CN117362439A - anti-IGF-1R IgG4 antibodies and their use in treating thyroid-related eye diseases - Google Patents

Abstract

The present disclosure relates to high affinity anti-IGF-1 r IgG4 antibodies and their use in the treatment of thyroid-related eye diseases. The antibody or antigen binding fragment thereof comprises: a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein VH comprises the sequence of SEQ ID NO:3 (CDR) -H1, having the sequence of SEQ ID NO:4 and CDR-H2 of SEQ ID NO: CDR-H3 of 5; and/or the VL comprises a sequence of SEQ ID NO:6, CDR-L1 of SEQ ID NO:7 and CDR-L2 of SEQ ID NO: CDR-L3 of 8; and wherein the antibody is an IgG4 isotype. The antibody of the invention has no extra cell killing effect and is suitable for the treatment of autoimmune diseases such as TED and the like.

Description

anti-IGF-1R IgG4 antibodies and their use in treating thyroid-related eye diseases

The application requires that the application date is 2022, 12 months and 5 days, and the application number is: 202211550458.5 Chinese patent application entitled "anti-IGF-1R IgG4 antibody and its use in treating thyroid-related eye diseases" priority.

Technical Field

The present disclosure relates to high affinity anti-IGF-1 r IgG4 antibodies and their use in the treatment of thyroid-related eye diseases.

Background

Autoimmune diseases are diseases caused by autoimmune tissue injury caused by immune reaction of an organism to autoantigens, such as systemic lupus erythematosus, rheumatoid arthritis, thyroid-related eye diseases (Thyroid Eye Disease, hereinafter abbreviated as 'TED'), and the like, and the pathogenic mechanism is mainly exposure and release of the autoantigens induced by various reasons, and are further recognized by antigen presenting cells (such as dendritic cells, macrophages, and the like) of an immune system, presented to T cells, activated, and the activated T cells and B cells interact, and promote proliferation, activation and secretion of autoantibodies by the B cells. Autoantibodies bind to autoantigens and deposit in tissues (e.g., kidneys, skin, etc.), thereby inducing complement activation cascade, which is the core pathogenesis of autoimmune diseases.

TED is an autoimmune disease involving the orbit and surrounding tissues, with active inflammation, edema, and soft tissue fibrosis in the inactive phase being the main manifestations, most common orbital disease in adults. Studies have shown that TED often occurs with Graves' disease, with an annual incidence of about 16/100000 in females and about 3/100000 in males. According to a cohort study of Bartalena, about 50% of patients with Graves present with ocular symptoms, with a definite TED accounting for about 20-30% and a progression to severe vision impairment (due to compression optic neuropathy or corneal ulcers) of about 3-5%.

The clinical course of TED was first described accurately by rule and Wilson, and was divided into active and inactive phases. The active period can last for 3 years, and inflammation, congestion and edema are used as main manifestations, and common symptoms comprise eyelid retraction (92%), eyeball protrusion (62%), extraocular dyskinesia (43%), orbital pain (30%), lacrimation (23%) and compression optic neuropathy (6%); this is followed by a period of inactivity, which is primarily characterized by fibrosis of the extraocular muscles and intraorbital soft tissues, resulting in reduced coordination of movement of the patient's ocular muscles, limited eye movement, and strabismus. The current decision criteria for TED activity is an international CAS (Clinical Activity Score) score, comprising a total of 7: eyelid red, eyelid swelling, conjunctival red, conjunctival swelling, inflammation of the verruca or folds, auto-onset pain of the orbit, and gaze pain, wherein each term is 1 minute, and the total score is 3 minutes or more, and is determined as active phase.

There are studies showing that the presence of highly expressed IGF-1 (insulin-like growth factor 1) receptor on extraocular myofibroblasts in TED patients, and that the over-expressed thyrotropin receptor (thyroid stimulating hormone receptor, TSHR) and insulin-like growth factor 1receptor (IGF-1R) on orbital fibroblasts in TED patients can form bridged functional complexes that promote further secretion of inflammatory factors and hydrophilic glycosaminoglycans (GAGs) by orbital fibroblasts after activation by GD-IgG, exacerbating ocular inflammation and orbital tissue swelling. In addition, IGF-1R expression levels in T and B cells of TED patients were also significantly higher than normal. IGF-1R is an important autoantigen involved in the development of TED.

Currently, glucocorticoids are commonly used clinically to treat TED, but there are a number of side effects of glucocorticoids and 1/3 of patients respond poorly to this therapy. In addition to glucocorticoids, other biological agents are increasingly being used in TED therapies, such as Rituximab (anti-CD 20), to reduce autoimmune B cell production of autoimmune antibodies, but at the same time to bring about a risk of infection; tocilizumab (anti-IL-6) and Adaliumab (anti-TNF-alfa), while capable of alleviating inflammation in the acute phase, such as pain and oedema, do not alleviate herniation, nor do they alleviate symptoms of restricted extraocular movement.

The anti-IGF-1R antibody can bind IGF-1R, prevent the orbit fibroblast induced by IGF-1 and GD-IgG from releasing inflammatory cytokines, inhibit the orbit fibroblast from further differentiating into adipocytes and GAG secretion, and improve eye tissue lesions such as eyeball protrusion. The prior art, for example WO2004087756, describes that the therapeutic efficacy of anti-IGF-1R antibodies for the treatment of active TED has been well validated and applied clinically, but it is actually found in clinical practice that it risks inducing serious adverse events such as hearing impairment and hyperglycemia. This may be relevant in view of the fact that the development of these published IGF-1R antibodies (WO 2004087756, WO2006069202, WO 2003059951) has focused solely on tumor cell clearance.

For example, teprotumumab-trbwIs a specific IgG 1-type antibody to human IGF1-R, and does not recognize IGF1-R in rodents or other common animal models. Thus, neither the IGF-1 and/or TSH induced mouse thyroid-related eye disease (TAO, thyroid associated ophthalmopathy) model (2002-Animal Models of Thyroid-Associated Ophthalmopathy;2017-Review of Mouse Models of Graves Disease and Orbiopathy) nor the mouse TAO model over-expressing hTSHR (2019-An improved mouse model of Graves disease by once immunization with Ad-TSHR 289) was used for complete efficacy validation experiments with tetumumab-trbw, and even more for evaluation of immune cell involvement during antibody therapy.

Literature reports have shown that teprotumumab-trbw can bind to IGF1-R on orbital fibroblasts of TAO patients and block IGF 1-R-mediated intracellular signaling (2018-IGF-Ireceptor and thyroid-associated ophthalmopathy), blocking proliferation of fibroblasts and remodeling of local tissue structures (remodelling). IGF-1 and TSH may stimulate orbital fibroblasts to secrete inflammatory cytokines, such as IL-6 and IL-8.IL-6 can promote the expression of IL-2R on the surface of T cells, and enhance the mitogenic effect of IL-1 and TNF on Th cells; inducing synthesis of acute phase reaction proteins in acute inflammatory reaction, promoting proliferation and differentiation of B cells and producing antibodies; can also effectively promote TNF and IL-1 induced cachexia. IL-8 is a cytokine belonging to the chemokine family, and its main biological activity is the attraction and activation of neutrophils, which can lead to and exacerbate local inflammatory reactions, and in addition IL-8 has a certain effect on eosinophils, basophils and lymphocytes.

However, there remains a need in the art for therapeutic antibodies for the treatment of thyroid-related eye diseases.

Disclosure of Invention

The present invention is based in part on the following findings of the inventors. The inventors found that in addition to proliferation and remodeling of fibroblasts in the orbital tissue of thyroid-related eye patients, there was also massive immune cell infiltration during the acute inflammatory phase. Antibodies of the prior art, such as teprotumumab-trbw, bind to IGF-1R through an antigen binding region, blocking IGF-1R mediated intracellular signaling and proliferation; while the IgG-1Fc region of teprotumumab-trbw can bind complement, producing complement-dependent cytotoxicity (complement dependent cytotoxicity, CDC); and binding to FcR of T lymphocytes, mediating antibody-dependent cell-mediated cytotoxicity (ADCC), resulting in destruction of orbital fibroblasts, exacerbating the inflammatory response in the orbit part. Thus, the design of therapeutic antibodies for thyroid-related eye diseases requires consideration of both the effect of the antibodies on orbital fibroblasts and the effect of surrounding immune cells. The inventors found that when converting an existing IgG1 antibody such as teprotumumab-trbw for treating thyroid-related eye disease into an IgG4 antibody, the IgG4 antibody is more suitable for treating thyroid-related eye disease, ADCC effect is extremely weak during treatment, and no additional cell killing effect is generated.

The present invention relates to high affinity anti-human insulin-like growth factor 1receptor (Insulin Like Growth Factor. Sup. 1receptor, IGF-1R) IgG4 antibodies and their use in the treatment of thyroid-related eye diseases. The invention also relates to a pharmaceutical combination comprising an anti-IGF-1 r IgG4 antibody and at least one additional therapeutic agent.

In one aspect, the invention provides an antibody or antigen-binding fragment thereof having binding specificity for IGF-1R, wherein the antibody or antigen-binding fragment thereof comprises the following Complementarity Determining Regions (CDRs): a variant of CDR1 or a sequence thereof, a variant of CDR2 or a sequence thereof, and a variant of CDR3 or a sequence thereof contained in the heavy chain variable region (VH) shown in SEQ ID NO. 11; and/or

A variant of CDR1 or a sequence thereof, a variant of CDR2 or a sequence thereof, and a variant of CDR3 or a sequence thereof contained in the light chain variable region (VL) shown in SEQ ID NO. 12; wherein the variant of the sequence is a CDR having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) as compared to the CDR from which it was derived.

In one embodiment, the substitution is a conservative substitution.

In one embodiment, the CDRs are defined according to the IMGT numbering system.

In one embodiment, the antibody is an IgG4 isotype.

In one aspect, the invention provides an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises:

heavy chain variable region (VH) and/or light chain variable region (VL), wherein defined by the IMGT numbering system:

the VH comprises a sequence comprising SEQ ID NO:3, comprising the sequence SEQ ID NO:4 and a CDR2 comprising the sequence SEQ ID NO: CDR3 of 5; and/or the number of the groups of groups,

the VL comprises a sequence comprising SEQ ID NO:6, CDR-L1 comprising the sequence SEQ ID NO:7 and a CDR-L2 comprising the sequence SEQ ID NO: CDR3 of 8.

In one aspect, the invention provides an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising the sequence set forth in SEQ ID NO. 11, and/or a light chain variable region comprising the sequence set forth in SEQ ID NO. 12; and the antibody is of the IgG4 isotype.

In one aspect, the invention provides an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain comprising the sequence shown in SEQ ID NO. 1, and/or a light chain comprising the sequence shown in SEQ ID NO. 2; and the antibody is of the IgG4 isotype.

In one aspect, the invention provides an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is a murine antibody, chimeric antibody, or humanized antibody.

In one aspect, the invention provides an antibody or antigen binding fragment thereof, wherein the antibody or antigen binding fragment thereof has less Fc-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) effect than the IgG1 isotype.

In another aspect, the invention also provides an isolated nucleic acid molecule encoding an antibody or antigen binding fragment thereof, a heavy and/or light chain thereof, or a heavy and/or light chain variable region thereof as described herein.

In another aspect, the invention also provides a vector comprising an isolated nucleic acid molecule as described herein. In one embodiment, the vector is a cloning vector or an expression vector.

In another aspect, the invention also provides a host cell comprising an isolated nucleic acid molecule as described herein or a vector as described herein.

In another aspect, the invention also provides a method of making an antibody or antigen-binding fragment thereof described herein, comprising culturing a host cell described herein under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

In another aspect, the invention also provides a multispecific antibody comprising a first antibody, or fragment thereof, and a further antibody, or fragment thereof, or antibody analog; wherein the first antibody or fragment thereof is an antibody or antigen-binding fragment thereof that specifically binds IGF-1R as described herein. In one embodiment, the multispecific antibody is a bispecific antibody or a trispecific antibody or a tetraspecific antibody.

In another aspect, the invention also provides a conjugate comprising an antibody or antigen-binding fragment thereof and a coupling moiety, wherein the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof described herein and the coupling moiety is a detectable label.

In one embodiment, the detectable label may be selected from the group consisting of a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.

In one embodiment, the coupling moiety may be a pharmaceutical, such as selected from a chemotherapeutic agent, radionuclide, or toxin.

In another aspect, the invention also provides a cell expressing a Chimeric Antigen Receptor (CAR) comprising or expressing an antibody or antigen binding fragment thereof as described herein, a nucleic acid as described herein or a vector as described herein.

In one embodiment, the cells may be derived from immune cells. The antigen binding fragment may be selected from scFv.

In one embodiment, the cells may be derived from T lymphocytes, NK cells, monocytes, macrophages or dendritic cells, and any combination thereof.

In another aspect, the invention also provides an oncolytic virus comprising a nucleic acid as described herein or a vector as described herein.

In another aspect, the invention also provides a fusion protein comprising an antibody or antigen-binding fragment thereof and a non-immunoglobulin moiety, wherein the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof as described herein. In one embodiment, the non-immunoglobulin moiety may be a polypeptide. In one embodiment, the polypeptide is a cytokine. In one embodiment, the cytokine is selected from the group consisting of IL-2, IL-15, IL-7, IL-12, IL-18, IL-21, TNF-alpha, and IFN gamma.

In another aspect, the invention also provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described herein, an isolated nucleic acid molecule as described herein, a vector as described herein, a host cell as described herein, a multispecific antibody as described herein, a conjugate as described herein, a CAR cell as described herein, an oncolytic virus as described herein, and/or a fusion protein as described herein. The pharmaceutical composition may comprise one or more thyroid stimulating hormone receptor TSHR inhibitors. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier and/or excipient.

In a further aspect, the invention provides the use of an anti-IGF-1R antibody of the IgG4 type in combination with one or more thyroid stimulating hormone receptor TSHR inhibitors for the treatment of thyroid eye disease.

In a further aspect, the invention also provides the use of an anti-IGF-1R antibody of the IgG4 type and one or more thyroid stimulating hormone receptor TSHR inhibitors in the manufacture of a medicament for the treatment of thyroid-related eye diseases.

In another aspect, the invention also provides a combination of an anti-IGF-1R antibody of the IgG4 type and one or more thyroid stimulating hormone receptor TSHR inhibitors for use in the treatment of thyroid-related eye diseases.

In embodiments of aspects, the anti-IGF-1R antibody of IgG4 type is an antibody described herein or an antigen-binding fragment thereof.

In embodiments of the aspects, the thyroid stimulating hormone receptor TSHR inhibitor comprises a small molecule inhibitor and/or an antibody to TSHR.

In yet another aspect, the invention provides the use of an antibody or antigen binding fragment thereof as described herein, an isolated nucleic acid molecule as described herein, a vector as described herein, a host cell as described herein, a multispecific antibody as described herein, a conjugate as described herein, a CAR cell as described herein, an oncolytic virus as described herein, a fusion protein as described herein, a pharmaceutical composition as described herein, in the manufacture of a medicament for the prevention and/or treatment of thyroid-associated ocular disease.

In yet another aspect, the invention provides an antibody or antigen binding fragment thereof as described herein, an isolated nucleic acid molecule as described herein, a vector as described herein, a host cell as described herein, a multispecific antibody as described herein, a conjugate as described herein, a CAR cell as described herein, an oncolytic virus as described herein, a fusion protein as described herein, a pharmaceutical composition as described herein for use in preventing and/or treating a thyroid-associated ocular disease.

In yet another aspect, the invention provides a method for preventing and/or treating thyroid-related eye disease in a subject, the method comprising administering to a subject in need thereof an antibody or antigen-binding fragment thereof as described herein, an isolated nucleic acid molecule as described herein, a vector as described herein, a host cell as described herein, a multispecific antibody as described herein, a conjugate as described herein, a CAR cell as described herein, an oncolytic virus as described herein, a fusion protein as described herein, a pharmaceutical composition as described herein. In one embodiment, the subject is a mammal. In one embodiment, the subject is a human. In one embodiment, the subject contains a thyroid-related eye disease.

In embodiments herein, the thyroid-related eye disease is selected from the group consisting of herniation, photophobia, lacrimation, pain, redness of the eye, swelling of the eye, dyskinesia, presbyopia, eyelid insufficiency, vision loss, and blindness induced by compression of the optic nerve.

Advantageous effects of the invention

Compared with the prior art, the antibody or the fragment thereof has the following beneficial effects:

(1) The antibody of the invention not only can specifically recognize/bind IGF-1R and block the downstream signal path, but also can be used for preventing or treating thyroid-related eye diseases.

(2) The IgG4 antibody targeting human IGF-1R provided by the invention has extremely weak ADCC effect and no extra cell killing effect, and is more suitable for the treatment of autoimmune diseases such as TED. Thus, the antibodies of the invention (particularly humanized antibodies) are of great clinical value.

Drawings

FIG. 1 shows the identification of anti-human IGF-1R mab 6 (IgG 4) and anti-human IGF-1R mab 6 (IgG 1) proteins by electrophoresis.

FIG. 2 affinity assay results for human IGF-1R for antibody 6 (IgG 4) and antibody 6 (IgG 1).

FIG. 3. Antibody 6 (IgG 4) and antibody 6 (IgG 1) compete for binding to the antigenic site of human IGF-1R.

FIG. 4 effects of antibody 6 (IgG 4) and antibody 6 (IgG 1) on IGF-1/IGF-1R mediated proliferation of FDC-P1 cells.

FIG. 5A comparison of ADCC effect of antibody 6 (IgG 4) and antibody 6 (IgG 1).

Fig. 6.The cell culture chamber co-cultures human orbital fibroblasts and PBMC immune cells, mimicking physiological conditions during disease treatment. An upper cell for PBMC culture; a lower chamber for performing the wall culture of the orbital fibroblasts; the cells are separated by a Polycarbonate (PC) film.

Fig. 7. Secretion of cytokines by human orbital fibroblasts under stimulation of TSH and IGF-1.

Fig. 8.Effects of PMBC and anti-human IGF-1R antibodies of different Fc subtypes on human orbital fibroblasts secreting TGF- β in cell culture chamber models.

Fig. 9.In the cell culture chamber model, TSH and IGF-1 are involved in the secretion of human orbital fibroblast TGF-beta and are affected by PMBC and anti-human IGF-1R antibodies of different Fc subtypes.

Figure 10 human orbital fibroblasts have IGF-1R expression.

Fig. 11.In the cell culture chamber model, the effect of anti-human IGF-1R antibodies on human orbital fibroblast proliferation.

Fig. 12.In cell culture chamber model, the effect of anti-human IGF-1R antibodies on human orbital fibroblast apoptosis.

Detailed Description

Definition of terms

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Moreover, the procedures of cell culture, biochemistry, nucleic acid chemistry, immunology laboratories and the like as used herein are all conventional procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

Chinese grammar has no single-plural rules, and nouns as used herein refer to both singular and plural.

As used herein, the term "antibody" refers to an immunoglobulin molecule that is typically composed of two pairs of polypeptide chains, each pair having one Light Chain (LC) and one Heavy Chain (HC). Antibody light chains can be classified as kappa (kappa) and lambda (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotypes of antibodies are defined as IgM, igD, igG, igA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also comprises a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH 1, CH2 and CH 3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant domains are not directly involved in binding of antibodies to antigens, but exhibit a variety of effector functions, such as may mediate binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). VH and VL regions can also be subdivided into regions of high variability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FR). Each VH and VL is prepared from the following sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 consist of 3 CDRs and 4 FRs arranged from amino-terminus to carboxy-terminus. The variable regions (VH and VL) of each heavy/light chain pair form antigen binding sites, respectively. The assignment of amino acids to regions or domains can be carried out by Kabat, sequences of Proteins of Immunological Interest (National Institutes of Health, bethesda, md. (1987 and 1991)), or Chothia & Lesk (1987) J.mol.biol.196:901-917; chothia et al (1989) Nature 342:878-883.

As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in an antibody variable region that are responsible for antigen binding. The exact boundaries of these amino acid residues may be defined according to various numbering systems known in the art, for example according to the Kabat numbering system (Kabat et al, sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md, 1991), the Chothia numbering system (Chothia & Lesk (1987) J.mol. Biol.196:901-917; chothia et al (1989) Nature 342:878-883) or the IMGT numbering system (Lefranc et al, dev. Company. Immunol.27:55-77,2003). For a given antibody, one skilled in the art will readily identify the CDRs defined by each numbering system. Also, the correspondence between the different numbering systems is well known to those skilled in the art (see, e.g., lefranc et al, dev. Comparat. Immunol.27:55-77,2003). In this context, the allocation of amino acids in each region or domain may also be defined according to the IMGT numbering system.

In the present invention, the CDRs contained in the antibodies or antigen binding fragments thereof of the present invention can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained in an antibody or antigen binding fragment thereof of the invention are preferably determined by the Kabat or IMGT numbering system.

As used herein, the term "framework region" or "FR" residues refer to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

As used herein, the term "germline antibody gene" is an immunoglobulin sequence encoded by a non-lymphocyte that does not undergo a maturation process that results in the genetic rearrangement and maturation of the expressed specific immunoglobulin. One advantage provided by the various embodiments of the present invention derives from the recognition that germline antibody genes retain more of the important amino acid sequence structure characteristic of an individual animal species than mature antibody genes. And thus less recognized by the species as foreign when applied therapeutically to the species.

The term "antibody" is not limited by any particular method of producing an antibody. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibodies may be of different isotypes, for example, igG (e.g., igG1, igG2, igG3, or IgG4 subclasses), igA1, igA2, igD, igE, or IgM antibodies.

As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide fragment of an antibody, e.g., a polypeptide fragment of a full-length antibody, that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody for specific binding to an antigen, also referred to as an "antigen-binding portion. Non-limiting examples of antigen binding fragments that can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies include Fab, fab ', F (ab') 2, fd, fv, dAb and Complementarity Determining Region (CDR) fragments, single chain antibodies (e.g., scFv), chimeric antibodies, diabodies, linear antibodies (e.g., from Ablynx), nanobodies (e.g., from Ablynx), domain antibodies (e.g., from Domains), and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen binding capacity to the polypeptide.

As used herein, the term "full length antibody" means an antibody consisting of two "full length heavy chains" and two "full length light chains". Wherein "full length heavy chain" refers to a polypeptide chain consisting of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a Hinge Region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the N-to C-terminal direction; and, when the full length antibody is an IgE isotype, optionally further comprises a heavy chain constant region CH4 domain. Preferably, a "full length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2 and CH3 in the N-to C-terminal direction. A "full length light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-to C-terminal direction. The two pairs of full length antibody chains are linked together by a disulfide bond between CL and CH1 and a disulfide bond between HR of the two full length heavy chains. The full length antibodies of the invention may be from a single species, e.g., human; chimeric or humanized antibodies are also possible. The full length antibodies of the invention comprise two antigen binding sites formed by VH and VL pairs, respectively, which specifically recognize/bind the same antigen.

As used herein, the term "Fd fragment" means an antibody fragment consisting of VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of a VH domain (Ward et al Nature 341:544 546 (1989)); the term "Fab fragment" means an antibody fragment consisting of VL, VH, CL and CH1 domains; the term "F (ab') 2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab 'fragment" means the fragment obtained after reduction of the disulfide bond joining the two heavy chain fragments of the F (ab') 2 fragment, consisting of one complete light and heavy chain Fd fragment (consisting of VH and CH1 domains).

As used herein, the term "Fv fragment" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. Fv fragments are generally considered to be the smallest antibody fragment that forms the complete antigen binding site. It is believed that six CDRs confer antigen binding specificity to the antibody. However, even one variable region (e.g., fd fragment, which contains only three CDRs specific for an antigen) is able to recognize and bind antigen, although its affinity may be lower than the complete binding site.

As used herein, the term "Fc fragment" means an antibody fragment formed by disulfide bonding of the second and third constant regions of a first heavy chain of an antibody with the second and third constant regions of a second heavy chain. The Fc fragment of an antibody has a number of different functions, but does not participate in antigen binding.

As used herein, the term "scFv" refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH domains are linked by a linker (linker) (see, e.g., bird et al, science 242:423-426 (1988); huston et al, proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckaphun, the Pharmacology of Monoclonal Antibodies, volume 113, roseburg and Moore, springer-Verlag, new York, pages 269-315 (1994)). Such scFv molecules may have the general structure: NH 2-VL-linker-VH-COOH or NH 2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) 4 may be used, but variants thereof may also be used (Holliger et al (1993), proc.Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present invention are described by Alfthan et al (1995), protein Eng.8:725-731, choi et al (2001), eur.J.Immunol.31:94-106, hu et al (1996), cancer Res.56:3055-3061, kipriyanov et al (1999), J.mol.biol.293:41-56 and Roovers et al (2001), cancer Immunol. In some cases, disulfide bonds may also exist between VH and VL of scFv. As used herein, the term "di-scFv" refers to an antibody fragment formed by the ligation of two scFv.

As used herein, the term "diabody" means that its VH and VL domains are expressed on a single polypeptide chain, but uses a linker that is too short to allow pairing between two domains of the same chain, forcing the domains to pair with complementary domains of the other chain and creating two antigen binding sites (see, e.g., holliger p. Et al, proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak R.J. Et al, structures 2:1121-1123 (1994)).

As used herein, "antibody analog" refers to an antigen that binds as specifically as an antibody, but without an antibody structure. They are generally artificial peptides or proteins, with a molar mass of about 3 to 20kDa. For example, ankyrin repeat protein (DARPin) and fynomer. The engineered ankyrin repeat protein (DARPin) may be linked to an IgG antibody, scFv-Fc antibody fragment, or a combination thereof, such as CN104341529a. Fynomer against IL-17a binds to an anti-IL-6R antibody, e.g., WO2015141862A1.

Each of the above antibody fragments retains the ability to specifically bind to the same antigen to which the full-length antibody binds and/or competes with the full-length antibody for specific binding to the antigen.

Antigen-binding fragments of antibodies (e.g., the antibody fragments described above) can be obtained from a given antibody (e.g., an antibody provided by the invention) using conventional techniques known to those of skill in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and specifically screened for antigen-binding fragments in the same manner as used for intact antibodies.

In this context, unless the context clearly indicates otherwise, when referring to the term "antibody" it includes not only whole antibodies, but also antigen-binding fragments of antibodies.

As used herein, the terms "monoclonal antibody," "mAb," and "mAb" have the same meaning and are used interchangeably to refer to an antibody or a fragment of an antibody from a population of highly homologous antibody molecules, i.e., a population of identical antibody molecules except for natural mutations that may occur spontaneously. Monoclonal antibodies have a high specificity for a single epitope on an antigen. Polyclonal antibodies are relative to monoclonal antibodies, which typically comprise at least 2 or more different antibodies, which typically recognize different epitopes on an antigen. Furthermore, the modifier "monoclonal" merely indicates the character of the antibody as being obtained from a population of highly homologous antibodies, and is not to be construed as requiring preparation of the antibody by any particular method.

Monoclonal antibodies of the invention may be prepared by a variety of techniques, such as hybridoma techniques (see, e.g., kohler et al Nature,256:495, 1975), recombinant DNA techniques (see, e.g., U.S. patent application 4,816,567), or phage antibody library techniques (see, e.g., clackson et al Nature352:624-628,1991, or Marks et al J.mol.biol.222:581-597, 1991).

For example, monoclonal antibodies can be prepared as follows. Mice or other suitable host animals are first immunized with the immunogen (with adjuvant if necessary). Upon immunization, the animals will produce lymphocytes in vivo that secrete antibodies that specifically bind to the immunogen. Alternatively, lymphocytes can be obtained by in vitro immunization. Lymphocytes of interest are collected and fused with myeloma cells using a suitable fusion agent, such as PEG, to obtain hybridoma cells (Goding, monoclonal Antibodies: principles and Practice, pp.59-103,Academic Press,1996). The hybridoma cells prepared as described above may be inoculated into a suitable culture medium for growth, and the culture medium for growing the hybridoma cells is used for detecting the production of the monoclonal antibody against the specific antigen. Methods for determining the binding specificity of monoclonal antibodies produced by hybridoma cells include, for example, immunoprecipitation or in vitro binding assays, such as Radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA). For example, the affinity of monoclonal antibodies can be determined using the Scatchard assay described by Munson et al, anal biochem.107:220 (1980). After determining the specificity, affinity and reactivity of the antibodies produced by the hybridomas, the cell lines of interest can be subcloned by standard limiting dilution methods as described (Goding, monoclonal Antibodies: principles and Practice, pp.59-103,Academic Press,1996). Suitable culture media may be DMEM or RPMI-1640, and the like. In addition, hybridoma cells can also be grown in animals as ascites tumors. Monoclonal antibodies secreted by subcloned cells can be separated from cell culture fluid, ascites fluid or serum by conventional immunoglobulin purification methods, such as protein a sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

Monoclonal antibodies can also be obtained by genetic engineering recombinant techniques. DNA molecules encoding the heavy and light chain genes of the monoclonal antibodies can be isolated from hybridoma cells by PCR amplification using nucleic acid primers that specifically bind the heavy and light chain genes of the monoclonal antibodies. The resulting DNA molecule is inserted into an expression vector, and then a host cell (e.g., an E.coli cell, COS cell, CHO cell, or other myeloma cell that does not produce immunoglobulin) is transfected and cultured under appropriate conditions to obtain a recombinantly expressed antibody of interest.

Antibodies can be purified by well-known techniques, such as affinity chromatography using protein a or protein G. Subsequently or alternatively, a specific antigen (the target molecule recognized by the antibody) or an epitope thereof may be immobilized on the column, and the immunospecific antibody may be purified by immunoaffinity chromatography. Purification of immunoglobulins can be referred to, for example, by D.Wilkinson (The scientific, inc., philadelphia Pa., vol.14, no.8 (Apr.17, 2000), pp.25-28).

As used herein "% identity" between two sequences refers to a function of the number of identical positions shared by the sequences (i.e.,% homology = number of identical positions/number of total positions x 100), wherein the gaps need to be introduced when performing optimal alignment of the two sequences, taking into account the number of gaps and the length of each gap. Sequence comparison and determination of% identity between two sequences can be accomplished using mathematical algorithms. For example, the% identity between two amino acid sequences can be determined using the algorithm of E.Meyers and W.Miller (Comput. Appl. Biosci., 4:11-17 (1988)).

As used herein, sequences having "% identity" retain important biological activity, such as antibody binding specificity, of the sequences to which they are compared or from which they are derived. Sequences having one or several amino acid substitutions, deletions or additions, or any combination thereof, retain important biological activity, such as antibody binding specificity, of the sequences to which they are compared or from which they are derived. Nucleotide sequences having "% identity" or nucleotide sequences differing by no more than 3, 6, 15, 30 or 45 nucleotides are capable of performing a function similar to the nucleotide sequence from which they are compared or derived, e.g., the expressed protein is capable of specifically binding to the same antigen or molecule.

As used herein, the term "chimeric antibody" refers to an antibody in which a portion of the light chain or/and heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass) and another portion of the light chain or/and heavy chain is derived from another antibody (which may be derived from the same or a different species or belong to the same or a different antibody class or subclass), but which retains binding activity for the antigen of interest in any event (u.s.p4, 816,567; morrison et al, proc.Natl. Acad.Sci.USA,81:6851 6855 (1984)).

As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence is modified to increase homology with the sequence of a human antibody. Typically, all or part of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody) and all or part of the non-CDR regions (e.g., variable region FR and/or constant regions) are derived from a human immunoglobulin (acceptor antibody). Humanized antibodies generally retain the desired properties of the donor antibody including, but not limited to, antigen specificity, affinity, reactivity, ability to enhance immune cell activity, ability to enhance immune responses, and the like. The donor antibody can be a mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibody having the desired properties (e.g., antigen specificity, affinity, reactivity, ability to enhance immune cell activity, and/or ability to enhance an immune response).

Humanized antibodies are particularly advantageous because they are capable of retaining the desired properties of non-human donor antibodies (e.g., murine antibodies) and are also capable of effectively reducing the immunogenicity of non-human donor antibodies (e.g., murine antibodies) in human subjects. However, due to matching problems between CDRs of a donor antibody and FRs of a recipient antibody, the desired properties of a humanized antibody (e.g., antigen specificity, affinity, reactivity, ability to enhance immune cell activity, and/or ability to enhance immune response) are typically lower than non-human donor antibodies (e.g., murine antibodies).

Thus, although researchers in the field have developed intensive studies on the humanization of antibodies and made some progress (see, e.g., jones et al, nature,321:522 525 (1986), reichmann et al, nature,332:323-329 (1988), presta, curr. Op. Structure. Biol.,2:593-596 (1992), clark, immunol. Today 21:397 402 (2000)), how to adequately humanize a donor antibody so that the resulting humanized antibody has as high a degree of humanization as possible while retaining as much of the intended properties of the donor antibody, the prior art does not provide exhaustive guidance. The skilled artisan needs to fumbly, explore and engineer specific donor antibodies, with the great effort of inventiveness being possible to obtain, humanized antibodies that have both a high degree of humanization (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% degree of humanization) and retain the desired properties of the specific donor antibody.

In the present invention, in order for the humanized antibody to retain the properties of the donor antibody (including, for example, antigen specificity, affinity, reactivity, ability to enhance immune cell activity, and/or ability to enhance immune response) as much as possible, the Framework Region (FR) in the humanized antibody of the present invention may contain both amino acid residues of the human acceptor antibody and amino acid residues of the corresponding non-human donor antibody.

In this application, the expected properties of the antibodies of the invention include: (1) Specifically recognizing/binding IGF-1R (particularly human IGF-1R); (2) inhibit and/or block binding of IGF-1 to IGF-1R; (3) Inhibit and/or block intracellular signaling mediated by IGF-1 binding IGF-1R; (4) Preventing IGF-1 induced orbital fibroblasts from releasing inflammatory cytokines, inhibiting the orbital fibroblasts from further differentiating into adipocytes and GAG secretion, and improving ocular tissue lesions such as eyeball protrusion; (5) ADCC is reduced or eliminated and local inflammatory response of the orbit is reduced compared to existing anti-IGF-1R IgG1 type antibodies. The humanized antibodies of the invention retain one or more of the above-described desirable properties of the parent antibody (murine antibody or murine-human chimeric antibody).

The chimeric or humanized antibody of the present invention can be prepared according to the sequence of the murine monoclonal antibody prepared as described above. DNA encoding the heavy and light chains can be obtained from a murine hybridoma of interest and engineered to contain non-murine (e.g., human) immunoglobulin sequences using standard molecular biology techniques.

To prepare chimeric antibodies, the murine immunoglobulin variable region can be linked to a human immunoglobulin constant region using methods known in the art (see, e.g., U.S. Pat. No.4,816,567 to Capilli et al). For example, DNA encoding VH is operably linked to another DNA molecule encoding a heavy chain constant region to obtain a full length heavy chain gene, or DNA encoding VL is operably linked to another DNA molecule encoding a light chain constant region CL to obtain a full length light chain gene (as well as Fab light chain gene). The sequences of human heavy and light chain constant region genes are known in the art (see, e.g., kabat, E.A. et al (1991) Sequences of Proteins of Immunological Interest, fifth edition, U.S. device of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments comprising these regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgG1 (e.g., uniprot ID P01857), igG2 (e.g., uniprot ID P01859), igG3 (e.g., uniprot ID P01860), igG4 (e.g., uniprot ID P01861), igA, igE, igM, or IgD constant region, but generally an IgG1 or IgG4 constant region is preferred. The light chain constant region may be a kappa or lambda constant region, but is generally preferred.

To prepare humanized antibodies, murine CDR regions can be inserted into a human framework sequence using methods known in the art (see U.S. Pat. No.5,225,539 to Winter; U.S. Pat. No.5,530,101 to Queen et al; U.S. Pat. Nos.5,585,089; 5,693,762 and 6,180,370; and Lo, benny, K.C., antibody Engineering: methods and Protocols, volume 248,Humana Press,New Jersey,2004). Alternatively, transgenic animals can also be utilized that are capable of producing no endogenous immunoglobulins upon immunization, and that are capable of producing a complete human antibody repertoire (see, e.g., jakobovits et al, 1993,Proc.Natl.Acad.Sci.USA 90:2551;Jakobovits et al, 1993,Nature 362:255-258; bruggermann et al, 1993,Year in Immunology 7:33; and Duchoal et al, 1992,Nature 355:258;Lonberg et al (1994) Nature 368 (6474): 856-859; WO 02/43478). Other methods of antibody humanization include phage display techniques (Hoogenboom et al, 1991, J. Mol. Biol.227:381; marks et al, J. Mol. Biol.1991, 222:581-597; vaughan et al, 1996,Nature Biotech 14:309).

As used herein, the term "degree of humanization" is an indicator for evaluating the number of non-human amino acid residues in a humanized antibody. The degree of humanization of a humanized antibody can be predicted, for example, by the IMGT website DomainGapAlign, to predict the homology of the variable region sequence to the human V domain.

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed. The strength or affinity of a specific binding interaction can be expressed in terms of the equilibrium dissociation constant (KD) of the interaction. In the present invention, the term "KD" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and antigen. In certain embodiments, an antibody that specifically binds to (or has specificity for) an antigen means that the antibody binds to or binds to an antigen in an amount of less than about 10 ^-9 M, e.g. less than about 10 ^-9 M、10 ^-10 M、10 ^-11 M or 10 ^-12 M or less affinity (KD) binds the antigen. In certain embodiments, when KD is less than or equal to 10X 10 ^-8 M (preferably KD.ltoreq.5X10) ^-9 M), the antibodies or antigen binding fragments thereof of the invention are believed to specifically bind PD-1.

The specific binding properties between two molecules can be determined using methods well known in the art. One method involves measuring the rate of antigen binding site/antigen complex formation and dissociation. "binding Rate constant" (k) _a Or k _on ) And "dissociation rate constant" (k) _dis Or k _off ) Both can be calculated from the concentration and the actual rate of association and dissociation (see Malmqvist M, nature,1993, 361:186-187). The kdis/kon ratio is equal to the dissociation constant KD (see Davies et al, annual Rev Biochem,1990; 59:439-473). KD, kon and k can be measured by any effective methoddis value. In certain embodiments, the dissociation constant may be measured using bioluminescence interferometry (e.g., forteBio oct method). In addition to this, the dissociation constants can be measured using surface plasmon resonance techniques (e.g., biacore) or Kineca.

As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes, such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC), or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, papilloma vacuolation virus (e.g., SV 40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin.

As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell such as e.g. escherichia coli or bacillus subtilis, a fungal cell such as e.g. yeast cells or aspergillus, an insect cell such as e.g. S2 drosophila cells or Sf9, or an animal cell such as e.g. fibroblasts, CHO cells, COS cells, NSO cells, heLa cells, BHK cells, HEK 293 cells or human cells.

As used herein, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matched positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 out of 10 positions of two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions in total are matched). Typically, the comparison is made when two sequences are aligned to produce maximum identity. Such alignment may be conveniently performed using, for example, a computer program such as the Align program (DNAstar, inc.) Needleman et al (1970) j.mol.biol.48: 443-453. Furthermore, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J MoI biol.48:444-453 (1970)) algorithm that has been incorporated into the GAP program of the GCG software package (available on www.gcg.com), using the Blossum62 matrix or PAM250 matrix, and GAP weights (GAP weights) of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the desired properties of a protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions may be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions that replace an amino acid residue with an amino acid residue having a similar side chain, such as substitutions with residues that are physically or functionally similar (e.g., of similar size, shape, charge, chemical nature, including the ability to form covalent or hydrogen bonds, etc.) to the corresponding amino acid residue. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of amino acids are well known in the art (see, e.g., brummell et al, biochem.32:1180-1187 (1993); kobayashi et al Protein Eng.12 (10): 879-884 (1999); and Burks et al Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

The twenty conventional amino acids referred to herein are written following conventional usage. See, e.g., immunology-a Synthesis (second edition, e.s. golub and d.r. gren, sinauer Associates, sunderland, mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally indicated by single-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and active ingredient, which is well known in the art (see, e.g., remington's Pharmaceutical sciences, gennaro AR, 19 th edition pennsylvania: mack Publishing Company, 1995), and includes, but is not limited to: pH modifiers, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugar, naCl, and the like. Agents that delay absorption include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (e.g., buffered saline), alcohols and polyols (e.g., glycerol), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like. Stabilizers have the meaning commonly understood by those skilled in the art and are capable of stabilizing the desired activity of the active ingredient in a medicament, including but not limited to sodium glutamate, gelatin, SPGA, saccharides (e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin or casein) or degradation products thereof (e.g., lactalbumin hydrolysate), and the like.

As used herein, the term "preventing" refers to a method performed to prevent or delay the occurrence of a disease or disorder or symptom (e.g., a tumor, infection, or autoimmune disease) in a subject. As used herein, the term "treatment" refers to a method that is performed in order to obtain beneficial or desired clinical results. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., no longer worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and diminishment of symptoms (whether partial or total), whether detectable or undetectable. Furthermore, "treatment" may also refer to an extension of survival compared to the expected survival (if not treated).

As used herein, the term "subject" refers to a mammal, such as a primate mammal, e.g., a human. In certain embodiments, the subject (e.g., human) has, or is at risk of having, an infection or an autoimmune disease.

As used herein, the term "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, the desired effect. For example, a disease-preventing effective amount refers to an amount sufficient to prevent, or delay the onset of a disease; a therapeutically effective amount refers to an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Determination of such effective amounts is well within the ability of those skilled in the art. For example, the amount effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, and the like.

As used herein, the term "immune cell" includes cells, such as lymphocytes, e.g., B cells and T cells, of hematopoietic origin and that play a role in an immune response; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils and granulocytes.

As used herein, the term "immune response" refers to the action of immune cells (e.g., lymphocytes, antigen presenting cells, phagocytes, or granulocytes) and soluble macromolecules (including antibodies, cytokines, and complement) produced by immune cells or liver that result in selective damage, destruction, or clearance of invasive pathogens, pathogen-infected cells or tissues, cancer cells, or normal human cells or tissues in the case of autoimmune or pathological inflammation. In the present invention, the term "antigen-specific T cell response" refers to an immune response generated by a T cell that results when the T cell is stimulated by the T cell-specific antigen. Non-limiting examples of responses generated by T cells upon antigen-specific stimulation include proliferation of T cells and production of cytokines (e.g., IL-2).

As used herein, the term "effector function" refers to those biological activities attributable to the Fc region of an antibody (either the native sequence Fc region or the amino acid sequence variant Fc region), and which vary with the antibody isotype. Examples of antibody effector functions include, but are not limited to: fc receptor binding affinity, antibody-dependent cell-mediated cytotoxicity (ADCC), complement Dependent Cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), down-regulation of cell surface receptors (e.g., B cell receptors), B cell activation, cytokine secretion, half-life/clearance of antibodies and antigen-antibody complexes, and the like. Methods for altering the effector function of antibodies are known in the art, for example by introducing mutations in the Fc region.

As used herein, the term "antibody-dependent cell-mediated cytotoxicity (ADCC)" refers to a form of cytotoxicity whereby Ig binds specifically to antigen-attached target cells by binding to Fc receptors (FcR) present on cytotoxic cells such as Natural Killer (NK) cells, neutrophils or macrophages, and then kills the target cells by secreting cytotoxins. Methods for detecting ADCC activity of antibodies are known in the art and can be assessed, for example, by measuring the binding activity between the antibody to be tested and an Fc receptor (e.g., CD16 a).

As used herein, the term "Complement Dependent Cytotoxicity (CDC)" refers to the activation of the cytotoxic form of the complement cascade by binding complement component C1q to antibody Fc. Methods for detecting CDC activity of antibodies are known in the art and can be assessed, for example, by measuring the binding activity between the antibody to be tested and an Fc receptor (e.g., C1 q).

The term "pharmaceutically acceptable" refers to a molecule that does not produce adverse, allergic or other untoward reactions when administered to an animal or human in a suitable manner. Specific examples of some substances that may be pharmaceutically acceptable carriers or components thereof include saccharides (e.g., lactose), starches, celluloses and derivatives thereof, vegetable oils, gelatins, polyols (e.g., propylene glycol), alginic acid and the like.

Antibodies of the invention

The inventors found that tepromumab-trbw directly mediates the killing effect of macrophages or NK cells on orbital fibroblasts, and that tepromumab-trbw IgG1 Fc, as the Fc with the highest affinity for fcγr, can also bind to fcrs of a variety of cells including neutrophils, eosinophils, basophils, and mast cells, producing a biological effect. For example, neutrophils mostly have high expression of fcyriia to And a small expression of FcR1 (Wang Yu,friederike, expression, roller, and Regulation of Neutrophil Fc. Gamma. Receptors in Immunology VOLUME, 2019, www.frontiers in. Org/arotides/10.3389/fimmeu.2019.01958) may mediate up-regulation of TNF-alpha Expression, as well as up-regulation of IFN-gamma and G-CSF more than 10-fold.

The inventors pass through in the examples of the present applicationCell culture chamber models have found that the ADCC effect on orbital fibroblasts can be directly achieved by using teprotumumab-trbw, or indirectly achieved by eliciting an immune response in the surrounding immune cells of the orbital fibroblasts, exacerbating local inflammatory responses and tissue damage. Surprisingly, the present invention overcomes this side effect of prior art antibodies by significantly reducing the immune response of an antibody in the form of IgG4 after conversion of teprotumumab-trbw to an antibody in the form of IgG4 (retaining the 6 heavy and light chain CDRs of teprotumumab-trbw). The antibodies of the invention comprise VH and VL. VH may comprise a polypeptide comprising the sequence of SEQ ID NO:3 (CDR) -H1, comprising the sequence SEQ ID NO:4 and a CDR-H2 comprising the sequence SEQ ID NO: CDR-H3 of 5. VL may comprise a sequence comprising SEQ ID NO:6, CDR-L1 comprising the sequence SEQ ID NO:7 and a CDR-L2 comprising the sequence SEQ ID NO: CDR-L3 of 8. The antibody may be of the IgG4 isotype. The antibodies of the invention may be suitably truncated into fragments, but the fragments should retain the CDR sequences of the invention.

Pharmaceutical composition

The antibodies of the invention may be formulated in combination, e.g., in pharmaceutical compositions, with other active ingredients, e.g., drugs for the treatment of thyroid-related eye diseases, such as thyroid stimulating hormone receptor TSHR inhibitors, TSH inhibitors, IGF-1R inhibitors, or IGF-1 inhibitors. The pharmaceutical composition may comprise a pharmaceutically acceptable carrier and/or excipient. The pharmaceutical composition may be in the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained release formulation, etc. Can be according to conventional techniques such as Remington: the pharmaceutical compositions are formulated as disclosed in The Science and Practice of Pharmacy,19 edition, gennaro, mack Publishing co., easton, PA, 1995. The pharmaceutical compositions of the present invention may, for example, include diluents, fillers, salts, buffers, stabilizers (e.g., sugars or protein-free amino acids), preservatives, tissue fixatives, solubilizers, and/or other materials suitable for use in the pharmaceutical compositions.

Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic agents, antioxidants and absorption delaying agents, and the like physiologically compatible with the compounds of the present invention. Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the present invention include water, saline, phosphate buffered saline, ethanol, dextrose, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, carboxymethyl cellulose gum solutions, tragacanth, and injectable organic esters, such as ethyl oleate, and/or various buffers. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Proper fluidity can be maintained, for example, by the use of a coating material, such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. The pharmaceutical compositions of the invention may also include pharmaceutically acceptable antioxidants, for example, (1) water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) Oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelators such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

The pharmaceutical compositions of the present invention may also include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, glycerol, or sodium chloride in the composition.

The pharmaceutical compositions of the present invention may also contain one or more adjuvants suitable for the chosen route of administration, such as preserving, wetting, emulsifying, dispersing, preserving or buffering agents, which may improve the shelf life or effectiveness of the pharmaceutical composition. The compounds of the present invention may be prepared with carriers that protect the compounds from rapid release, such as controlled release dosage forms, including implants, transdermal patches, and microencapsulated delivery systems. Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid, alone or with waxes, or other materials known in the art. Methods for preparing such formulations are generally known to those skilled in the art. Sterile injectable solutions may be prepared by incorporating the required amount of antibody in an appropriate solvent with one or a combination of the required components (e.g., as enumerated above) followed by sterile microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients (e.g., from those enumerated above). In the case of sterile powders for the preparation of sterile injectable solutions, examples of methods of preparation are vacuum drying and freeze-drying (lyophilization) which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The actual dosage level of the active ingredient (e.g., an antibody described herein) in the pharmaceutical composition can be varied to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, or mode of administration without toxicity to the patient. The dosage level selected will depend on a variety of pharmacokinetic factors including the activity of the particular composition of the present invention or its amide employed, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

The pharmaceutical composition may be administered by any suitable route and pattern. In one embodiment, the pharmaceutical composition of the invention is administered parenterally. As used herein, the term "parenteral administration" refers to modes of administration other than enteral and topical administration, typically by injection, including epicutaneous, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural, and intrasternal injection and infusion.

Method and use

The invention also provides methods and uses of using the anti-IGF-1R antibodies or pharmaceutical compositions described herein. The anti-IGF-1R antibodies or pharmaceutical compositions described herein can be used to prevent and/or treat thyroid-related eye diseases. For example, the present invention provides a method for preventing and/or treating thyroid-related eye disease in a subject, the method comprising administering an antibody or antigen-binding fragment thereof or a pharmaceutical composition as described herein to a subject in need thereof.

The invention also provides the use of an antibody or pharmaceutical composition as described herein in the manufacture of a medicament for the treatment of thyroid-related eye diseases.

In this context, the thyroid-related eye disease may be selected from the group consisting of herniation, photophobia, lacrimation, pain, redness of the eye, swelling of the eye, dyskinesia, presbyopia, eyelid insufficiency, vision loss, and blindness caused by compression of the optic nerve.

Sequence information

The information of the sequences involved in the invention is described in the following table, and specific sequences are shown in the sequence table.

Sequence(s)
		SEQ ID NO:1	Antibody 6 (IgG 4) heavy chain
SEQ ID NO:2	Antibody 6 (IgG 4) light chain
		SEQ ID NO:3	Antibody 6 (IgG 4) heavy chain CDR1
SEQ ID NO:4	Antibody 6 (IgG 4) heavy chain CDR2
		SEQ ID NO:5	Antibody 6 (IgG 4) heavy chain CDR3
SEQ ID NO:6	Antibody 6 (IgG 4) light chain CDR1
		SEQ ID NO:7	Antibody 6 (IgG 4) light chain CDR1
SEQ ID NO:8	Antibody 6 (IgG 4) light chain CDR1
		SEQ ID NO:9	Antibody 6 (IgG 1) heavy chain
SEQ ID NO:10	Antibody 6 (IgG 1) light chain
		SEQ ID NO:11	Antibody 6 (IgG 4) heavy chain variable region VH
SEQ ID NO:12	Antibody 6 (IgG 4) light chain variable region VL

Abbreviations

Complementarity determining regions in CDR immunoglobulin variable regions

FR antibody framework region: amino acid residues other than CDR residues in the variable region of an antibody

VH antibody heavy chain variable regions

VL antibody light chain variable regions

IgG immunoglobulin G

The immunoglobulin alignment and numbering system proposed by Elvin a.kabat for Kabat (see, e.g., kabat et al Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md, 1991).

IMGT is based on the numbering system of the international immunogenetics information system (The international ImMunoGeneTics Information (IMGT)) initiated by Lefranc et al, see Lefranc et al, dev.

Specific receptors on HLA-DR lymphocytes

KD equilibrium dissociation constant

Ka binding rate constant

Kd dissociation rate constant

ADCC antibody-dependent cytotoxicity

ADCP antibody dependent cell-mediated phagocytosis

Kon binding Rate

Kdif dissociation rate

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are only for illustrating the present invention and are not to be construed as limiting the scope of the present invention. Various objects and advantageous aspects of the invention will become apparent to those skilled in the art from the following detailed description of the preferred embodiments and the accompanying drawings. The specific techniques or conditions are not noted in the examples, and are carried out according to techniques or conditions described in the literature in the art (for example, refer to J. Sam Brookfield et al, ind. Molecular cloning Experimental guidelines, third edition, scientific Press) or according to the product specifications. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Example 1: expression of anti-IGF-1R antibodies, antibody 6 (IgG 4) and antibody 6 (IgG 1)

The anti-IGF-1R antibodies antibody 6 (IgG 4) and antibody 6 (IgG 1) were expressed in the Chinese hamster ovary cell CHO cell transient system with the following sequence information:

the full length of the heavy chain of the antibody 6 (IgG 4) is shown as SEQ ID NO. 1.

The full length of the antibody 6 (IgG 4) light chain is shown as SEQ ID NO. 2.

The CDR1 of the heavy chain of the antibody 6 (IgG 4) is shown as SEQ ID NO:3, and is positioned at amino acids 26-33 of the heavy chain: GFTFSSYG; the CDR2 of the heavy chain is shown as SEQ ID NO. 4, and is positioned at amino acids 51-58 of the heavy chain: IWFGSST; and the heavy chain CDR3 is shown in SEQ ID NO. 5, at amino acids 97-107 of the heavy chain: ARELGRRYFDL, wherein the CDRs are defined according to the IMGT numbering system.

Antibody 6 (IgG 4) light chain CDR1 is shown in SEQ ID NO:6 and is positioned at amino acids 27-32 of the light chain: QSVSSY; light chain CDR2 is shown in SEQ ID NO. 7 and is located at amino acids 49-52 of the light chain: YDAS; and light chain CDR3 is shown in SEQ ID NO. 8, at amino acids 89-98 of the light chain: QQRSKWPPWT, wherein the CDRs are defined according to the IMGT numbering system.

The full length of the heavy chain of the antibody 6 (IgG 1) is shown as SEQ ID NO. 9.

The full length of the antibody 6 (IgG 1) light chain is shown in SEQ ID NO. 10.

The variable region VH of the heavy chain of antibody 6 (IgG 4) is shown in SEQ ID NO. 11.

Antibody 6 (IgG 4) light chain variable region VL is shown in SEQ ID NO. 12.

CHO cell starting cell concentration was adjusted to 2×10 ⁶ Expression was collected after 7 days of culture at each cell/mlThe supernatant was isolated and purified using conventional affinity purification methods to obtain anti-human IGF-1R mAb. The separated and purified proteins were subjected to 12% SDS-PAGE purity detection, and the detection results are shown in FIG. 1.

Example 2: affinity detection of anti-IGF-1R antibodies, i.e., antibody 6 (IgG 4) and antibody 6 (IgG 1) for human IGF-1R

The affinity of antibody 6 (IgG 4) and antibody 6 (IgG 1) for Human IGF-1R (Human IGF-1R, ACRO, cat# IGRH 5229) was tested using the Biacore T200 biomolecular interaction analysis system. The antibody concentration gradient was 800,400,200,100,50,25,12.5nM, and the reaction was carried out at a flow rate of 30. Mu.L.min-1 for 120s, and the dissociation 420s,10 mmol.L-1 glycine (pH 1.5) was regenerated for 30s. By Biacore ^TM The T200 Evaluation Software 3.1.1 software analyzes the data and calculates the binding constant (ka), dissociation constant (KD) and affinity constant (KD). The results are shown in Table 1 and FIG. 2 below.

TABLE 1

Example 3: competitive binding assays of anti-IGF-1R antibodies, i.e., antibody 6 (IgG 4) and antibody 6 (IgG 1), to human IGF-1R antigen recognition sites

The antigen binding site differences of antibody 6 (IgG 4) and antibody 6 (IgG 1) to human IGF-1R were detected using the Biacore T200 biomolecular interaction analysis system. Coupling IGF-1R to a chip at a flow rate of 10 mu L min-1, adding antibody 6 (IgG 1) to bind IGF-1R to saturation, eluting to equilibrium by using running buffer, and adding antibody 6 (IgG 4) to detect whether binding rise exists and judge whether the same epitope exists; the experiment was verified in both positive and negative ways. As a result, as shown in FIG. 3, the antibodies 6 (IgG 4) and 6 (IgG 1) were identical to the antigen binding sites for human IGF-1R.

Example 4 Effect of anti-IGF-1R antibodies, namely antibody 6 (IgG 4) and antibody 6 (IgG 1), on IGF-1/IGF-1R mediated proliferation of FDC-P1 cells

FDC-P1 cells in logarithmic growth phase were adjusted to 4X 10 in density ⁵ Each cell/mL was inoculated into a 96-well plate in an amount of 50. Mu.L/well, and 10ng/mL IGF-1 (GenScript, cat# was addedZ03017) and adding antibody 6 (IgG 4) and antibody 6 (IgG 1) in multiple concentration gradients, incubating in 96-well plate incubator (37deg.C/5% CO 2) for 48 hr, taking out, and adding CellWorking solution is incubated for 5-10 minutes at room temperature, and chemiluminescent values are read by using an enzyme-labeled instrument. The results of the experiment are shown in FIG. 4, where the inhibition of IGF-1/IGF-1R mediated cell proliferation by antibody 6 (IgG 4) and antibody 6 (IgG 1) is similar.

Example 5 comparison of ADCC Activity of anti-IGF-1R antibodies, namely antibody 6 (IgG 4) and antibody 6 (IgG 1)

The ADCC activities of antibody 6 (IgG 4) and antibody 6 (IgG 1) were compared by a luciferase assay system using MCF-7 cells expressing IGF-1R on the cell surface as target cells and GS-J2C/CD16A cells as effector cells. MCF-7 cells grown in log phase were adjusted to a density of 2.5X10 ⁵ Individual cells/mL, inoculated in a 96-well plate in an amount of 40. Mu.L/well, incubated for 30min with 8 concentration gradients of antibody 6 (IgG 4) and antibody 6 (IgG 1), and effector cells GS-J2C/CD16A (1.5X10) ⁶ Individual cells/mL, 40 μl/well), the incubation was continued for 6 hours. After incubation is completed, adding working solution of luciferase detection reagent, usingThe plate reader reads the chemiluminescent values and performs data analysis. The experimental results are shown in fig. 5, the ADCC effect of the antibody 6 (IgG 4) is very weak, no extra cell killing effect exists, and the antibody is more suitable for the treatment of autoimmune diseases such as TED.

Example 6 anti-IGF-1R antibodies, antibody 6 (IgG 4) and antibody 6 (IgG 1), indirectly affect the secretory function of orbital fibroblasts by immune cells

By usingThe cell culture chamber co-cultures orbital fibroblasts and immune cells, which were separately cultured with Polycarbonate (PC) membrane, mimicking the relationship of orbital tissue and blood circulation (fig. 6). In clinical applications, teprotumumab-trbw is injected intravenously and undergoes pharmacological effects in the periorbital tissues. To mimic pathophysiological conditions during disease treatment, the test design is to add antibody 6 (IgG 4) and antibody 6 (IgG 1) to the culture chamber of immune cells; whereas IGF-1 and TSH were added to the fibroblast culture chamber. Because the semipermeable membrane can only limit the migration and infiltration of PBMC, and has no physical pore size limitation on protein molecules and hormones, the concentration titer of the medicine gradually tends to be balanced after the antibodies and the hormones are respectively added into the upper and lower cells.

The test procedure is briefly described as follows:

anti-human CD3 antibodies were purchased from Invitrogen (cat No. 16-0037-85), and human peripheral blood mononuclear cells (Peripheral blood mononuclear cell, PBMC) were purchased from Shanghai Australian organism (cat No. PB 004F-C). Human orbital fibroblasts and human orbital fibroblast culture media were purchased from Shanghai pure biosciences, inc. IGF-1 was purchased from Biyun Tian Biotechnology (cat. No. P5502) and TSH was purchased from Parker (cat. No. hor-001-a). The human interleukin-6 Elisa kit (cat No. SEKH 0013), the human tumor necrosis factor-. Alpha.Elisa kit (cat No. SEKH 0047) and the human transforming growth factor-. Beta.1 Elisa kit (cat No. SEKH 0316) were all purchased from Soy Biotechnology Co. anti-IGF-1 antibodies, antibody 6 (IgG 1) and antibody 6 (IgG 4), were expressed and purified by the company inc.

Pbmc cells can be cultured according to the description of the supplier (shanghai pure biosciences limited). The details are as follows.

1.1: one anti-hCD3 (OKT 3) branch was taken at a concentration of 1mg/mL.

1.2: mu.L of PBS was added thereto at a concentration of 999. Mu.L, and the mixture was diluted to 1. Mu.g/mL.

1.3: a24-well plate was used, and diluted anti-hCD3 (OKT 3), 300. Mu.L/well, and 3 wells were plated for each well.

1.4: put into an incubator at 37 ℃ and incubate for 2 hours.

1.5: two hours later, the supernatant was discarded, the wells were gently washed with PBS and immediately resuscitated PBMC were added. ( And (3) injection: if PBMC is not recovered, the plate incubated for 2 hours at 37℃in 2.4 steps can be placed at 4℃ )

1.6: the water bath is heated to 37 ℃ in advance, and the RPMI-1640 culture medium is balanced to 37 ℃.

1.7: 10mL of RPMI-1640 medium was placed in a 15mL centrifuge tube.

1.8: PBMCs were removed from the liquid nitrogen and placed on ice and quickly thawed to leave pieces of ice in a 37 ℃ water bath.

1.9: the thawed PBMC were transferred to 10mL RPMI-1640 medium, 500g, and centrifuged at 20℃for 10min.

1.10: the supernatant was discarded, 10mL of RPMI-1640 medium was resuspended, and centrifuged at 500g at 20℃for 10 minutes.

1.11:10mL RPMI-1640 medium was resuspended and counted.

1.12: part of the cells was diluted to 2X 10 with RPMI-1640 medium ⁶ Per mL, 1.5 mL/well, 3 wells added to plate wells of a 24-well plate with anti-hCD 3.

1.13：37℃5％CO ₂ Culturing for 24 hours.

2. Human orbital fibroblasts can be cultured according to the description of the supplier (Shanghai pure Biotechnology Co.). The details are as follows.

2.1: passaging 2 times, cells grown to 90%, and prepared for digestion.

2.2: 10mL of PBS was added to the T75 bottle of the cells, and after one wash pass, the PBS was poured out.

2.3: 10mL of PBS was added again, and after washing the cells, the PBS was poured out.

2.4: 4mL of 0.25% pancreatin was added, and pancreatin was immediately poured out after rinsing the cells.

2.5: 4mL of 0.25% pancreatin was added again, and pancreatin was immediately poured out after rinsing the cells. Pancreatin digestion was observed under a microscope.

2.6: immediately after cell rounding, 10mL OF complete medium was added, digestion was stopped and the cell walls were gently blown with a pipette, and the cells were blown down.

2.7: the cell suspension was transferred to a 15mL centrifuge tube, mixed well and counted.

2.8: diluting the cell suspension with OF medium to 1.25X10 ⁵ /mL。

3. Human eyesOrbital fibroblast and human PMBCThe cell co-culture model can be based onProduct specifications are established. The details are as follows.

3.1: the upper chamber was charged with 0.2mL of culture medium and 1X10 ⁵ Human PMBC.

3.2: the lower chamber was charged with 0.8mL of culture medium and 5X10 ⁵ Human orbital fibroblasts.

3.3: anti-IGF-1 antibody, antibody 6 (IgG 1) and antibody 6 (IgG 4) were diluted to 1mg/mL with PBS and added to the upper culture broth alone or to the upper and lower chamber culture broth, respectively, at a final concentration of 50. Mu.g/mL. The 24-well plate was placed at 37℃with 5% CO ₂ After 2 hours of reaction in the cell culture incubator, TSH and/or IGF-1 was added.

3.4: TSH (10. Mu.g/ml) was diluted 10-fold and 5-fold to TSH (1. Mu.g/ml) concentrations, respectively, TSH (2. Mu.g/ml), IGF-1 (200. Mu.g/ml) was diluted 10-fold and 5-fold to IGF-1 (20. Mu.g/ml) concentrations, respectively, IGF-1 (40. Mu.g/ml), TSH (10. Mu.g/ml) and IGF-1 (200. Mu.g/ml) were diluted 2.5-fold and mixed at equal volumes to prepare a solution of TSH (2. Mu.g/ml) +IGF-1 (40. Mu.g/ml). Added to the lower chamber culture medium at concentrations of 10ng/mL TSH and 20ng/mL TSH, and IGF-1 200ng/mL and 400ng/mL respectively. 37 ℃,5% CO ₂ The culture was continued for 24 hours in the cell culture tank.

4. Human transforming growth factor-beta 1 can be detected according to the human transforming growth factor-beta 1Elisa kit. The details are as follows.

4.1: the sample was activated, 80. Mu.l of standard/sample diluent+100. Mu.l of sample, 10. Mu.l of 1N HCl was added, and the mixture was left to stand at 4℃for 60 minutes.

4.2: add 10. Mu.l 1N NaCL and mix well.

4.3: after the microplate was taken out from the refrigerator at 4℃and equilibrated to room temperature, the plate was washed 3 times with a washer.

4.4: the standard substance is dissolved by a standard substance/sample diluent, and the mixture is placed for 15min after preparation, and is gently mixed until the mixture is used, and the concentration of the mother solution is 2000pg/ml.

4.5: 1000pg/ml was taken as the highest point of the standard well, and 2-fold dilutions were performed sequentially, diluting to 15.625pg/ml.

4.6: 100. Mu.l of the gradient diluted standard (1000 pg/ml-15.625 pg/ml) was added to each well. 100 μl of the activated sample was added to the sample well and the side wall of the plate was tapped and mixed well.

4.7: cover the plate membrane and incubate at 37℃for 90 minutes.

4.8: the plate was washed 4 times with a washer.

4.9: 100 μl of diluted biotinylated antibody working solution (1:100) was added to all wells, covered with cover plate membrane and incubated at 37deg.C for 60 min.

4.10: the plate was washed 4 times with a washer.

4.11: to all wells 100. Mu.l of diluted enzyme conjugate working solution (1:100) was added, covered with cover plate membrane and incubated for 30 min at 37 ℃.

4.12: the plate was washed 5 times with a washer.

4.13: mu.l of the color-developing solution was added to each well, and the cover film was covered and incubated at 37℃for 15 minutes.

4.14: mu.l of Stop buffer was added to each well.

4.15: readings were taken at OD450 nm and OD630 nm.

Figure 7 shows that human orbital fibroblasts produce mainly TGF- β and small amounts of IL-6 under stimulation of Thyroid Stimulating Hormone (TSH) and insulin-like growth factor (IGF-1). TNF-alpha is rarely produced. Although IGF-1R expression is not high on normal orbital fibroblasts, stimulation of TSH and/or IGF-1 still allows more than 2-fold production and release of TGF-beta.

TGF-beta is a very complex cytokine involved in regulating cell growth and differentiation, apoptosis, cell motility, extracellular matrix production, angiogenesis, inflammatory responses and cellular immunity. Can transform the phenotype of normal fibroblast, and in vitro experiments show that under the coaction of EGF, the adherent growth characteristics of the fibroblast can be changed to obtain invasive growth capacity, and the inhibition effect of density dependence in growth is lost. (Morikawa M, derynck R, miyazono K.TGF-. Beta.and the TGF-. Beta.family: context-Dependent Roles in Cell and Tissue Physiolog.Cold Spring Harb Perspect biol.2016May 2;8 (5): a 021873.). More recent studies have shown that TGF- β plays an important role in the development of inflammation, as well as in the process of tissue fibrosis. It is speculated that TGF-. Beta.also plays a role in the development and progression of TAO.

FIG. 8 shows that anti-IGF-1R antibody was added to the upper medium, incubated for 24 hours, and the concentration of TGF-beta in the medium was measured. The results showed a 2-3 fold increase in TGF-beta concentration in antibody 6 (IgG 1) -treated cell culture broth. The antibodies do not directly stimulate the orbital fibroblasts to secrete TGF-beta, and thus elevated TGF-beta may be derived from PBMC, and cytokines produced by PBMC activation act on the orbital fibroblasts. This hypothesis can be confirmed by the IgG4-Fc group: antibody 6 (IgG 4) treated cells, although also having an elevated TGF-. Beta.concentration, were significantly lower than antibody 6 (IgG 1) treated cells, which was associated with a weaker binding of IgG4-Fc to FcγR than to IgG 1-Fc. Thus, it was demonstrated that different Fc subtypes can indirectly act on the projections of orbital fibroblasts. The TGF-beta produced by antibody 6 (IgG 4) is relatively low and can alleviate the transformation and inflammation degree of cells.

The test of fig. 9 is more closely related to the complex reality of pathological conditions. Antibodies against IGF-1R were added to the upper and lower media at consistent concentrations. After incubation for 2 hours, TSH and IGF-1 were added to the lower chamber and after further incubation for 24 hours, the TGF- β concentration in the culture broth was measured. The antibodies do not directly stimulate the orbital cells to secrete TGF-beta, but can prevent IGF-1 from stimulating the orbital cells, and reduce the increase of TGF-beta secretion caused by IGF-1 and TSH stimulating the orbital fibroblasts. The results show that antibody 6 (IgG 1) treated cells did not reduce TGF- β production, whereas antibody 6 (IgG 4) reduced TGF- β by 50%. It is suggested that IgG4-Fc brings about a more remarkable effect of inhibiting proliferation and transformation of fibroblasts.

Example 7: anti-IGF-1R antibodies, antibody 6 (IgG 4) and antibody 6 (IgG 1), indirectly affect orbital fibroblast proliferation function through immune cells

Anti-human IgG biotin was purchased from bailibo (cat No. F030822). Annexin V-PE/7 AAD apoptosis assay kit (cat# CA 1030-100) and CCK8 cell proliferation kit (cat# CA 1210-100) were purchased from Soxhobao biotechnology Co.

The detection of cell proliferation by the cck8 kit is performed according to the instructions of the kit. The details are as follows.

1.1: taking 100T OF CCK8 reagent, taking out 1mL, adding 10mL OF OF culture medium, mixing uniformly and keeping away from light for later use.

1.2: in B row and C row of two 24-hole platesAfter 100. Mu.L (note not to be aspirated into the cells) of the supernatant of (A) will be +.>Move to a new 24-well plate to complement RPMI-1640 for use.

1.3: row a supernatants from two 24-well plates were transferred to two 24-well plates and 300 μl CCK8 working fluid was added.

1.4: the lower chamber supernatants in rows B and C of two 24-well plates were transferred to 300. Mu.L of CCK8 working fluid in the corresponding 24-well plates.

1.5: 300 μl of CCK8 working fluid was placed in D2 wells of a 24-well plate as a blank.

1.6: the 24-well plate was placed at 37℃with 5% CO ₂ The cell culture was incubated for 1.5 hours and 2.5 hours for OD450 and OD630, respectively.

2. Apoptosis assays were performed according to the instructions of the Annexin V-PE/7 AAD apoptosis assay kit (cat. No. CA 1030-100). The details are as follows.

2.1: the supernatant OF the CCK-detected OF cells was discarded, and after washing twice gently with 400. Mu.L OF PBS, 400. Mu.L OF pancreatin was added, the supernatant was discarded, and the cells were observed under a microscope to wait for rounding.

2.2: after rounding the cells, the digestion was terminated by adding 600. Mu.L of PBS and after gently blowing the cells from the bottom of the cell plate, transferring the cells to the corresponding cell pellet after centrifugation of the cell supernatant, and mixing well. 300. Mu.L was removed for apoptosis and another 300. Mu.L was subjected to FACS.

2.3: the cell suspension, prepared for apoptosis, was pelleted at 1000rpm for 5 min.

2.4: the supernatant was removed and resuspended with 100. Mu.L of binding buffer.

2.5: mu.L of Annexin V-PE was added, and the mixture was incubated at room temperature for 5 minutes in the dark.

2.6: 10. Mu.L of 7AAD was added, and 400. Mu.L of PBS was added, and the flow test was immediately performed.

3. Flow cytometry for detecting expression of IGF-1R on surface of human orbital fibroblast

3.1: after human orbital fibroblasts were gently washed twice with 400 μl PBS, 400 μl pancreatin was added, the supernatant was discarded, and the waiting cells were observed under a microscope to be rounded.

3.2: after rounding the cells, the digestion was terminated by adding 600. Mu.L of PBS and after gently blowing the cells from the bottom of the cell plate, transferring the cells to the corresponding cell pellet after centrifugation of the cell supernatant, and mixing well. 300. Mu.L was removed for apoptosis and another 300. Mu.L was subjected to FACS.

3.3: 300g of the cell suspension ready for apoptosis was centrifuged for 2 minutes to pellet the cells.

3.4: resuspension with 15 μl of anti-human IgG biotin and incubation at 4 ℃ for 30 min. After the incubation, adding 500 mu L of PBS into each tube, mixing the mixture upside down, fully cleaning the mixture, and centrifuging the mixture for 2 minutes at 300 g; after centrifugation, the supernatant was aspirated, 15. Mu.L of SA-PE was added and incubated at 4deg.C for 30 min in the absence of light: after the incubation is finished, 200 mu L of PBS is added into each tube, and the mixture is uniformly mixed upside down to be fully washed, and 300g of the mixture is centrifuged for 2 minutes; the supernatant was aspirated, resuspended in 200 μl PBS, and pipetted and flow-on-machine detected.

The high expression of insulin-like growth factor 1 receptor (IGF-1R) on orbital fibroblasts in TAO patients and abnormal proliferation under local IGF-1 stimulation is an important pathological feature of the disease. There was also a small amount of IGF-1R expression on orbital fibroblasts in healthy subjects, but there was no significant upregulation of IGF-1R expression under the stimulation of TSH and IGF-1 (FIG. 10 and Table 2). Thus blocking IGF-1 mediation by anti-IGF-1R antibodies The phenomenon of orbital cell proliferation was difficult to see on orbital fibroblasts in healthy people (fig. 11). However, inIn the in vitro model of cell culture chamber, when anti-IGF-1R antibody was added to the culture medium, antibody 6 (IgG 1) treated cells showed slight proliferation of lower orbital fibroblasts, and antibody 6 (IgG 4) group had some proliferation. This phenomenon is consistent with the trend observed in example 6 for TGF- β secretion. Thus, low expression of IGF-1R by orbital cell proliferation is not associated with IGF-1R, but rather is due to immune cell indirect effects. Orbital fibroblasts from TAO patients are reported to be IGF-1 dependent cells, which can cause apoptosis after anti-IGF-1R antibody treatment. However, in this experiment, this phenomenon was not observed (fig. 12 and table 3). Thus, anti-IGF-1R antibody-induced apoptosis of TAO fibroblasts should be based on high IGF-1R expression, and thus it is speculated that anti-IGF-1R antibody-mediated apoptosis is not very pronounced in low IGF-1R-expressed TAO orbital tissues. However, most of the cell surfaces in orbital tissues have TGF- β receptors, and in TAO pathological conditions, orbital tissues may be more affected and more widely affected by TGF- β and other cytokines. This is also the theoretical and practical basis for the combination of IGF-1R antibodies with other drugs for the treatment of TAO.

Table 2: numerical value of flow cytometry

Table 3: numerical value of flow cytometry

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Although the invention has been described in connection with several embodiments described, with a certain length and some details, it is not intended to be limited to any such details or embodiments or to any particular embodiment, but rather should be construed with reference to the appended claims, in order to provide as broad an interpretation of such claims as possible in accordance with the prior art, and thus to effectively encompass the intended scope of the invention. It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims (19)

1. An antibody or antigen-binding fragment thereof having binding specificity for IGF-1R, wherein the antibody or antigen-binding fragment thereof comprises:

heavy chain variable region (VH) and/or light chain variable region (VL), wherein defined by the IMGT numbering system:

the VH comprises a sequence comprising SEQ ID NO:3 (CDR) -H1, comprising the sequence SEQ ID NO:4 and a CDR-H2 comprising the sequence SEQ ID NO: CDR-H3 of 5; and/or the number of the groups of groups,

The VL comprises a sequence comprising SEQ ID NO:6, CDR-L1 comprising the sequence SEQ ID NO:7 and a CDR-L2 comprising the sequence SEQ ID NO: CDR-L3 of 8; and is also provided with

Wherein the antibody is an IgG4 isotype.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises:

a heavy chain variable region (VH) comprising the sequence shown in SEQ ID No. 11, and/or a light chain variable region (VL) comprising the sequence shown in SEQ ID No. 12, wherein defined by the IMGT numbering system.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody comprises a heavy chain comprising the sequence set forth in SEQ ID No. 1 and/or a light chain comprising the sequence set forth in SEQ ID No. 2.

4. The antibody or antigen-binding fragment thereof of any one of claims 1-3, wherein the antibody or antigen-binding fragment thereof is a murine antibody, chimeric antibody, or humanized antibody.

5. The antibody or antigen-binding fragment thereof of any one of claims 1-4, wherein the antibody or antigen-binding fragment thereof has a lower Fc-mediated immune cell activation effect, and an antibody-dependent cell-mediated cytotoxicity (ADCC) effect than an IgG1 isotype.

6. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof, heavy and/or light chain thereof, or heavy and/or light chain variable region thereof of any one of claims 1-5.

7. A vector comprising the isolated nucleic acid molecule of claim 6; preferably, the vector is a cloning vector or an expression vector.

8. A host cell comprising the isolated nucleic acid molecule of claim 6 or the vector of claim 7.

9. A method of making the antibody or antigen-binding fragment thereof of any one of claims 1-5, comprising culturing the host cell of claim 8 under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

10. A multispecific antibody comprising a first antibody or fragment thereof, and a further antibody or fragment thereof, or antibody analog; wherein the first antibody or fragment thereof is the antibody or antigen-binding fragment thereof of any one of claims 1-5 that specifically binds IGF-1R; preferably, the multispecific antibody is a bispecific or trispecific or tetraspecific antibody.

11. A conjugate comprising an antibody or antigen-binding fragment thereof, and a coupling moiety, wherein the antibody or antigen-binding fragment thereof is any one of claims 1-5, the coupling moiety is a detectable label, such as a radioisotope, fluorescent substance, luminescent substance, colored substance, or enzyme, or the coupling moiety is a pharmaceutical, such as a chemotherapeutic agent, radionuclide, or toxin.

12. A cell expressing a Chimeric Antigen Receptor (CAR) comprising or expressing the antibody or antigen-binding fragment thereof of any one of claims 1-5, the nucleic acid of claim 6 or the vector of claim 7; preferably, the cells are derived from immune cells, or the antigen binding fragment is selected from scFv; more preferably, the cells are derived from T lymphocytes, NK cells, monocytes, macrophages or dendritic cells and any combination thereof.

13. An oncolytic virus comprising the nucleic acid of claim 6 or the vector of claim 7.

14. A fusion protein comprising an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof of any one of claims 1-5, and a non-immunoglobulin moiety that is a polypeptide, such as a cytokine or antagonistic polypeptide thereof, and an antibody, preferably a cytokine selected from the group consisting of IL-2, IL-15, IL-7, IL-12, IL-18, IL-21, TNF-a, or ifnγ.

15. A pharmaceutical composition comprising the antibody or antigen binding fragment thereof of any one of claims 1-5, the isolated nucleic acid molecule of claim 6, the vector of claim 7, the host cell of claim 8, the multispecific antibody of claim 10, the conjugate of claim 11, the CAR cell of claim 12, the oncolytic virus of claim 13, and/or the fusion protein of claim 14, preferably the pharmaceutical composition further comprises one or more thyroid stimulating hormone receptor TSHR inhibitors and/or a pharmaceutically acceptable carrier and/or excipient.

16. Use of the antibody or antigen binding fragment thereof of any one of claims 1-5, the isolated nucleic acid molecule of claim 6, the vector of claim 7, the host cell of claim 8, the multispecific antibody of claim 10, the conjugate of claim 11, the CAR cell of claim 12, the oncolytic virus of claim 13, the fusion protein of claim 14, the pharmaceutical composition of claim 15 in the manufacture of a medicament for the prevention and/or treatment of thyroid-related eye diseases.

17. The use of claim 16, wherein the thyroid-related eye condition comprises herniation, photophobia, lacrimation, pain, redness of the eye, swelling of the eye, dyskinesia, double vision, eyelid insufficiency, vision loss, and/or blindness caused by compression of the optic nerve.

18. A method for preventing and/or treating thyroid-related eye disease in a subject, the method comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition of claim 15, wherein the subject is a mammal; preferably, the subject is a human.

19. A combination of an antibody according to claim 1 and one or more thyroid stimulating hormone receptor TSHR inhibitors for use in the treatment of thyroid eye diseases, preferably wherein the thyroid stimulating hormone receptor TSHR inhibitor is a small molecule inhibitor or an antibody against TSHR, preferably wherein the thyroid related eye diseases comprise herniation, photophobia, lacrimation, pain, redness, swelling of the eye, dyskinesia, presbyopia, eyelid insufficiency, vision loss and/or blindness induced by compression of the optic nerve.