229, containing positions 31-932 of SEQ ID NO: 45) is used as an immunogen.

[0134] Immunogens are typically used for antibody preparation by immunizing a suitable target (e.g., a human or transgenic animal expressing at least one human immunoglobulin locus). A suitable immunogenic preparation may contain, for example, recombinantly expressed or chemically synthesized polypeptides (e.g., a fragment of human IGF1R). The preparation may further contain an adjuvant, such as a Freund complete or incomplete adjuvant, or a similar immunostimulant.

[0135] Polyclonal antibodies can be prepared as described above by immunizing a suitable target with IGF1R polypeptide or its antigenic peptide (e.g., a part of IGF1R, such as the extracellular region) as an immunogen. The antibody titer in the immunized target can be monitored over time using standard techniques such as enzyme-linked immunosorbent assay (ELISA) with the immobilized IGF1R polypeptide or peptide. If desired, the antibody molecule can be isolated from a mammal (e.g., from blood) and further purified by well-known techniques such as protein A chromatography or protein G chromatography to obtain the IgG fraction. At an appropriate time after immunization, for example, when the titer of specific antibodies is at its maximum, antibody-producing cells can be obtained from the target organism and used to prepare monoclonal antibodies using standard techniques such as the hybridoma technique originally described by Kohler et al. (Nature 256:495-497, 1975), the human B-cell hybridoma technique (Kozbor et al., Immunol. Today 4:72, 1983), the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96, 1985), or the trioma technique. Techniques for producing hybridomas are well known (generally, see Current Protocols in Immunology, 1994, Coligan et al. (Eds.), John Wiley & Sons, Inc., New York, NY). For example, hybridoma cells that produce monoclonal antibodies can be detected by screening hybridoma culture supernatants for antibodies that bind to a target polypeptide or epitope using a standard ELISA assay.

[0136] Variants of antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding a human antibody, a humanized antibody, or a chimeric antibody, or an antibody or antigen-binding fragment described herein, or by peptide synthesis. Such variants include, for example, deletions, insertions, or substitutions of residues within the amino acid sequence that produces the antigen-binding site or antigen-binding domain of the antibody. In a population of such variants, some antibodies or antigen-binding fragments exhibit increased affinity for a target protein, such as IGF1R. Any combination of deletions, insertions, and / or combinations can be realized in an antibody or antigen-binding fragment with increased binding affinity to the target. Antibodies or antigen-binding fragments can be modified, or novel post-translational modifications can be introduced, by changing the amino acids introduced into the antibody or antigen-binding fragment, such as changing the number of glycosylation sites (e.g., increasing or decreasing them), changing the type of glycosylation sites (e.g., altering the amino acid sequence so that different sugars are bound by enzymes present in the cell), or introducing novel glycosylation sites.

[0137] The antibodies disclosed herein may originate from any species of animal, including mammals. Non-limiting examples of natural antibodies include antibodies derived from humans, primates (e.g., monkeys and apes), cattle, pigs, horses, sheep, camelids (e.g., camels and llamas), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits), including transgenic rodents genetically modified to produce human antibodies.

[0138] Examples of human antibodies and humanized antibodies include antibodies having variable and constant regions derived from (or having the same amino acid sequence as) human germline immunoglobulin sequences. Examples of human antibodies include amino acid residues within the CDR that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro, or somatic mutations in vivo).

[0139] Humanized antibodies typically have a human framework (FR) into which a non-human CDR has been transplanted. Therefore, a humanized antibody has one or more amino acid sequences introduced from a non-human source. These non-human amino acid residues are often called “import” residues, and typically, these are derived from “import” variable domains. Humanization can essentially be carried out by substituting, for example, a rodent CDR or CDR sequence with the corresponding sequence in a human antibody. These methods are described, for example, in Jones et al. "Replacing the complementarity-determining regions in a human antibody with those from a mouse." Nature 321.6069(1986):522, Riechmann et al. "Reshaping human antibodies for therapy." Nature 332.6162(1988):323, and Dall'Acqua et al. "Antibody humanization by framework shuffling." Methods 36.1(2005):43-60, each of which is incorporated herein by reference in its entirety. Thus, a “humanized” antibody is a chimeric antibody in which a portion considerably smaller than the intact human V domain is substituted with a corresponding sequence derived from a non-human species. In practice, humanized antibodies are typically mouse antibodies in which several CDR residues and several FR residues are substituted with residues derived from similar sites within the human antibody.

[0140] The selection of human VH and VL domains used in the production of humanized antibodies is crucial for reducing immunogenicity. Following the so-called "best-fit" method, the V domain sequence of the mouse antibody is screened against an entire library of known human domain sequences. Then, the human sequence that most closely matches the mouse sequence is accepted as the human FR for the humanized antibody (Sims et al. et al. "A humanized CD18 antibody can block function without cell destruction." The Journal of Immunology 151.4(1993):2296-2308, Chothia, et al., "Canonical structures for the hypervariable regions of immunoglobulins." Journal of molecular biology 196.4(1987):901917).

[0141] Furthermore, it is important to humanize antibodies while maintaining high specificity and affinity for antigens, as well as other desirable biological properties. To achieve this goal, humanized antibodies can be prepared by analytical processes of parental sequences and various conceptual humanization products using three-dimensional models of parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and well known to those skilled in the art. Computer programs are available that can illustrate and display the possible three-dimensional structures of selected candidate immunoglobulin sequences. By observing these displays, it is possible to analyze the roles that residues can play in the functionalization of candidate immunoglobulin sequences, i.e., the residues that affect the candidate immunoglobulin's ability to bind to its antigen. In this way, FR residues can be selected and combined from recipient and import sequences to achieve desired antibody properties, such as increased affinity for the target antigen.

[0142] Typically, amino acid sequence variants of human, humanized, or chimeric anti-IGF1R antibodies contain amino acid sequences that have at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the sequences present in the light or heavy chain of the original antibody.

[0143] In some embodiments, antibodies are generated using mice (e.g., RenMab mice) having humanized heavy chain immunoglobulin loci and humanized κ chain immunoglobulin loci. A heavy chain immunoglobulin locus is a chromosomal region containing the gene for the heavy chain of an antibody. The locus can include, for example, the human IGHV (variable) gene, the human IGHD (variability) gene, the human IGHJ (binding) gene, and the mouse heavy chain constant domain gene. A κ chain immunoglobulin locus is a chromosomal region containing the gene encoding the light chain (κ chain) of an antibody. Examples of κ chain immunoglobulin loci include the human IGKV (variable) gene, the human IGKJ (binding) gene, and the mouse light chain constant domain gene. A detailed description of RenMab mice can be found in PCT / CN2020 / 075698, which is incorporated herein by reference in its entirety. The antibodies generated by the mice have full human VH, full human VL, and mouse constant regions. In some embodiments, human VH and human VL are linked to the constant region of human IgG (e.g., IgG1, IgG2, IgG3, and IgG4).

[0144] In some embodiments, mice having humanized heavy chain immunoglobulin loci and humanized κ chain immunoglobulin loci (e.g., RenLite TMAntibodies are generated using mice. A heavy chain immunoglobulin locus is a chromosomal region containing the gene for the heavy chain of an antibody. Examples of loci include the human IGHV (variable) gene, the human IGHD (diversity) gene, the human IGHJ (binding) gene, and the mouse heavy chain constant domain gene. A κ chain immunoglobulin locus is a chromosomal region containing the gene encoding a common light chain. Examples of κ chain immunoglobulin loci include the human IGKV (variable) gene, the human IGKJ (binding) gene, and the mouse light chain constant domain gene. RenLite TM A detailed description of the mouse can be found in PCT / CN2021 / 097652, which is incorporated herein by reference in its entirety.

[0145] Identity or homology to the original sequence is typically the percentage of amino acid residues present in a candidate sequence that is identical to a sequence present in a human antibody or fragment, humanized antibody or fragment, or chimeric anti-IGF1R antibody or fragment, after aligning the sequences, introducing gaps where necessary, and achieving the maximum percentage of sequence identity, without considering conservative substitutions as part of the sequence identity.

[0146] Further modifications can be made to anti-IGF1R antibodies or antigen-binding fragments. For example, cysteine ​​residues can be introduced into the Fc region to enable interchain disulfide bond formation within this region. Homodimeric antibodies thus produced may have some kind of extended in vitro and / or in vivo half-life. For example, homodimeric antibodies with extended in vitro and / or in vivo half-lives can also be prepared using heterobifunctional crosslinking agents, as described in Wolff et al. ("Monoclonal antibody homodimers: enhanced antitumor activity in nude mice." Cancer Research 53.11(1993):2560-2565). Alternatively, antibodies with a double Fc region can be recombinant.

[0147] In some embodiments, covalent modifications can be added to anti-IGF1R antibodies or their antigen-binding fragments. These covalent modifications can be added by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage. Other types of covalent modifications of antibodies or antibody fragments are introduced into the molecule by reacting targeted amino acid residues of the antibody or fragment with an organic derivatizing agent that can react with selected side chains or N- or C-terminal residues.

[0148] In some embodiments, antibody variants are provided having carbohydrate structures lacking fucose (directly or indirectly) bound to the Fc region. For example, the amount of fucose in such an antibody composition may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycans located at Asn297 relative to the total amount of all sugar structures (e.g., complexes, hybrids, and high-mannose structures) bound to Asn297, measured by MALDI-TOF mass spectrometry as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at position 297 in the Fc region (position 314 in the Eu numbering of Fc region residues, or position 314 in the Kabat numbering), however, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in the antibody. Such fucosylated mutants may have improved ADCC function. In some embodiments, to reduce glycan heterogeneity, the Fc region of the antibody can be further recombined, and asparagine at position 297 can be replaced with alanine (N297A).

[0149] In some embodiments, to enhance production efficiency by avoiding Fab-arm exchange, the Fc region of the antibody is further recombined, replacing the serine at position 228 (EU numbering) of IgG4 with proline (S228P). A detailed description of the S228 mutation is, for example, found in Silva et al. "The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation." Journal of Biological Chemistry 290.9(2015):5462-5469, which is incorporated in its entirety by reference.

[0150] Recombination vectors This disclosure also provides recombinant vectors (e.g., expression vectors) containing isolated polynucleotides disclosed herein (e.g., polynucleotides encoding polypeptides disclosed herein), host cells into which the recombinant vectors have been introduced (i.e., such host cells contain polynucleotides and / or the polynucleotide-containing vectors), and the production of recombinant antibody polypeptides or fragments thereof by recombinant technology.

[0151] As used herein, “vector” is any construct that, when introduced into a host cell, can deliver one or more polynucleotides of interest to the host cell. An “expression vector” can deliver and express one or more polynucleotides of interest as encoded polypeptides within the host cell into which the expression vector has been introduced. Thus, within the expression vector, the polynucleotides of interest are positioned for expression within the vector by being operably bound to regulatory elements such as promoters, enhancers, and / or poly-A tails, either within the vector or in the host cell genome, at, near, or adjacent to the integration site of the polynucleotides of interest, so that the polynucleotides of interest are translated within the host cell into which the expression vector has been introduced.

[0152] Vectors can be introduced into host cells by methods well known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., by recombinant viruses). Therefore, non-limiting examples of vectors include viral vectors (that can be used to generate recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensers.

[0153] In some embodiments, polynucleotides disclosed herein (e.g., polynucleotides encoding polypeptides disclosed herein) are introduced using a viral expression system (e.g., a smallpox or other poxvirus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (deficient) replicable virus or a non-replicable virus. In the latter case, viral replication generally occurs only in complementary viral packaging cells. Suitable systems include, for example, Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA 86:317-321, Flexner et al., 1989, Ann. NYAcad Sci. 569:86-103, Flexner et al., 1990, Vaccine, 8:17-21, U.S. Patent Nos. 4,603,112, 4,769,330, and 5,017,487, WO 89 / 01973, U.S. Patent No. 4,777,127, GB 2,200,651, EP 0,345,242, WO 91 / 02805, Berkner-Biotechniques, 6:616-627, 1988, Rosenfeld et al. This is disclosed in al., 1991, Science, 252:431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, 91:215-219; Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci. USA, 90:11498-11502; Guzman et al., 1993, Circulation, 88:2838-2848; and Guzman et al., 1993, Cir. Res., 73:1202-1207. Techniques for incorporating DNA into such expression systems are well known to those skilled in the art. DNA may also be “naked,” as described, for example, in Ulmer et al., 1993, Science, 259:1745-1749 and Cohen, 1993, Science, 259:1691-1692. The uptake of naked DNA can be increased by coating the DNA with biodegradable beads that are efficiently transported into cells.

[0154] For expression, a DNA insert containing a polynucleotide encoding an antibody or polypeptide, as disclosed herein, can be operably ligated to a suitable promoter (e.g., a heterologous promoter), such as, to name a few, the phage λPL promoter, the E. coli lac, trp, and tac promoters, the SV40 early and late promoters, and the promoter of a retroviral LTR. Other suitable promoters are known to those skilled in the art. In some embodiments, the promoter is the cytomegalovirus (CMV) promoter. The expression construct may further contain sites for transcription start and end, and within the transcription region, a ribosome-binding site for translation. The coding portion of the mature transcript expressed by the construct may include a translation start at the beginning and a stop codon (UAA, UGA, or UAG) located approximately at the end of the polypeptide being translated.

[0155] As shown, the expression vector may contain at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell cultures, and tetracycline or ampicillin resistance genes for Escherichia coli and other bacterial cultures. Representative examples of suitable hosts include, but are not limited to, bacterial cells such as Escherichia coli, Streptomyces, and Salmonella typhimurium cells, fungal cells such as yeast cells, insect cells such as Drosophila S2 and Spodoptera litura Sf9 cells, animal cells such as CHO, COS, Bowes melanoma, and HK 293 cells, and plant cells. Suitable culture media and conditions for the host cells described herein are well known in the art.

[0156] Non-restrictive vectors for bacterial use include pQE70, pQE60, and pQE-9 from Qiagen, pBS vector, Phagescript vector, Bluescript vector, pNH8A, pNH16a, pNH18A, and pNH46A from Stratagene, and ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5 from Pharmacia. Non-restrictive eukaryotic cell vectors include pWLNEO, pSV2CAT, pOG44, pXT1, and pSG from Stratagene, and pSVK3, pBPV, pMSG, and pSVL from Pharmacia. Other suitable vectors will be readily apparent to those skilled in the art.

[0157] Suitable non-limiting bacterial promoters for use include the E. coli lacI and lacZ promoters, T3 and T7 promoters, gpt promoter, λPR and PL promoters, and trp promoter. Suitable eukaryotic cell promoters include the CMV pre-early promoter, HSV thymidine kinase promoter, early and late SV40 promoters, retroviral LTR promoters such as those for Rous sarcoma virus (RSV), and metallothionein promoters such as the mouse metallothionein-I promoter.

[0158] In the yeast Saccharomyces cerevisiae, several vectors containing constitutional or inducible promoters, such as α-factor, alcohol oxidase, and PGH, can be used.

[0159] The construct can be introduced into host cells by calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986), which are incorporated herein by reference.

[0160] Transcription of the antibody-encoding DNA in more eukaryotes can be increased by inserting enhancer sequences into vectors. Enhancers are typically cis-acting elements of DNA, approximately 10–300 bp in length, that enhance the transcriptional activity of promoters in a given host cell type. Examples of enhancers include the SV40 enhancer, located behind the origin of replication at base pairs 100–270, the cytomegalovirus early promoter enhancer, the polyoma enhancer behind the origin of replication, and the adenovirus enhancer.

[0161] Appropriate secretory signals can be incorporated into expressed polypeptides to induce the secretion of translated proteins into the endoplasmic reticulum lumen, periplasmic space, or extracellular environment. These signals can be endogenous to the polypeptide, or they can be heterologous signals.

[0162] Polypeptides (e.g., antibodies) can be expressed in modified forms such as fusion proteins (e.g., GST fusions) or by histidine tagging, and may include not only secretory signals but also additional heterologous functional regions. For example, additional amino acids, particularly charged amino acid regions, can be added to the N-terminus of a polypeptide to improve stability and endurance in host cells during purification or subsequent handling and storage. Peptide moieties can also be added to polypeptides to facilitate purification. Such regions can be removed before the final preparation of the polypeptide. Adding peptide moieties to polypeptides to induce secretion or excretion, improve stability, and facilitate purification is a well-known and common technique, particularly in the art.

[0163] Treatment method The antibodies or antigen-binding fragments thereof described herein can be used for a variety of therapeutic purposes.

[0164] In one embodiment, the present disclosure provides a method for treating cancer in a subject, a method for reducing the rate of increase in tumor volume in a subject over time, a method for reducing the risk of metastasis, or a method for reducing the risk of further metastasis in a subject. In some embodiments, the treatment can interrupt, slow, stop, or inhibit the progression of cancer. In some embodiments, the treatment can result in a reduction of the number, severity, and / or duration of one or more symptoms of cancer in a subject.

[0165] In one embodiment, the disclosure provides a method for treating an eye disease (e.g., thyroid eye disease (TED) or thyroid-associated eye disease) in a subject, a method for reducing the rate of disease progression over time in a subject, and a method for reducing the risk of developing an eye disease in a subject. In some embodiments, the treatment can interrupt, slow, block, or inhibit the progression of the eye disease. In some embodiments, the treatment can result in a reduction in the number, severity, and / or duration of one or more symptoms of the eye disease in a subject.

[0166] In some embodiments, the methods for treating eye diseases described herein are combined with one or more additional treatments. Examples of treatments include, but are not limited to, eyelid surgery, eye muscle surgery, and orbital decompression surgery.

[0167] In one embodiment, the disclosure provides a method for treating bone disease in a subject, a method for reducing the rate of disease progression in a subject over time, and a method for reducing the risk of developing bone disease in a subject. In some embodiments, the treatment can halt, slow, stop, or inhibit the progression of bone disease. In some embodiments, the treatment can result in a reduction of the number, severity, and / or duration of one or more symptoms of bone disease in a subject.

[0168] In one embodiment, the disclosure provides methods for treating IGF1R-related diseases in subjects, methods for reducing the rate of disease progression over time in subjects, and methods for reducing the risk of developing diseases in subjects. In some embodiments, the treatment can interrupt, slow, block, or inhibit the progression of IGF1R-related diseases. In some embodiments, the treatment can result in a reduction in the number, severity, and / or duration of one or more symptoms of IGF1R-related diseases in subjects. As used herein, IGF1R-related diseases are diseases caused by or associated with the activation of the IGF1R signaling pathway. In some embodiments, IGF1R-related diseases are cancers. In some embodiments, IGF1R-related diseases are eye diseases (e.g., thyroid eye disease (TED) or thyroid-associated eye disease). In some embodiments, IGF1R-related diseases are bone diseases.

[0169] In one embodiment, the present disclosure is characterized by a method comprising administering a therapeutically effective amount of the antibody or antigen-binding fragment thereof disclosed herein to a subject in need (e.g., a subject having cancer, or identified or diagnosed as having cancer), such as lung cancer, non-small cell lung cancer (NSCLC), or small cell lung cancer (SCLC). In some embodiments, the subject has a solid tumor or an advanced solid tumor.

[0170] In some embodiments, anti-IGF1R antibodies are designed to treat non-small cell lung cancer (NSCLC) and / or small cell lung cancer (SCLC).

[0171] In some embodiments, the cancer is non-small cell lung cancer.

[0172] In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk of cancer. Patients at risk of cancer can be identified by various methods well known in the art.

[0173] In some embodiments, the present disclosure relates to a method for treating an autoimmune disease or inflammation, the method comprising administering to a subject an effective amount of a composition comprising an antibody or antigen-binding fragment thereof as described herein, or an antibody-drug conjugate as described herein.

[0174] In one embodiment, the present disclosure provides methods for treating, preventing, or reducing the risk of developing diseases associated with abnormal or undesirable immune responses, such as autoimmune diseases. These autoimmune diseases include, but are not limited to, Graves' disease. In some embodiments, antibodies or antigen-binding fragments can be used to treat inflammation. In some embodiments, anti-IGF1R antibodies are designed to treat other diseases or disorders, such as microbial infections and allergic disorders.

[0175] In some embodiments, the present disclosure relates to a method for inhibiting an immune response in a subject, the method comprising administering to the subject an effective amount of a composition comprising an antibody or antigen-binding fragment thereof as described herein, or an antibody-drug conjugate as described herein. In some embodiments, the subject has an autoimmune disease.

[0176] As used herein, “effective dose” means an amount or dosage sufficient to produce a beneficial or desired outcome, including interrupting, slowing, blocking, or inhibiting the progression of a disease, such as cancer. The effective dose varies depending on the age and weight of the person to whom the antibody, antigen-binding fragment, polynucleotide encoding the antibody, vector containing the polynucleotide, and / or composition thereof is administered, the severity of the symptoms, and the route of administration, and therefore the dosage can be determined on an individual basis.

[0177] An effective dose can be administered in one or more doses. For example, an effective dose of antibody or antigen-binding fragment is sufficient to alleviate, halt, stabilize, reverse, inhibit, slow, and / or delay the progression of autoimmune disease or cancer in a patient, or sufficient to alleviate, halt, stabilize, reverse, slow, and / or delay the proliferation of cells (e.g., biopsy cells, any of the cancer cells described herein, or cell lines (e.g., cancer cell lines)) in vitro. As is understood in the art, an effective dose of antibody or antigen-binding fragment may vary depending on other factors, in particular, the patient's medical history, as well as the type (and / or dose) of antibody used.

[0178] The effective doses and schedules for administering the antibodies, polynucleotides encoding the antibodies, and / or compositions disclosed herein can be determined experimentally, and making such determinations is within the scope of the art. Those skilled in the art will understand that the dose to be administered will vary depending, for example, on the mammal receiving the antibodies, polynucleotides encoding the antibodies, and / or compositions disclosed herein, the route of administration, the specific type of antibody, the polynucleotide encoding the antibody, the antigen-binding fragment, and / or the compositions disclosed herein used, and other agents administered to the mammal.

[0179] The typical daily dose of an effective antibody is 1 mg / kg to 10 mg / kg (mg per kg of patient's body weight).

[0180] In any of the methods described herein, at least one antibody, its antigen-binding fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding fragments, or pharmaceutical compositions described herein) and optionally at least one additional therapeutic agent can be administered to a target. In some embodiments, at least two different antibodies and / or antigen-binding fragments are administered in the same composition (e.g., a liquid composition). In some embodiments, at least one antibody or antigen-binding fragment and at least one additional therapeutic agent are administered in the same composition (e.g., a liquid composition). In some embodiments, at least one antibody or antigen-binding fragment and at least one additional therapeutic agent are administered in two different compositions (e.g., a liquid composition containing at least one antibody or antigen-binding fragment and a solid oral composition containing at least one additional therapeutic agent). In some embodiments, at least one additional therapeutic agent is administered as a pill, tablet, or capsule. In some embodiments, at least one additional therapeutic agent is administered as a sustained-release oral formulation.

[0181] In some embodiments, one or more additional therapeutic agents can be administered to a subject before or after administration of at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein). In some embodiments, one or more additional therapeutic agents and at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) are administered to a subject such that the biological activity periods of the one or more additional therapeutic agents and the biological activity periods of at least one antibody or antigen-binding fragment (e.g., any of the antibodies or antigen-binding fragments described herein) overlap within the subject.

[0182] In some embodiments, a subject may be administered at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) over a long period of time. A skilled medical professional may determine the length of the treatment period using any of the methods described herein to diagnose or track the effectiveness of the treatment (e.g., observe at least one symptom of cancer). As described herein, a skilled medical professional may also change (e.g., increase or decrease) the identity and number of antibodies or antigen-binding antibody fragments (and / or one or more additional therapeutic agents) administered to the subject, and may adjust (e.g., increase or decrease) the dose or frequency of administration of at least one antibody or antigen-binding antibody fragment (and / or one or more additional therapeutic agents) to the subject based on an assessment of the effectiveness of the treatment (e.g., using any of the methods described herein and known in the art).

[0183] In some embodiments, one or more additional therapeutic agents can be administered to the subject. In some embodiments, the additional therapeutic agent is an anti-CD3 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-HER2 antibody, or an anti-EGFR antibody.

[0184] Pharmaceutical composition and route of administration Pharmaceutical compositions containing at least one (e.g., 1, 2, 3, or 4) of the antibodies or antigen-binding fragments described herein are also provided herein. Two or more (e.g., 2, 3, or 4) of any of the antibodies or antigen-binding fragments described herein may be present in any combination in the pharmaceutical composition. The pharmaceutical composition may be formulated in any form well known in the art.

[0185] Pharmaceutical compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). Compositions may include sterile diluents (e.g., sterile water or saline), non-volatile oils or other synthetic solvents, antioxidants such as chelating agents, buffers, and isotonic agents such as sugars, polyhydric alcohols, or salts, or any combination thereof. Liposome suspensions can also be used as pharmaceutically acceptable carriers. Composition preparations can be formulated and encapsulated in ampoules, disposable syringes, or multi-dose vials. If necessary (e.g., in injectable formulations), adequate fluidity can be maintained by coatings such as lecithin or surfactants. Absorption of antibodies or their antigen-binding fragments can be extended by including absorption-delaying agents. Alternatively, controlled release can be achieved by implants and microencapsulated delivery systems that may contain biodegradable, biocompatible polymers.

[0186] Compositions containing one or more antibodies or antigen-binding fragments described herein can be formulated in unit dosage forms (i.e., physically distinct units containing a predetermined amount of the active compound to facilitate administration and ensure uniformity of dose) for parenteral administration (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, or intraperitoneal).

[0187] Pharmaceutical compositions for parenteral administration are preferably sterile, substantially isotonic, and manufactured under Good Manufacturing Practice (GMP) conditions. Pharmaceutical compositions may be supplied in unit dosage forms (i.e., doses for single administration). Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants. Formulation depends on the chosen route of administration. For injection, antibodies can be formulated in aqueous solution, preferably in a physiologically compatible buffer, to reduce discomfort at the injection site. The solution may contain compounding agents such as suspending agents, stabilizers, and / or dispersants. Alternatively, antibodies may be in lyophilized form for preparation with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0188] The toxicity and therapeutic effect of a composition can be determined by standard pharmaceutical procedures in cell culture media or experimental animals (e.g., monkeys). For example, the LD50 (lethal dose for 50% of the population) and ED50 (effective dose for 50% of the population) can be determined, and the therapeutic index is the ratio of LD50:ED50. Drugs exhibiting a high therapeutic index are preferred. If a drug exhibits undesirable side effects, care must be taken to minimize the potential harm (i.e., mitigate the undesirable side effects). Toxicity and therapeutic effect can be determined by other standard pharmaceutical procedures.

[0189] Data obtained from cell culture assays and animal studies can be used in the formulation of appropriate doses of any given agent for use in subjects (e.g., humans). A therapeutically effective dose of one or more antibodies (e.g., 1, 2, 3, or 4) or their antigen-binding fragments (e.g., any of the antibodies or antibody fragments described herein) is the amount that treats the disease (e.g., kills cancer cells) or reduces the severity, frequency, and / or duration of one or more symptoms of the disease in a subject (e.g., a human subject identified as having cancer) or a subject identified as being at risk of developing the disease (e.g., a subject who has previously had cancer but is now cured), or the amount that reduces the severity, frequency, and / or duration of one or more symptoms of the disease in a subject (e.g., a human). The efficacy and administration of any of the antibodies or antigen-binding fragments described herein can be determined by a healthcare professional or veterinary professional using methods well known in the art, in addition to observing one or more symptoms of the disease in a subject (e.g., a human). Certain factors may influence the dose and timing required to effectively treat the subject (e.g., severity of the disease or disability, previous treatments, the subject's overall health and / or age, and the presence of other diseases).

[0190] Exemplary doses include milligrams or micrograms of either the antibody or antigen-binding fragment described herein per kilogram of the subject's body weight (e.g., approximately 1 mg / kg to approximately 10 mg / kg). While these doses cover a wide range, those skilled in the art will understand that the efficacy and effective dose of therapeutic agents containing antibodies and their antigen-binding fragments can be determined by methods well known in the art. Typically, a relatively low dose is administered first, and the dose can be subsequently and gradually increased by the healthcare professional or veterinary professional (in the case of therapeutic use), or the researcher (if still working in the development stage), until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular subject depends on a variety of factors, including the activity of the specific compound used, the subject's age, body weight, overall health, sex, and diet, administration time, route of administration, excretion rate, and the half-life of the antibody or antibody fragment in the body.

[0191] The pharmaceutical composition may be included in a container, pack, or dispenser, along with instructions for administration. This disclosure also provides methods for producing antibodies or their antigen-binding fragments for various applications described herein. [Examples]

[0192] The present invention will be further illustrated by the following embodiments, which are not intended to limit the scope of the invention as described in the claims.

[0193] Example 1. Generation of anti-IGF1R antibody To generate antibodies against human IGF1R, RenMab mice were immunized with His-tagged human IGF1R protein (ACRO Biosystems Inc., catalog number: IGR-H5229, containing positions 31-932 of SEQ ID NO: 45). The antibody immune response was monitored by antigen-specific immunoassay.

[0194] RenMab mice possess both humanized heavy chain immunoglobulin loci and humanized κ-chain immunoglobulin loci. The heavy chain immunoglobulin loci are chromosomal regions containing genes for the heavy chain of antibodies. These loci include IGHV (variable), IGHD (variability), IGHJ (binding), and heavy chain constant domain genes. The κ-chain immunoglobulin loci are chromosomal regions containing genes encoding the light chain (κ chain) of antibodies. These loci include IGKV (variable), IGKJ (binding), and light chain constant domain genes. A detailed description of RenMab mice can be found in PCT / CN2020 / 075698, which is incorporated herein by reference in its entirety.

[0195] A total of three immunizations were performed. Immunizations were administered at two-week intervals. One week after the last immunization, post-orbital blood was collected, and the antibody titer in the serum was determined by fluorescence-activated cell sorting (FACS). Two weeks later, mice with high titers were selected and subjected to impulse immunization. His-tagged human IGF1R protein or CHO-S cells expressing human IGF1R protein were used for impulse immunization via intraperitoneal injection and tail vein injection, respectively.

[0196] If a desirable immune response was achieved, antigen-specific immune cells were isolated from immunized mice to further obtain anti-IGF1R antibodies or light and heavy chain variable region sequences of anti-IGF1R antibodies. For example, plasma cells secreting antigen-specific monoclonal antibodies were screened and discovered using single-cell technology (e.g., using Beacon® Optofluidic System, Berkeley Lights Inc.), and antibody variable region sequences were obtained using reverse transcription and PCR sequencing. The obtained variable region sequences were cloned into vectors containing sequences encoding the human IgG1 constant region for antibody expression. The binding activity of the expressed antibodies to IGF1R was validated using FACS. Exemplary antibodies obtained included 12A4, 12D1, 10D1, and 10G1.

[0197] The sequences of VH and VL CDR1-3 of 12A4, 12D1, 10D1, and 10G1 are shown in Figure 1 or Figure 2, and their VH and VL regions are shown in Figure 3.

[0198] The constant region can also contain several other mutations. For example, when the YTE mutation (M252Y / S254T / T256E according to EU numbering) is introduced into the Fc region of 12A4, the resulting antibody is named 12A4-YTE. When the QL mutation (T250Q / M428L according to EU numbering) is introduced into the Fc region of 12A4, the resulting antibody is named 12A4-QL. The sequences of the light chain constant region and the heavy chain constant region with mutations are shown in SEQ ID NOs. 50-53, respectively.

[0199] Example 2. Binding activity of anti-IGF1R antibody Both INSR (insulin receptor) and IGF1R are members of the receptor tyrosine kinase (RTK) superfamily. The binding activity of anti-IGF1R antibodies to human IGF1R, human INSR, and monkey IGF1R was investigated by flow cytometry. Briefly, CHO-S-hIGF1R cells (expressing human IGF1R (SEQ ID NO: 45)), CHO-S-fasIGF1R cells (expressing cynomolgus monkey IGF1R (SEQ ID NO: 46)), and CHO-S-hINSR (expressing SEQ ID NO: 47) were placed in 96-well plates in a 2x10⁶ format. 5 Cells were seeded at a density of 10 cells / well. Purified anti-IGF1R antibody at a concentration of 20 μg / mL was added to each well and incubated at 4°C for 30 minutes. Next, after a single wash with PBS, the cells were incubated with the secondary antibody Alexa Fluor® 647 anti-human IgG Fcγ (Jackson ImmunoResearch Laboratories, Inc., catalog number: 109-606-170) at 4°C for 15 minutes, and flow cytometry analysis was performed. Human IgG1 was used as the ISO control. The results are shown in the table below.

[0200] [Table 1]

[0201] Teprotumumab is a human IgG1 monoclonal antibody targeting IGF1R, developed by Genmab in collaboration with River Vision (now Horizon Pharma) for the treatment of orbitophthalmos and scleroderma. The VH and VL sequences of teprotumumab are shown in SEQ ID NOs: 48 and 49, respectively.

[0202] The results showed that all four antibodies, 12A4, 12D1, 10D1, and 10G1, exhibited binding activity to human and monkey IGF1R, but not to human INSR.

[0203] Example 3. Binding affinity of anti-IGF1R antibody Biacore is equipped with a protein A sensor chip pre-immobilized with anti-IGF1R antibodies: His-tagged human IGF1R protein (hIGF1R-His, ACRO Biosystems Inc., catalog number: IGR-H5229) and His-tagged cynomolgus monkey IGF1R protein (cIGF1R-His, ACRO Biosystems Inc., catalog number: IGR-C5225). TM (Biacore, Inc., Piscataway, New Jersey) Measurements were taken using surface plasmon resonance (SPR) with an 8K biosensor.

[0204] Purified anti-IGF1R antibody was captured on a Protein A tip (Series S Sensor Tip Protein A) for detection. 1 μg / mL or 10 μg / mL of purified anti-IGF1R antibody was loaded at 10 μL / min and bound to a gradient concentration of recombinant hIGF1R-His or cIGF1R-His (400 nM, 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM). The flow rate was 30 μL / min, and the binding and dissociation times were set to 180 seconds and 800 seconds or 1200 seconds, respectively. After the final injection of each titration, the tip was refilled with glycine solution (pH 2.0) at 30 μL / min for 30 seconds.

[0205] Biacore TM Using 8K Evaluation software 3.0, the entire dataset is fitted to a 1:1 Langmuir coupled model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994. Methods Enzymology 6.99-110) to determine the motor assembly velocity (k on ) and dissociation rate (k off ) was obtained simultaneously. The affinity value was obtained using the velocity constant (KD=k off / k on This was estimated from the quotient of ).

[0206] As those skilled in the art will understand, the same method, with appropriate adjustments to parameters (e.g., antibody concentration), was performed for each test antibody. The results for each test antibody are summarized in the table below.

[0207] [Table 2]

[0208] The results showed that the anti-IGF1R antibodies 12A4, 12D1, 10D1, and 10G1 exhibited good binding affinity to human IGF1R and monkey IGF1R.

[0209] Example 4. Blocking of the binding of human IGF-1 / 2 to IGF1R. A blockade assay was performed to determine whether the anti-IGF1R antibody could block the binding of IGF1R to its ligands, IGF-1 and IGF-2. Specifically, microwell plates were coated with 100 μl / well of 5 μg / mL hIGF1R-His dissolved in pH 9.6 carbonate buffer and left to stand overnight at 4°C. The plates were blocked with 200 μl / well of 1% bovine serum albumin and incubated at 37°C for 2 hours. Serially diluted anti-IGF1R antibodies (0.005–10 μg / mL + buffer blank, 3-fold serial dilutions) were added to the plates at 100 μL / well. After incubation for 1.5 hours, biotinylated human IGF-I protein (Acro Biosystems, catalog number: IG1-H82F7) or biotinylated human IGF-II protein (Acro Biosystems, catalog number: IG2-H82Q6) was replaced and added to the plate at 100 μL / well. After incubation at 37°C for 1.5 hours, 100 μL / well of 1:3000 dilution of HRP streptavidin (Biolegend, catalog number: 405210) was added to the plate and incubated for 1 hour. The plate was washed 5 times, 100 μL of TMB was added to each well and incubated for 10 minutes. Next, 100 μL of 1 M phosphate was added to each well, and the ELISA plate was read at wavelengths of 450 / 570 nm. A fitting curve was obtained using Log(antibody concentration (M)) on the X axis and chemiluminescence on the Y axis. The IC50 values ​​were calculated, and the results are shown in the table below.

[0210] [Table 3]

[0211] 12A4, 12D1, 10D1, and 10G1 all strongly inhibited the binding of IGF-1 / 2 to IGF1R.

[0212] Example 5. Internalization of IGF1R by anti-IGF1R induction Anti-IGF1R antibody (3 μg / mL) and pHAb-AffiniPure Fab goat anti-human IgG secondary antibody were added to NCI-H838 cells (human lung cells, Procell Life Science & Technology Co., Ltd., catalog number: CL-0406). After incubation for 3 hours, the cells were centrifuged and washed with PBS. MFI was detected by flow cytometry, and EC50 was calculated using serially diluted antibodies.

[0213] The results are shown in Figure 4 and Table 4 below, demonstrating that 12A4, 12D1, 10D1, and 10G1 showed comparable endocytosis rates in NCI-H838 cells compared to positive control teprotumumab analogs.

[0214] [Table 4]

[0215] Example 6. Epitope analysis of anti-IGF1R antibody To determine whether two anti-IGF1R antibodies target the same or overlapping epitopes, Biacor TM An epitope-binding assay was performed using an 8K biosensor. A 1× HBS-EP+ buffer (10 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), 150 mM NaCl, 3 mM ethylenediaminetetraacetic acid (EDTA), and 0.05% P20, pH 7.4), diluted from 10× HBS-EP+ buffer, was used as the running buffer throughout the experiment. Approximately 50 RU of hIGF1R-His was captured at a flow rate of 10 μL / min, and 200 nM test antibody (sample 1) was injected at a flow rate of 30 μL / min to bind the ligand. Another comparison antibody (sample 2) was injected under the same conditions to determine whether the binding of different antibodies interfered with each other. Binding time was 180 seconds per antibody.

[0216] The binding values ​​for each antibody were obtained using Biacore Insight Evaluation (version: 2.0.15.12933). To quantify the interference of one antibody's binding to another, the binding ratio was calculated and each pair of antibodies was compared. The binding ratio was defined by dividing the binding value of the second antibody (sample 2) by the binding value of the first antibody (sample 1). The threshold for determining blockade or non-blockade was 0.3. The results are summarized in the table below, showing that 12A4, 12D1, 10D1, 10G1, and the positive control recognized the same or overlapping epitopes.

[0217] [Table 5]

[0218] Example 7. Inhibition of IGF-1-induced proliferation NCI-H838 cells were seeded at 5,000 cells / well in 96-well plates. After incubation for 24 hours, serially diluted anti-IGF1R antibody (maximum concentration: 10 μg / mL, 5-fold dilution, 8 gradients) was added to each well, and the plates were incubated at 4°C for 1 hour before adding IGF-1 protein. After incubation for 72 hours, the cells were harvested, mixed with CellCounting-Lite 2.0 luminescent cell viability assay (Vazyme, catalog number: DD1101), and tested with a microplate reader. The IC50 was calculated, and the results are shown in the table below.

[0219] [Table 6]

[0220] The results showed that 12A4, 12D1, and 10D1 exhibited near-complete inhibition, with maximum inhibition exceeding 100%.

[0221] Example 8. Inhibition of IGF1R autophosphorylation Experiments were conducted to test whether an anti-IGF1R antibody can inhibit IGF1R phosphorylation. Specifically, CHO-S-hIGF1R cells (2.4 × 10⁶) were used. 6 Cells (10000 cells / mL) were added to a 96-well plate. Anti-IGF1R antibody was added and incubated at 37°C for 15 minutes. Then, IGF-1 protein was added to each well and incubated for 10 minutes. After centrifugation for 5 minutes, the supernatant was removed. 50 μL of lysis buffer (containing protease and phosphatase inhibitors) was added to each well and incubated on ice for 30 minutes. After washing with PBS and centrifugation for 5 minutes, the samples were transferred to EILSA plates. Phosphorylated IGF1R levels were measured using a phosphotyrosine IGF1 receptor ELISA kit (Abcam, catalog number: ab279824) according to the manufacturer's instructions. The mean absorbance of each antibody was measured and the IC50 was calculated.

[0222] The results are shown in Figures 5A and 5B and Table 7 below, demonstrating that 12A4, 12D1, 10D1, and 10G1 exhibited equivalent inhibitory effects on IGF1R phosphorylation (e.g., within 2x IC50) compared to the positive control teprotumumab analog.

[0223] [Table 7]

[0224] Example 9. Inhibition of IGF-1-induced OPN secretion Thyroid eye disease (TED) is characterized by swelling and inflammation of the orbital and periorbital tissues, with a wide range of symptoms. Crosstalk between TSHR and IGF1R has been studied from the perspective of TED pathogenesis. Activation of both TSHR and IGF1R in orbital fibroblasts and fibroblasts leads to their differentiation into adipocytes that can secrete inflammatory cytokines and hyaluronic acid and proliferate. In human osteosarcoma U2OS-TSHR cells (expressing both IGF1R and TSHR), TSHR binds to IGF1R to induce upregulation of genes related to osteoblast activity (e.g., osteopontin (OPN)).

[0225] Experiments were conducted to test whether an anti-IGF1R antibody can inhibit IGF-1-induced OPN secretion. Specifically, U2OS-TSHR cells (3 × 10⁶) were used. 4 U-2OS cells (ATCC, catalog number: HTB-96, endogenously expressing high levels of human IGF-1R) were obtained by transfecting them with a plasmid expressing human TSHR (sequence number 54) at a concentration of 100 cells / mL and added to a 24-well plate. Anti-IGF1R antibody or human IgG1 (as ISO control) was added and incubated at 37°C for 24 hours. Next, IGF-1 protein was added to each well to induce OPN secretion for 24 hours. Cells were harvested and centrifuged for 7 days. OPN secretion levels were measured using the Human Osteopontin (OPN) Quantikine ELISA kit (R&D systems, catalog number: PDOST00) according to the manufacturer's instructions. The ELISA plate was read at a wavelength of 450 nm. A fitting curve was obtained using Log(antibody concentration (M)) on the X axis and human OPN level (ng / mL) on the Y axis. The IC50 values ​​were calculated, and the results are shown in Figure 6 and Table 8 below.

[0226] [Table 8]

[0227] The results showed that 12A4, 12D1, 10D1, and 10G1 all effectively inhibited IGF-1-stimulated OPN secretion. Specifically, 12A4, 10D1, and 10G1 showed comparable inhibitory effects to the positive control teprotumumab analog.

[0228] Example 10. Pharmacokinetic (PK) analysis The pharmacokinetic clearance rate of anti-IGF1R antibody was measured in B-hFcRn mice (Biocytogen, catalog number: 110001). Specifically, mice were placed in different groups (3 mice per group) and administered anti-IGF1R antibody at a dose of 3 mg / kg by intravenous injection. Serum samples were collected 4 days before administration, and 15 minutes, 1 day, 2 days, 5 days, 7 days, 14 days, 21 days, 28 days, 35 days, and 42 days after administration.

[0229] Serum levels of human antibodies were measured using sandwich ELISA. Briefly, goat anti-human IgG(H+L) (Jackson ImmunoResearch Inc., catalog number: 109-005-088) was diluted to a final concentration of 2000 ng / mL, added to a 96-well plate (ELISA plate) at 100 μL / well, and then incubated overnight at 4°C. After incubation, the plate was conditioned in PBS-T buffer (Tween TM The plates were washed four times with PBS supplemented with 20. Regions without antibody binding were blocked with 2% BSA (bovine serum albumin) for 2 hours at 37°C. The plates were then washed four times with PBS-T buffer. After washing, 100 μL of serum sample was added to each well. The wells were sealed and incubated at 37°C for 1 hour. After washing the plates in a plate washer, 100 μL / well of peroxidase AffiniPure F(ab')2 fragment goat anti-human IgG, Fcγ fragment specificity (Jackson ImmunoResearch Inc., catalog number: 109-036-098) was added to each well of the plate and incubated at 37°C for 1 hour. After washing the plates, tetramethylbenzidine (TMB) solution was added as a substrate to a 96-well plate at 100 μL / well. After incubation in the dark at room temperature, 100 μL of stop solution (Beyotime, catalog number: P0215) was added to each well. Using a microplate reader, absorbance values ​​for each well were read at wavelengths of 450 nm and 630 nm. Analysis software: Gen5 TMThe data was analyzed using the absorbance values ​​of the calibration samples prepared with each test product, and the corresponding concentrations, to determine four parameters (i.e., T 1 / 2 , C max AUC 0~42日 A standard curve with , and CL was constructed. The antibody concentration of each serum sample was calculated using the standard curve. A drug concentration-time curve was constructed using the calculated sample concentrations at each time point. Phoenix TM Pharmacokinetic parameters were calculated using WinNolin 8.3. The results are shown in Table 9 below.

[0230] [Table 9]

[0231] Four days prior to antibody administration, the antibody concentration was detected as 0 μg / mL (results not shown). As shown in Figure 7 and Table 9, the results were consistent with typical pharmacokinetic characteristics, showing that the concentration of the antibody in the serum of hFcRn mice decreased over time after injection of the anti-IGF1R antibody. The test antibody had a longer half-life and lower clearance than the positive control teprotumumab analog.

[0232] Example 11. Stability of anti-IGF1R antibody Experiments were conducted to test the stability of anti-IGF1R antibodies. Anti-IGF1R antibodies were converted to a buffer solution (20 mM His, 140 mM Arg-HCl, pH 5.5) using UF / DF, with concentrations exceeding 100 mg / mL. The antibodies were then stored under pressure at 40°C for 4 weeks. Potential molecular variants were identified using SEC-HPLC (TOSOH TSKgel G3000 SWXL, 7.8 mm × 30 cm, 5 μm).

[0233] The results are shown in Table 10 below. No significant decreasing trend was observed in the evaluation of the main peaks, demonstrating that 12A4, 12D1, 10G1, and 10D1 have good stability at high concentrations.

[0234] [Table 10]

[0235] Example 12. ADCC Activity Assay Experiments were conducted to test the ADCC (antibody-dependent cell-mediated cytotoxicity) activity of anti-IGF1R antibodies. Specifically, effector cells FcR-TANK and target cells Du145 were incubated with serially diluted anti-IGF1R antibodies at an E:T ratio of 2:1 for 24 hours, and the supernatant was collected to test for cell toxicity. Lactate dehydrogenase (LDH) activity released from the cytosol of damaged cells was measured by a non-radioactive colorimetric assay using a cytotoxicity detection kit (Roche, catalog number: 04744926001). Antibodies with ADCC activity that targeted unrelated targets were used as reference antibodies. The results are shown in Figure 8, indicating that all tested anti-IGF1R antibodies did not show ADCC effect.

[0236] Furthermore, cytokine INF-γ release levels in the supernatant from the ADCC assay were tested by ELISA. The results are shown in Figure 9. While there was no change in cytokine levels in the 12A4-YTE and 12D1-QL treatment groups, the teprotumumab analog treatment group showed an increase in INF-γ levels, indicating that 12A4-YTE and 12D1-QL have a better safety profile.

[0237] Example 13. HA (Hyaluronic Acid) Release Test TED is a debilitating disorder characterized by the accumulation of adipocytes and hyaluronic acid (HA) in the orbital cavity. HA production by orbital fibroblasts leads to a marked increase in tissue mass and forward movement of the eye. Fibroblasts isolated from the periorbital cavity of TED patients were used to test the effect of anti-IGF1R antibody on HA release. Specifically, fibroblasts were cultured until confluent. Next, the cells were seeded into 96-well plates. The following day, serially diluted anti-IGF1R antibody (5-fold, maximum concentration 50 μg / mL) was added to each well. Human IgG1 was used as an ISO control. Next, IGF-1 was added to each well. After incubation, the supernatant from the plates was tested with a HA ELISA kit to detect HA release. Figure 10 shows that 12A4-YTE and 12D1-QL significantly decreased HA release in a dose-dependent manner, with 12A4-YTE providing the greatest suppression.

[0238] In a similar experiment, fibroblasts were seeded into 96-well plates. The following day, serially diluted anti-IGF1R antibody (3-fold, maximum concentration 2 μg / mL) was added to each well. After incubation with IGF-1, the supernatant from the plates was tested with a HA ELISA kit to detect HA release. As shown in Figure 11, compared to the positive control teprotumumab analog, 12A4-YTE is more effective in reducing HA release levels, indicating great potential for TED therapy.

[0239] Other embodiments Although the invention has been described in conjunction with its detailed description, it should be understood that the above description is for purposes of illustration only and is not intended to limit the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

1. An antibody or antigen-binding fragment thereof that binds to the insulin-like growth factor 1 receptor (IGF1R), A heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH CDR1, the VH CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH CDR2, and the VH CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH CDR3, and A light chain variable region (VL) comprising CDR1, 2, and 3, wherein the VL CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL CDR1, the VL CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL CDR2, and the VL CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL CDR3. The amino acid sequences of the selected VH CDR1, 2, and 3, and the amino acid sequences of the selected VL CDR1, 2, and 3, are one of the following, and are antibodies or antigen-binding fragments thereof. (1) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 1, 2, and 3, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 4, 5, and 6, respectively. (2) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs. 25, 26, and 27, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs. 4, 5, and 6, respectively. (3) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 7, 8, and 9, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 10, 11, and 12, respectively. (4) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 28, 29, and 30, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 10, 11, and 12, respectively. (5) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 13, 14, and 15, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 16, 17, and 18, respectively. (6) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 31, 32, and 33, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 16, 17, and 18, respectively. (7) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 19, 20, and 21, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 22, 23, and 24. (8) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 34, 35, and 36, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 22, 23, and 24, respectively.

2. Based on the definition of Kabat, the antibody or antigen-binding fragment thereof according to claim 1, wherein the VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 4, 5, and 6, respectively.

3. Based on the definition of Kabat, the antibody or antigen-binding fragment thereof according to claim 1, wherein the VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 7, 8, and 9, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 10, 11, and 12, respectively.

4. Based on the definition of Kabat, the antibody or antigen-binding fragment thereof according to claim 1, wherein the VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 13, 14, and 15, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 16, 17, and 18, respectively.

5. Based on the definition of Kabat, the antibody or antigen-binding fragment thereof according to claim 1, wherein the VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 19, 20, and 21, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 22, 23, and 24, respectively.

6. Based on Chothia's definition, the antibody or antigen-binding fragment thereof according to claim 1, wherein VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 25, 26, and 27, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 4, 5, and 6, respectively.

7. Based on the definition of Chothia, the antibody or antigen-binding fragment thereof according to claim 1, wherein VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 28, 29, and 30, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 10, 11, and 12, respectively.

8. Based on the definition of Chothia, the antibody or antigen-binding fragment thereof according to claim 1, wherein VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 31, 32, and 33, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 16, 17, and 18, respectively.

9. Based on Chothia's definition, the antibody or antigen-binding fragment thereof according to claim 1, wherein VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 34, 35, and 36, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 22, 23, and 24, respectively.

10. The antibody or antigen-binding fragment according to any one of claims 1 to 9, wherein the antibody or antigen-binding fragment specifically binds to human IGF1R or monkey IGF1R.

11. The antibody or antigen-binding fragment according to any one of claims 1 to 10, wherein the antibody or antigen-binding fragment is a human antibody or its antigen-binding fragment, or a humanized antibody or its antigen-binding fragment (for example, a human IgG1 antibody or a human IgG4 antibody).

12. The antibody or antigen-binding fragment according to any one of claims 1 to 11, wherein the antibody or antigen-binding fragment is a single-chain variable fragment (scFv) or a multispecific antibody (e.g., a bispecific antibody).

13. A nucleic acid containing a polynucleotide that codes for a polypeptide including the following: (1) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which include the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3, wherein the VH binds to IGF1R when paired with a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO: 38, (2) An immunoglobulin light chain or fragment thereof comprising a VL containing CDR1, 2, and 3, respectively, which contain the amino acid sequences shown in SEQ ID NOs: 4, 5, and 6, wherein the VL binds to IGF1R when paired with a VH containing the amino acid sequence shown in SEQ ID NO: 37, (3) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which include the amino acid sequences shown in SEQ ID NOs. 25, 26, and 27, wherein the VH binds to IGF1R when paired with a light chain variable region (VL) which includes the amino acid sequence shown in SEQ ID NO.

38. (4) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which include the amino acid sequences shown in SEQ ID NOs: 7, 8, and 9, wherein the VH binds to IGF1R when paired with a light chain variable region (VL) which includes the amino acid sequence shown in SEQ ID NO: 40, (5) An immunoglobulin light chain or fragment thereof comprising a VL containing CDR1, 2, and 3, respectively, which contain the amino acid sequences shown in SEQ ID NOs: 10, 11, and 12, wherein the VL binds to IGF1R when paired with a VH containing the amino acid sequence shown in SEQ ID NO: 39, (6) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which include the amino acid sequences shown in SEQ ID NOs: 28, 29, and 30, wherein the VH binds to IGF1R when paired with a light chain variable region (VL) which includes the amino acid sequence shown in SEQ ID NO: 40, (7) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which include the amino acid sequences shown in SEQ ID NOs: 13, 14, and 15, wherein the VH binds to IGF1R when paired with a light chain variable region (VL) which includes the amino acid sequence shown in SEQ ID NO:

42. (8) An immunoglobulin light chain or fragment thereof comprising a VL containing CDR1, 2, and 3, respectively, which contain the amino acid sequences shown in SEQ ID NOs: 16, 17, and 18, wherein the VL binds to IGF1R when paired with a VH containing the amino acid sequence shown in SEQ ID NO: 41, (9) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which include the amino acid sequences shown in SEQ ID NOs: 31, 32, and 33, wherein the VH binds to IGF1R when paired with a light chain variable region (VL) which includes the amino acid sequence shown in SEQ ID NO:

42. (10) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which include the amino acid sequences shown in SEQ ID NOs: 19, 20, and 21, wherein the VH binds to IGF1R when paired with a light chain variable region (VL) which includes the amino acid sequence shown in SEQ ID NO:

44. (11) An immunoglobulin light chain or fragment thereof comprising a VL containing CDR1, 2, and 3, respectively, which contain the amino acid sequences shown in SEQ ID NOs: 22, 23, and 24, wherein the VL binds to IGF1R when paired with a VH containing the amino acid sequence shown in SEQ ID NO: 43, or (12) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which include the amino acid sequences shown in SEQ ID NOs: 34, 35, and 36, wherein the VH binds to IGF1R when paired with a light chain variable region (VL) which includes the amino acid sequence shown in SEQ ID NO:

44.

14. The nucleic acid according to claim 13, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide, and the polypeptide comprises an immunoglobulin heavy chain or fragment thereof, comprising a VH containing CDR1, 2, and 3, which respectively contain the amino acid sequences shown in SEQ ID NOs. 1, 2, and 3, or SEQ ID NOs. 25, 26, and 27.

15. The nucleic acid according to claim 13, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide, and the polypeptide comprises an immunoglobulin light chain or fragment thereof, comprising a VL containing CDR1, 2, and 3, which respectively contain the amino acid sequences shown in SEQ ID NOs: 4, 5, and 6.

16. The nucleic acid according to claim 13, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide, and the polypeptide comprises an immunoglobulin heavy chain or fragment thereof, comprising a VH containing CDR1, 2, and 3, which respectively contain the amino acid sequences shown in SEQ ID NOs. 7, 8, and 9, or SEQ ID NOs. 28, 29, and 30.

17. The nucleic acid according to claim 13, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide, and the polypeptide comprises an immunoglobulin light chain or fragment thereof, comprising a VL containing CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs. 10, 11, and 12, respectively.

18. The nucleic acid according to claim 13, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide, and the polypeptide comprises an immunoglobulin heavy chain or fragment thereof, comprising a VH containing CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs. 13, 14, and 15, or SEQ ID NOs. 31, 32, and 33, respectively.

19. The nucleic acid according to claim 13, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide, and the polypeptide comprises an immunoglobulin light chain or fragment thereof, comprising a VL containing CDR1, 2, and 3, which respectively contain the amino acid sequences shown in SEQ ID NOs. 16, 17, and 18.

20. The nucleic acid according to claim 13, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide, and the polypeptide comprises an immunoglobulin heavy chain or fragment thereof, comprising a VH containing CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs. 19, 20, and 21, or SEQ ID NOs. 34, 35, and 36, respectively.

21. The nucleic acid according to claim 13, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide, and the polypeptide comprises an immunoglobulin light chain or fragment thereof, comprising a VL containing CDR1, 2, and 3, which respectively contain the amino acid sequences shown in SEQ ID NOs. 22, 23, and 24.

22. The nucleic acid according to any one of claims 13 to 21, wherein the VH specifically binds to human IGF1R or monkey IGF1R when pairing with VL, or the VL specifically binds to human IGF1R or monkey IGF1R when pairing with VH.

23. The nucleic acid according to any one of claims 13 to 22, wherein the immunoglobulin heavy chain or fragment thereof is a human immunoglobulin heavy chain or fragment thereof or a humanized immunoglobulin heavy chain or fragment thereof (for example, a human IgG1 heavy chain or fragment thereof), and the immunoglobulin light chain or fragment thereof is a human immunoglobulin light chain or fragment thereof or a humanized immunoglobulin light chain or fragment thereof.

24. The nucleic acid according to any one of claims 13 to 23, wherein the nucleic acid encodes a single-stranded variable fragment (scFv), a multispecific antibody (e.g., a bispecific antibody), or a chimeric antigen receptor (CAR).

25. The nucleic acid according to any one of claims 13 to 24, wherein the nucleic acid is cDNA.

26. A vector comprising one or more nucleic acids as described in any one of claims 13 to 25.

27. A vector comprising two nucleic acids according to any one of claims 13 to 25, wherein the vector encodes the VH region and the VL region that bind together to IGF1R.

28. A pair of vectors, each comprising one of the nucleic acids described in any one of claims 13 to 25, wherein the pair of vectors encode a pair of VH and VL regions that bind together to IGF1R.

29. A cell comprising the vector according to claim 26 or 27, or the pair of vectors according to claim 28.

30. The cell according to claim 29, wherein the cell is a CHO cell.

31. A cell comprising one or more nucleic acids as described in any one of claims 13 to 25.

32. A cell comprising two nucleic acids according to any one of claims 13 to 25.

33. The cell according to claim 32, wherein the two nucleic acids encode a pair of VH and VL regions that bind together to IGF1R.

34. A method for producing an antibody or an antigen-binding fragment thereof, wherein the method is: (a) Culturing the cells according to any one of claims 29 to 33 under conditions sufficient for the cells to produce the antibody or the antigen-binding fragment, (b) Recovering the antibody or antigen-binding fragment produced by the cells, including, method.

35. An antibody that binds to IGF1R or an antigen-binding fragment thereof, An antibody or antigen-binding fragment comprising a heavy chain variable region (VH) containing an amino acid sequence that is at least 80% identical to a selected VH sequence, and a light chain variable region (VL) containing an amino acid sequence that is at least 80% identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following. (1) The selected VH sequence is sequence number 37, and the selected VL sequence is sequence number 38. (2) The selected VH sequence is sequence number 39, and the selected VL sequence is sequence number 40. (3) The selected VH sequence is sequence number 41, the selected VL sequence is sequence number 42, and (4) The selected VH sequence is sequence number 43, and the selected VL sequence is sequence number 44.

36. The antibody or antigen-binding fragment thereof according to claim 35, wherein VH comprises the sequence of SEQ ID NO: 37 and VL comprises the sequence of SEQ ID NO:

38.

37. The antibody or antigen-binding fragment thereof according to claim 35, wherein VH comprises the sequence of SEQ ID NO: 39 and VL comprises the sequence of SEQ ID NO:

40.

38. The antibody or antigen-binding fragment thereof according to claim 35, wherein VH comprises the sequence of SEQ ID NO: 41 and VL comprises the sequence of SEQ ID NO:

42.

39. The antibody or antigen-binding fragment thereof according to claim 35, wherein VH comprises the sequence of SEQ ID NO: 43 and VL comprises the sequence of SEQ ID NO:

44.

40. The antibody or antigen-binding fragment according to any one of claims 35 to 39, wherein the antibody or antigen-binding fragment specifically binds to human IGF1R or monkey IGF1R.

41. The antibody or antigen-binding fragment according to any one of claims 35 to 40, wherein the antibody or antigen-binding fragment is a human antibody or its antigen-binding fragment, or a humanized antibody or its antigen-binding fragment.

42. The antibody or antigen-binding fragment according to any one of claims 35 to 41, wherein the antibody or antigen-binding fragment is a single-chain variable fragment (scFv) or a multispecific antibody (e.g., a bispecific antibody).

43. An antibody or an antigen-binding fragment that cross-competes with the antibody or antigen-binding fragment described in any one of claims 1 to 12 and 35 to 42.

44. An antibody that binds to IGF1R or an antigen-binding fragment thereof, A heavy chain variable region (VH) containing VH CDR1, VH CDR2, and VH CDR3 which are identical to VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence, An antibody or antigen-binding fragment comprising a light chain variable region (VL) containing VL CDR1, VL CDR2, and VL CDR3 which are identical to VL CDR1, VL CDR2, and VL CDR3 of the selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following. (1) The selected VH sequence is sequence number 37, and the selected VL sequence is sequence number 38. (2) The selected VH sequence is sequence number 39, and the selected VL sequence is sequence number 40. (3) The selected VH sequence is sequence number 41, the selected VL sequence is sequence number 42, and (4) The selected VH sequence is sequence number 43, and the selected VL sequence is sequence number 44.

45. An antibody-drug conjugate comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 and 35 to 44, covalently bound to a therapeutic agent.

46. The antibody-drug conjugate according to claim 45, wherein the therapeutic agent is a cytotoxic agent or a cell proliferation inhibitor.

47. A method for treating a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 and 35 to 44, or an antibody-drug conjugate according to claim 45 or 46.

48. The method according to claim 47, wherein the subject has breast cancer, prostate cancer, or lung cancer.

49. The method according to claim 47 or 48, further comprising administering a therapeutically effective dose of an anti-CD3 antibody, an anti-PD1 antibody, an anti-PDL1 antibody, an anti-HER2 antibody, and / or an anti-EGFR antibody to the subject.

50. A method for reducing the rate of tumor growth, wherein the method is The process involves contacting tumor cells with a composition comprising an effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 and 35 to 44, or an antibody-drug conjugate according to claim 45 or 46. method.

51. A method for killing tumor cells, wherein the method is The process involves contacting tumor cells with a composition comprising an effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 and 35 to 44, or an antibody-drug conjugate according to claim 45 or 46. method.

52. A method for treating an eye disease or eye disorder, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 and 35 to 44, or an antibody-drug conjugate according to claim 45 or 46.

53. The method according to claim 52, wherein the subject has thyroid eye disease (TED).

54. A method for treating a subject having a bone disease or bone disorder, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 and 35 to 44, or an antibody-drug conjugate according to claim 45 or 46.

55. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 12 and claims 35 to 44, and a pharmaceutically acceptable carrier.

56. A pharmaceutical composition comprising an antibody-drug conjugate according to claim 45 or 46 and a pharmaceutically acceptable carrier.

57. A chimeric antigen receptor (CAR) comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 12 and 35 to 44.

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