Antibodies or their antigen-binding fragments

Abstract

The antibody or its antigen-binding fragment binds to human prolactin (hPRL) while also neutralizing its agonism against the human PRL receptor (hPRLR). Advantageously, this allows other hPRLR agonists to continue signaling. As a result, this antibody or its antigen-binding fragment is suitable for therapeutic use, for example, as part of a composition containing the antibody or its antigen-binding fragment. For instance, the antibody or its antigen-binding fragment may be used in the treatment of diseases or conditions modulated by PRL, such as in the treatment of pain.

Description

[Technical Field]

[0001] This invention relates to an antibody or an antigen-binding fragment thereof. More specifically, this invention relates to an antibody or an antigen-binding fragment thereof that binds to human prolactin (hPRL). [Background technology]

[0002] Prolactin (PRL) is a widely expressed polypeptide hormone that exerts multifaceted endocrine, parasecretory, and autocrine functions. PRL is produced by prolactin-secreting cells in the anterior pituitary gland, as well as by several extrapituitary tissues. PRL plays an important role in mammary gland formation and milk production, and therefore naturally increases during pregnancy and lactation. Apart from this, excessive PRL production is associated with galactorrhea, amenorrhea, breast pain, infertility, endometriosis, osteoporosis, breast and prostate cancer, erectile dysfunction, alopecia, and migraines.

[0003] Circulating PRL levels are higher in women than in men, increase during reproductive age, decrease after menopause, and increase again during pregnancy, suggesting regulation by female hormones. In addition, PRL has been shown to be an important contributor to sexual dimorphism in pain. Both PRL and the PRL receptor (PRLR) are expressed in trigeminal ganglion (TG) nociceptors in animals. PRLR is expressed at higher levels in TG nociceptors in female animals. PRL sensitizes female TG nociceptors but not male TG nociceptors, leading to the release of calcitonin gene-related peptide (CGRP), a peptide known to induce migraine attacks in humans. Topical application of PRL to the dura mater induces migraine-like pain in female animals but not in male animals. In addition, PRL sensitizes dorsal root ganglion (DRG) nociceptors in female animals but not in male animals. Locally applied and / or locally produced PRL induces nociceptive hypersensitivity and pain in female animals but not in male animals. Taken together, these data suggest that excessive PRL signaling may contribute to widespread female-dominant pain syndromes, and that blocking both pituitary and extrapituitary PRL may be clinically beneficial for treating pain in women.

[0004] The production and secretion of PRL in both the pituitary gland and extrapituitary tissues are regulated differently. Humans have a single gene encoding PRL and two different promoters that regulate PRL expression in the pituitary gland and extrapituitary tissues in different ways. Furthermore, dopamine regulates PRL secretion in the pituitary gland but not in the extrapituitary tissues.

[0005] Dopaminergic type 2 (D2) receptor agonists, such as cabergoline, which are clinically used to inhibit PRL release from the pituitary gland, have no usefulness in controlling PRL release from extrapituitary tissues.

[0006] Conventional attempts to develop therapeutic agents capable of blocking both pituitary and extrapituitary PRL responses have focused on PRLR antagonists, either peptides or anti-human PRLR (hPRLR) antibodies.

[0007] Peptide hPRLR antagonists have a very short half-life, as disclosed in WO2018049092, and therefore have not been able to be developed as therapeutic agents.

[0008] Antibodies that bind to hPRLR, such as those disclosed in WO2012163932A1 or WO2008022295A2, prevent hPRLR activation by hPRL. However, these hPRLR antibodies are not selective for hPRL because they interfere with ligand binding and / or hPRLR signaling activation. Therefore, they are likely to also block hPRLR activation by two other polypeptide hormones structurally related to hPRL, namely growth hormone and placental lactogen, thus strongly indicating the need for a selective hPRL blocker.

[0009] Antibodies targeting hPRL are commercially available as research tools. While they generally bind to hPRL, they do not necessarily neutralize hPRL, or more specifically, the action of hPRL in hPRLR. Furthermore, since all these antibodies are derived from mice, rabbits, or goats, they are not suitable for therapeutic use.

[0010] For example, CN113956356 relates to a mouse anti-PRL protein monoclonal antibody. Such mouse antibodies are not suitable for therapeutic use. More specifically, CN'356 is functionally different from the present invention in that Example 4 of CN'356 explicitly describes an IgG1-type mouse monoclonal antibody that is not suitable for therapeutic use. It is well known in the art that mouse mAbs cannot be used as therapeutic agents because patients rapidly develop anti-drug antibodies that neutralize them.

[0011] U.S. Patent No. 4,585,740 relates to a prolactin immunoassay using a synthetic peptide.

[0012] WO2009013621A2 relates to inhibitors of growth hormone and related hormones, as well as their use.

[0013] Currently, there are no available drugs that can single-handedly and completely neutralize the response to hPRL produced by both the subpituitary and extrapituitary pituitary glands. Current treatments, and / or conventional attempts to develop therapeutic agents to inhibit hPRL activity, have various drawbacks that prevent blocking both subpituitary and extrapituitary hPRL without inhibiting other hPRLR agonists. [Overview of the project] [Problems that the invention aims to solve]

[0014] The present invention relates to an antibody or its antigen-binding fragment. More specifically, the present invention relates to an antibody or its antigen-binding fragment that binds to hPRL. More specifically, the present invention describes a novel humanized anti-hPRL antibody or its antigen-binding fragment that has been demonstrated to have high affinity and selectivity for hPRL, as well as the ability to potently and selectively inhibit hPRL activation by hPRLR.

[0015] Advantageously, the humanized antibodies or their antigen-binding fragments described in this application have been developed for use as human therapeutic agents.

[0016] Advantageously, the antibody or its antigen-binding fragment specifically binds to hPRL, allowing other hPRLR agonists to continue signaling. [Means for solving the problem]

[0017] According to a first aspect of the present invention, an antibody or an antigen-binding fragment thereof that binds to hPRL is provided. The antibody of the present invention may be beneficial in the treatment of pain in women. This is because female patients are at a higher risk of experiencing many clinical pain syndromes. Functional pain syndromes (FPS) constitute a large subgroup of pain conditions defined by the absence of clear etiology or tissue damage. FPS is characterized by an abnormally high prevalence ratio in women compared to men. These include, but are not limited to, temporomandibular joint disorders, fibromyalgia, irritable bowel syndrome, chronic pelvic pain, interstitial cystitis, and migraines, as well as female-specific FPS, such as dysmenorrhea, endometriosis, and vulvodynia. Female FPS typically peaks during reproductive age, is often aggravated during the menstrual cycle and by stress, and regresses or disappears after menopause, which suggests that stress and / or female hormones may be involved in the sex differences of FPS.

[0018] hPRL may be described as ID 5617 in NCBI, which is incorporated herein by reference.

[0019] The antibody or its antigen-binding fragment may be an isolated antibody or its isolated antigen-binding fragment.

[0020] An antibody can be an immunoglobulin molecule containing two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds. The amino-terminal portion of each LC and HC contains a variable region of approximately 100-120 amino acids that is primarily responsible for antigen recognition via a complementarity-determining region (CDR) contained within it. The CDR contains scattered, more conserved regions called framework regions (FRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDRs and four FRs, arranged from the amino-terminal to the carboxyl-terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the LC are referred to as "LCDR1, LCDR2, and LCDR3," and the three CDRs of the HC are referred to as "HCDR1, HCDR2, and HCDR3." The CDRs contain most of the residues that form specific interactions with the antigen; that is, the CDRs contain most of the residues that come into contact with the antigen's residues. Therefore, the functional ability of an antibody to bind to a specific antigen is greatly influenced by the amino acid residues within the six CDRs. The assignment of amino acids to the CDR domains within the LCVR and HCVR regions of the antibody of the present invention is based on the well-known Kabat numbering rules (Kabat et al., 1971).

[0021] Advantageously, antibodies containing the above-mentioned CDR or their antigen-binding fragments neutralize hPRL. As used herein, the term “neutralizes” refers to the inhibition of PRLR activation by one of its natural agonists, specifically PRL. In particular, PRL activates PRLR in a concentration-dependent manner, eventually reaching a maximal plateau. PRLR activation in this specification is tracked by an increase in cytoplasmic phosphorylated STAT5, which indicates downstream signaling. The neutralizing potency of the antibody in vitro was evaluated by exposing PRL below its maximal activation concentration to a molar excess of the antibody. The results were then compared to a control with PRL alone to determine the percentage of neutralization. Lack of neutralization was evident by continuous PRLR activation and a consistent response to the agonist, which was independent of antibody concentration. A neutralizing antibody was defined as reducing PRLR activation by at least 75% compared to PRL alone. Preferably, the addition of the antibody or its antigen-binding fragment to hPRL completely neutralizes the hPRL-inducible activation of hPRL (a 95-100% reduction), resulting in a treatment condition that is indistinguishable from one without agonist administration. The strength of neutralization was evaluated by the concentration-response curve of the antibody exposed to PRL below a certain maximum concentration. 50 The values ​​were determined using a variable gradient model of inhibition.

[0022] As described above, antibodies containing the above-mentioned CDR or their antigen-binding fragments neutralize hPRL activation in hPRLR. The applicant presents that ligand-binding mAbs do not necessarily neutralize their action at the receptor or other downstream mediators. For neutralization to occur, the mAb must effectively interfere with the binding of the ligand to its receptor or downstream mediator in a manner that interferes with signaling further downstream within the cell. In contrast to the antibodies or antigen-binding fragments of the present invention, CN113956356 does not teach that the mAb of CN'356 has the ability to neutralize the activity of hPRL at hPRLR, even in principle. Those skilled in the art have no reason to expect that humanization of the CN'356 mAb will result in a drug for therapeutic use in humans that effectively neutralizes the activity of hPRL.

[0023] Those skilled in the art will understand that, in order to produce a PRL-binding antibody or its antigen-binding fragment, no function other than interacting with PRL, which is part of the antigen, is required. The disclosed antibody or its antigen-binding fragment not only binds specifically to the antigen with high affinity, but this binding also provides the further function of neutralizing the biological function of hPRL on its receptor. Therefore, the antibody or its antigen-binding fragment described herein can be used as a therapeutic agent to alter the functional activity of hPRL. A further advantage of this antibody or its antigen-binding fragment is that its structure is humanized, which is necessary to reduce the potential for immunogenicity when used as a therapeutic agent in human patients. The data described herein support that the humanized antibody sequence exhibits the desired neutralizing activity.

[0024] To our surprise, the applicant has found that the antibody or its antigen-binding fragment of the present invention offers the following advantages over CN113956356: functional blockade of hPRL's ability to activate its cognitive receptor, demonstrated specificity, higher affinity, and a humanized sequence that reduces immunogenicity in humans. On the other hand, those skilled in the art would not have been motivated to produce a humanized anti-hPRL antibody starting from a mouse antibody of CN'356. This is mainly due to the fact that antibodies on CN'356 have not been evaluated for their ability to alter hPRL-mediated hPRL activation. As those skilled in the art will understand, many antibodies that exhibit hPRL binding also do not possess potent neutralizing activity.

[0025] Advantageously, the antibody described in this application exhibits higher affinity for hPRL than CN'356. Furthermore, this application provides evidence that the antibody has specificity for hPRL, as well as for PRL from other species, in addition to the structurally similar proteins hGH and hPL.

[0026] Furthermore, CN'356 does not disclose any specific complementarity-determining regions (CDRs) or heavy / light chain (HC / VC) antibody sequences of the present invention. Structurally, CN'356 differs from the present invention in that it is mouse IgG1 having a complete mouse framework, CDRs, and HC / LC. More specifically, sequence alignment of the HCVR and LCVR of the antibody described in CN'356 with the HCVR and LCVR of the antibody described in this application does not show any CDRs with more than 43% sequence identity among those disclosed in this application, and one CDR, namely HCDR3 of HC (SEQ ID NO: 7), is a distinct mouse sequence and therefore not identical to the HCVR disclosed in CN'356. CN'356 demonstrates that its mouse antibody can only be used for immunodetection and immunoassays for staining hPRL, and does not demonstrate or suggest the use of its claimed mAb as a therapeutic agent or as potentially developed as such. Those skilled in the art will readily understand that when developing a humanized version of a mouse antibody for CN'356, such a humanized antibody is likely to lose affinity and require additional development, such as affinity maturation (Safdari et al., 2013, Biotechnol Genet Eng Rev).

[0027] Furthermore, antibodies with neutralizing function often exhibit varying degrees of activity due to variations in overall affinity. Taken together, this demonstrates the intensive efforts of the present invention to discover and develop the novel antibodies described herein for specific therapeutic applications. The expression "selected from" can be understood as meaning "selecting a portion of the amino acid sequence from a longer amino acid sequence described in the SEQ ID NO: 8." For example, the expression "LCDR1 has an amino acid sequence selected from SEQ ID NO: 8" means that LCDR1 may have an amino acid sequence identical to SEQ ID NO: 8, or an amino acid sequence that is a selected portion of the amino acid sequence from a longer amino acid sequence described in SEQ ID NO: 8.

[0028] In some cases, the antibody or its antigen-binding fragment contains an LC comprising one or more LC CDRs. The LC CDRs may be LCDR1, LCDR2, and / or LCDR3. LCDR1 may have an amino acid sequence selected from SEQ ID NO: 8. LCDR2 may have an amino acid sequence selected from SEQ ID NO: 9. LCDR3 may have an amino acid sequence selected from SEQ ID NO: 10. Preferably, LCDR1 has an amino acid sequence selected from SEQ ID NO: 8, LCDR2 has an amino acid sequence selected from SEQ ID NO: 9, and / or LCDR3 has an amino acid sequence selected from SEQ ID NO: 10.

[0029] In some cases, the antibody or its antigen-binding fragment contains an HC comprising one or more HC CDRs. The HC CDRs may be HCDR1, HCDR2, and HCDR3. HCDR1 may have an amino acid sequence selected from SEQ ID NO: 5. HCDR2 may have an amino acid sequence selected from SEQ ID NO: 6. HCDR3 may have an amino acid sequence selected from SEQ ID NO: 7. Preferably, HCDR1 has an amino acid sequence selected from SEQ ID NO: 5, HCDR2 has an amino acid sequence selected from SEQ ID NO: 6, and / or HCDR3 has an amino acid sequence selected from SEQ ID NO: 7.

[0030] In some cases, the CDR of the antibody of the present invention is defined according to Table 1, which lists the CDR numbering rules used to define the CDR of the antibody of the present invention.

[0031] [Table 1]

[0032] In some cases, the antibody or its antigen-binding fragment may contain LCVR. LCVR may have an amino acid sequence selected from SEQ ID NO: 4. In some cases, the antibody or its antigen-binding fragment may contain HCVR. HCVR may have an amino acid sequence selected from SEQ ID NO: 3.

[0033] In some cases, the antibody or its antigen-binding fragment binds to an epitope having an amino acid sequence selected from SEQ ID NO: 11. As used herein, the term “epitope” refers to the surface of the antigen in contact with the variable region of the antibody. In particular, the tertiary structure of hPRLs consists of bundled α-helices connected by unstructured loops. The hPRL epitopes described are discontinuous residue structural epitopes, meaning that the antibody interacts with a specific region of the hPRL tertiary structure. Thus, the hPRL epitopes consist of residues of separate α-helices that are in close proximity in three-dimensional space.

[0034] In some cases, the antibody or its antigen-binding fragment binds to a discontinuous epitope, where contact aa is located within SEQ ID NO: 11 and / or a specific aa is shown in Example 4. The applicant advantageously presents that immunization of mice with derivatives of PRL yields antibodies that bind to a number of discontinuous epitopes, different from those of the present invention.

[0035] In some cases, the antibody or its antigen-binding fragment is an antibody or antigen-binding fragment of immunoglobulin G (IgG), for example, an antibody or antigen-binding fragment of IgG of subclasses IgG1, IgG2, IgG3 and / or IgG4.

[0036] In some cases, the antibody or antigen-binding fragment may contain known Fc region mutations. For example, the antibody or antigen-binding fragment may be IgG4(S228P). In some cases, the antibody or antigen-binding fragment may contain reduced Fc effector function. For example, antibody or antigen-binding fragments include IgG1(L235E), IgG1(L234A / L235A), IgG4(S228P / L235E), IgG1(L234A / L235A / P329G), IgG1(P331S / L234E / L235F), IgG1(D265A), IgG1(G237A), IgG1(E318A), IgG1(E233P), IgG1(G236R / L328R), IgG1(A330L), IgG1(D270A), IgG1(K322A), IgG1(P329A), IgG1(P331A), IgG1(V2 Possible other IgG4 compounds include 64A), IgG1(F241A), IgG1(N297A / G / Q), IgG4(S228P / F234A / L235A); and those reported to have extended half-lives, such as IgG1(R435H), IgG1(N434A), IgG1(M252Y / S254T / T256E), IgG1(M428L / N434S), IgG1(T252L / T253S / T254F), IgG1(ΔE294 / T307P / N434Y), IgG1(T256N / A378V / S383N / N343Y), and IgG1(ΔE294).

[0037] In some cases, the antibody or its antigen-binding fragment binds to PRL with an affinity of approximately 0.6 nM. In this specification, “approximately” is understood to mean ±10%, preferably ±5%, preferably ±1%, and preferably ±0.1%.

[0038] Preferably, the antibody or its antigen-binding fragment binds to hPRL with an affinity of 0.621 nM.

[0039] In some cases, the antibody or its antigen-binding fragment binds to hPRL with an affinity of 0.2 nM to 1.1 nM. Preferably, the antibody or its antigen-binding fragment binds to hPRL with an affinity of 0.3 to 1 nM. More preferably, the antibody or its antigen-binding fragment binds to hPRL with an affinity of 0.4 nM to 0.9 nM. More preferably, the antibody or its antigen-binding fragment binds to hPRL with an affinity of 0.5 to 0.8 nM. More preferably, the antibody or its antigen-binding fragment binds to hPRL with an affinity of 0.6 to 0.7 nM.

[0040] In some cases, the antibody or its antigen-binding fragment binds to hPRL, and the affinity at that time is at least 1 nM. More preferably, the antibody or its antigen-binding fragment binds to hPRL, and the affinity at that time is less than 1 nM. More preferably, the antibody or its antigen-binding fragment binds to hPRL, and the affinity at that time is less than 0.7 nM.

[0041] The antibody or its antigen-binding fragment can bind to hPRL with an affinity including any range from a given endpoint.

[0042] In some cases, the antibody or its antigen-binding fragment binds to hPRL with an association rate k of 3.47×10 5 M -1 s -1 and / or a dissociation rate k of 2.15×10 on s -4 s -1 of off .

[0043] In some cases, the antibody or its antigen-binding fragment binds to hPRL with an association rate k of 1×10 5 M -1 s -1 to 6×10 5 M -1 s -1 and / or a dissociation rate k of 1×10 on s -4 s -1 to 4×10 -4 s -1 of offIt binds to hPRL. Preferably, the antibody or its antigen-binding fragment is 2 × 10 5 M -1 s -1 ~5×10 5 M -1 s -1 The meeting velocity k on , and / or 2.5 × 10 -4 s -1 ~3×10 -4 s -1 Dissociation rate k off It binds to hPRL. Preferably, the antibody or its antigen-binding fragment is 3 × 10 5 M -1 s -1 ~4×10 5 M -1 s -1 The meeting velocity k on , and / or 2 × 10 -4 s -1 ~2.5×10 -4 s -1 Dissociation rate k off Then it merges with hPRL.

[0044] Preferably, the antibody or its antigen-binding fragment is 3.1 × 10 5 M -1 s -1 ~3.9×10 5 M -1 s -1 The meeting velocity k on , and / or 1.7 × 10 -4 s -1 ~2.5×10 -4 s -1 Dissociation rate k off It binds to hPRL. More preferably, the antibody or its antigen-binding fragment is 3.2 × 10 5 M -1 s -1 ~3.8×10 5 M -1 s -1 The meeting velocity k on , and / or 1.8 × 10 -4 s -1 ~2.4×10 -4 s -1 Dissociation rate k offIt binds to hPRL. More preferably, the antibody or its antigen-binding fragment has an association rate k 5 M -1 s -1 ~3.7×10 5 M -1 s -1 for binding to hPRL, and / or a dissociation rate k on of 1.0×10 -4 s -1 ~2.3×10 -4 s -1 for binding to hPRL. More preferably, the antibody or its antigen-binding fragment has an association rate k off of 3.4×10 5 M -1 s -1 ~3.6×10 5 M -1 s -1 for binding to hPRL, and / or a dissociation rate k on of 2.0×10 -4 s -1 ~2.2×10 -4 s -1 for binding to hPRL. off

[0045] The antibody or its antigen-binding fragment can bind to hPRL at an association rate k on that includes any range from a given endpoint. The antibody or its antigen-binding fragment can bind to hPRL at a dissociation rate k off that includes any range from a given endpoint.

[0046] Optionally, the antibody or its antigen-binding fragment binds to PRL at an association rate k 5 of approximately 3.47×10<l000096>M -1 s on and / or a dissociation rate k -4 of approximately 2.15×10 -1 s off In this specification, "approximately" is understood to mean ±10%, preferably ±5%, preferably ±1%, preferably ±0.1%.

[0047] ​In some cases, antibodies or their antigen-binding fragments neutralize the PRL-inducible activation of PRLR and its downstream signaling pathways. As used herein, the term “neutralizes” refers to the inhibition of PRLR activation by one of its natural agonists, specifically PRL. In particular, PRL activates PRLR in a concentration-dependent manner, eventually reaching a maximal plateau. PRLR activation in this specification is tracked by an increase in cytoplasmic phosphorylated STAT5, indicating downstream signaling. The neutralizing potency of antibodies in vitro was evaluated by exposing PRL below its maximal activation concentration to a molar excess of the antibody. The results were then compared to a control with PRL alone to determine the percentage of neutralization. Lack of neutralization was evident by continuous PRLR activation and a consistent response to the agonist, which was independent of antibody concentration. A neutralizing antibody was defined as reducing PRLR activation by at least 75% compared to PRL alone. Preferably, the addition of the antibody or its antigen-binding fragment to hPRL completely neutralizes hPRLR activation (a 95-100% reduction), resulting in a treatment condition indistinguishable from one without agonist administration. The strength of neutralization was evaluated by the concentration-response curve of the antibody exposed to PRL below a certain maximum concentration. Antibody IC 50 The values ​​were determined using a variable gradient model of inhibition.

[0048] In some cases, an antibody or its antigen-binding fragment may bind to hPRL with high affinity, for example. As used herein, the term “binding” refers to a protein-protein interaction between an antibody or its antigen-binding fragment and its antigen, hPRL. “Protein-protein interaction” refers to the physical contact of two proteins facilitated by hydrophobic, electrostatic, and / or hydrogen interactions of amino acid side chains from two or more separate proteins. More specifically, the binding of an antibody or its antigen-binding fragment is primarily mediated and identified by a subset of amino acids within the LCDR and / or HCDR. Furthermore, the site of interaction on the antigen hPRL is defined as an epitope, as described above. In this specification, binding is evaluated by the strength or affinity of the interaction, determined by biolayer interferometry, surface plasmon resonance, or similar techniques. The strength of the interaction is expressed as the ratio of two reaction rates. In particular, the dissociation rate of the antigen from the antibody (k off The association rate (k) between the antibody and antigen against ) on ) quotient (KD=k off / k on ) and here, slower dissociation rates generally best indicate higher affinity. In this specification, "high affinity" is defined as 1 × 10⁻⁶ -9 M or less. Binding "specificity" or "selectivity," as used herein, refers to the ability of an antibody to distinguish hPRL from other reference antigens, e.g., PRL from other species and other hormonal agonists of hPRLR. In this specification, specificity is determined, as described above, using biolayer interference and / or neutralization of agonist-induced activation of the hPRLR signaling pathway. Those skilled in the art will refer, for example, to the following detailed description, and / or K D Considering Kumaraswamy and Tobias, 2015, Methods Mol Biol, which describes a method for determining K D The meaning and calculation will be easily understood.

[0049] In some cases, the antibody or its antigen-binding fragment may produce a 3.1 nM IC50. 50It neutralizes hPRL with lower in vitro efficacy than [another drug].

[0050] Preferably, the antibody or its antigen-binding fragment has a 10 nM IC50. 50 ICs with a minimum current of 5 nM 50 ICs with a minimum current of 4 nM 50 ICs with a frequency lower than, preferably 3.5 nM 50 ICs with a frequency lower than, preferably 3.4 nM 50 ICs with a frequency lower than, preferably 3.3 nM 50 ICs with a frequency lower than, preferably 3.2 nM 50 It neutralizes hPRL with lower in vitro efficacy than [another drug].

[0051] In some cases, the antibody or its antigen-binding fragment produces an IC50 of approximately 3.1 nM. 50 It neutralizes hPRL with in vitro efficacy. In this specification, “approximately” is understood to mean ±10%, preferably ±5%, preferably ±1%, and preferably ±0.1%.

[0052] Advantageously, the antibody or antigen-binding fragment of the present invention can provide an improved drug that neutralizes only hPRL function. As described above, known D2 receptor agonists, such as cabergoline, effectively inhibit pituitary secretion of hPRL but do not affect hPRL derived from extrapituitary tissue. While hPRLR mAbs advantageously block all sources of hPRL from activating hPRLR, their antagonistic effect is also thought to inhibit other hPRLR hormone agonists. The antibody or antigen-binding fragment of the present invention selectively neutralizes hPRL activation by hPRLR while allowing continued hPRLR activation by human growth hormone and human placental lactogen. Advantageously, this provides improved safety.

[0053] Depending on the case, LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and / or HCDR3 may include amino acid substitutions, deletions, or additions. Preferably, the amino acid substitutions, deletions, or additions are conservative sequence modifications. The term "conservative sequence modification" refers to an amino acid modification that does not significantly affect, or significantly alter, the binding properties of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. The modifications can be introduced into the antibodies of the present invention by standard methods known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in the CDR region of the antibody of the present invention can be substituted with other amino acid residues from the same side chain family, and the modified antibody can be tested for retention of function (e.g., the functions described above) using the functional assays described herein.

[0054] In some cases, LCDR1 has an amino acid sequence selected from SEQ ID NO: 8, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to SEQ ID NO: 8.

[0055] In some cases, LCDR2 has an amino acid sequence selected from SEQ ID NO: 9, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to SEQ ID NO: 9.

[0056] Depending on the case, LCDR3 may have an amino acid sequence selected from SEQ ID NO: 10, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to SEQ ID NO: 10.

[0057] In some cases, HCDR1 has an amino acid sequence selected from SEQ ID NO: 5, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to SEQ ID NO: 5.

[0058] In some cases, HCDR2 has an amino acid sequence selected from SEQ ID NO: 6, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to SEQ ID NO: 6.

[0059] In some cases, HCDR3 has an amino acid sequence selected from SEQ ID NO: 7, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to SEQ ID NO: 7.

[0060] In some cases, LCDR1 includes the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence that is at least 60% identical to SEQ ID NO: 8, preferably at least 65% identical, more preferably at least 70% identical, more preferably at least 75% identical, more preferably at least 80% identical, more preferably at least 85% identical, more preferably at least 90% identical, more preferably at least 95% identical, more preferably at least 98% identical, and more preferably at least 99% identical.

[0061] In some cases, LCDR2 includes the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence that is at least 60% identical to SEQ ID NO: 9, preferably at least 65% identical, more preferably at least 70% identical, more preferably at least 75% identical, more preferably at least 80% identical, more preferably at least 85% identical, more preferably at least 90% identical, more preferably at least 95% identical, more preferably at least 98% identical, and more preferably at least 99% identical.

[0062] In some cases, LCDR3 includes the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence that is at least 60% identical to SEQ ID NO: 10, preferably at least 65% identical to SEQ ID NO: 10, more preferably at least 70% identical, more preferably at least 75% identical, more preferably at least 80% identical, more preferably at least 85% identical, more preferably at least 90% identical, more preferably at least 95% identical, more preferably at least 98% identical, and more preferably at least 99% identical.

[0063] In some cases, HCDR1 includes the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence that is at least 60% identical to SEQ ID NO: 5, preferably at least 65% identical, more preferably at least 70% identical, more preferably at least 75% identical, more preferably at least 80% identical, more preferably at least 85% identical, more preferably at least 90% identical, more preferably at least 95% identical, more preferably at least 98% identical, and more preferably at least 99% identical.

[0064] In some cases, HCDR2 includes the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence that is at least 60% identical to SEQ ID NO: 6, preferably at least 65% identical, more preferably at least 70% identical, more preferably at least 75% identical, more preferably at least 80% identical, more preferably at least 85% identical, more preferably at least 90% identical, more preferably at least 95% identical, more preferably at least 98% identical, and more preferably at least 99% identical.

[0065] In some cases, HCDR3 includes the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence that is at least 60% identical to SEQ ID NO: 7, preferably at least 65% identical, more preferably at least 70% identical, more preferably at least 75% identical, more preferably at least 80% identical, more preferably at least 85% identical, more preferably at least 90% identical, more preferably at least 95% identical, more preferably at least 98% identical, and more preferably at least 99% identical.

[0066] Advantageously, LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and / or HCDR3 have amino acid sequences that are identical by at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, and 99%, including amino acid substitutions, deletions, or additions, as described above, while still retaining their functionality. Those skilled in the art will readily understand the degree of sequence variability / identity and that a single amino acid change in a CDR does not necessarily result in loss of binding to the target due to CDR plasticity (Townsend et al., 2015, PNAS). In another aspect of the present invention, a composition comprising the antibody or its antigen-binding fragment described herein is provided, for example, a pharmaceutical composition comprising the antibody or its antigen-binding fragment described herein.

[0067] Depending on the circumstances, the pharmaceutical composition may include pharmaceutically acceptable carriers, excipients, and / or diluents.

[0068] In another aspect of the present invention, a composition (e.g., a pharmaceutical composition) comprising the antibody or antigen-binding fragment described herein is provided for use in the treatment of a disease or condition modulated by hPRL. Optionally, the composition comprising the antibody or antigen-binding fragment described herein is for use in the treatment of pain. Preferably, the composition comprising the antibody or antigen-binding fragment described herein is for use in the treatment of acute pain, neuropathic peripheral pain, chronic pain, and / or osteoarthritis.

[0069] A composition comprising the antibody or antigen-binding fragment described herein for use in treatment may be administered to a subject or patient. The patient may be a female patient. The patient may also be a male patient. A pharmaceutical composition comprising an effective amount of the antibody or antigen-binding fragment may be administered to a patient, or to a patient, for example, a patient at risk of or exhibiting one of the diseases or disorders described herein, by parenteral routes (e.g., subcutaneous, intravenous, intraperitoneal, intramuscular, or percutaneous). "Effective amount" refers to the amount (in terms of dosage, duration of administration, and means of administration) required to achieve the desired therapeutic outcome. The effective amount of antibody may vary depending on factors such as the individual's disease state, age, sex, and weight, and the antibody's ability to induce the desired response in the individual. The effective amount is also the amount in which the therapeutically beneficial effect outweighs any toxic or adverse effects of the antibody of the present invention.

[0070] The antibodies of the present invention can be used in the treatment of patients. More specifically, the antibodies of the present invention are expected to treat or prevent diseases or conditions that may be affected by hPRL modulation, such as pain, such as acute pain, neuropathic peripheral pain, chronic pain, or osteoarthritis.

[0071] Where used interchangeably herein, “treatment” and / or “to treat” and / or “to treat” are intended to refer to all processes which may result in slowing, interrupting, inhibiting, controlling, stopping or reversing the progression of the disorder described herein, but not necessarily indicating the complete elimination of all disorder symptoms. Treatment includes the administration of antibodies of the present invention for the treatment of a disease or condition in a human who is expected to benefit from reduced hPRL activity, and includes: (a) inhibiting further progression of the disease; and (b) mitigating the disease, i.e., causing regression of the disease or disorder, or alleviating its symptoms or complications.

[0072] In accordance with the common sense in the art, the term “activity” is understood to mean a measure of biological function. In particular, as used herein, “activity” refers to the degree of biological function of hPRL, hPRLR, and antibodies or antigen-binding fragments. The biological functions of hPRLR include, but are not limited to, cell surface receptor signaling, activation of JAK2 kinase activity, JAK-STAT cascade activity, binding to PRL, binding to growth hormone, and binding to placental lactogens. The biological functions of hPRL include, but are not limited to, binding to PRLR and its cell surface receptor signaling and initiation of downstream pathways. The biological functions of antibodies or their antigen-binding fragments include, but are not limited to, binding to antigens, as described above. Furthermore, as used herein, the effect of antigen binding by antibodies or their antigen-binding fragments can neutralize or inhibit the normal activity of the antigen, as described above.

[0073] In line with the common sense in the art, the expression "efficacy" is understood to mean a measure of the degree to which an antibody or its antigen-binding fragment inhibits the hPRL-inducible activation of hPRLR. In particular, maximum hPRLR activity can be obtained by the addition of hPRL alone. Complete inhibition or neutralization of hPRL can be observed when the addition of the antibody yields a sample indistinguishable from a sample that has not been administered the hPRL agonist. A concentration-response curve can be created by plotting the percentage of maximum hPRLR activity against the antibody concentration, and this can then be used to further quantify the ability of the antibody or its antigen-binding fragment to neutralize hPRL agonism using concentration-based values ​​(IC). 50 ) can be determined.

[0074] In another aspect of the present invention, a composition comprising an antibody or antigen-binding fragment described herein is provided for use as a pharmaceutical.

[0075] In another aspect of the present invention, the use of the compositions described herein for use in the manufacture of a pharmaceutical, for example, a pharmaceutical for the treatment of a disease or condition modulated by hPRL. The pharmaceutical may, in some cases, be for the treatment of acute pain, neuropathic peripheral pain, chronic pain, and / or osteoarthritis.

[0076] Another aspect of the present invention provides a method for treating a disease or condition modulated by hPRL, comprising the step of administering an effective amount of the antibody or its antigen-binding fragment described herein to a patient. The treatment method may optionally be for treating acute pain, neuropathic peripheral pain, chronic pain, or osteoarthritis.

[0077] In another aspect of the present invention, antibodies or antigen-binding fragments or pharmaceutical compositions thereof described herein are provided for use in therapeutic purposes. In some embodiments, the present invention provides antibodies or pharmaceutical compositions thereof for use in the treatment of pain.

[0078] In another aspect of the present invention, nucleic acid molecules encoding the antibody of the present invention are provided. In one embodiment, the present invention provides a DNA molecule comprising a polynucleotide sequence encoding HC, where the amino acid sequence of HC is SEQ ID NO: 1. According to some such embodiments, the DNA molecule has a polynucleotide sequence given by SEQ ID NO: 12.

[0079] In another aspect of the present invention, a DNA molecule is provided comprising a polynucleotide sequence encoding LC, where the amino acid sequence of LC is Sequence ID No. 2. According to some such embodiments, the DNA molecule has a polynucleotide sequence given by Sequence ID No. 13.

[0080] In another aspect of the present invention, a DNA molecule is provided comprising a polynucleotide sequence encoding an HC having the amino acid sequence of SEQ ID NO: 1, and a polynucleotide sequence encoding an LC having the amino acid sequence of SEQ ID NO: 2. In a particular embodiment, the polynucleotide sequence encoding an HC having the amino acid sequence of SEQ ID NO: 12 is given by SEQ ID NO: 12, and the polynucleotide sequence encoding an LC having the amino acid sequence of SEQ ID NO: 2 is given by SEQ ID NO: 13.

[0081] In another aspect of the present invention, mammalian cells transformed with a DNA molecule are provided, which can express compounds comprising HC and LC of the present invention, where HC is given by SEQ ID NO: 1 and LC is given by SEQ ID NO: 2. The present invention also provides a process for producing compounds comprising HC and LC, which includes culturing mammalian cells under conditions such that the antibody of the present invention is expressed. The present invention also provides an antibody produced by the said process.

[0082] In another aspect of the present invention, an antibody or antigen-binding fragment is provided that contacts hPRL at an epitope, where the epitope comprises the following residues of SEQ ID NO: 7 (Thr); 10 (Arg); 58 (Tyr); 65 (Arg); 76 (Ser); 85 (Thr); 117 (Ser); and 125 (Ser). In such embodiments, the epitope is determined by crosslinked high-mass MALDI mass spectrometry and nLC-Q-Exactive Plus tandem mass spectrometry.

[0083] In a particularly preferred embodiment of the present invention, the antibody is antibody 37 or an antigen-binding fragment thereof. In this specification, “antibody 37” is understood to mean the antibody expressed and purified according to Example 1 below. The characteristics of “antibody 37” are described below as an example: Antibody 37 binds to hPRL. Antibody 37 contains LCs, including LCDR1, LCDR2, and LCDR3. LCDR1 has an amino acid sequence selected from SEQ ID NO: 8. LCDR2 has an amino acid sequence selected from SEQ ID NO: 9. LCDR3 has an amino acid sequence selected from SEQ ID NO: 10. Antibody 37 contains HCs, including HCDR1, HCDR2, and HCDR3. HCDR1 has an amino acid sequence selected from SEQ ID NO: 5. HCDR2 has an amino acid sequence selected from SEQ ID NO: 6. HCDR3 has an amino acid sequence selected from SEQ ID NO: 7. Antibody 37 contains LCVRs, having an amino acid sequence selected from SEQ ID NO: 4. Antibody 37 contains HCVRs, having an amino acid sequence selected from SEQ ID NO: 3. Antibody 37 binds to an epitope having an amino acid sequence selected from SEQ ID NO: 11. Antibody 37 is an antibody against IgG4(S228P).

[0084] Herein, embodiments of the present invention are described for illustrative purposes only, with reference to the accompanying drawings. [Brief explanation of the drawing]

[0085] [Figure 1] This line graph shows the binding and dissociation dynamics of antibody 37 that binds to hPRL at various concentrations. [Figure 2] This graph shows the hPRLR activity assay using antibody 37. [Modes for carrying out the invention]

[0086] method The antibody or antigen-binding fragment of the present invention can be prepared and purified using known methods. For example, cDNA sequences encoding HC (amino acid sequence given by SEQ ID NO: 1) and LC (amino acid sequence given by SEQ ID NO: 2) can be cloned into a glutamine synthetase (GS) expression vector and manipulated. Subsequently, the manipulated immunoglobulin expression vector can be stably transfected into CHO cells. As those skilled in the art will understand, mammalian expression of antibodies is typically thought to result in glycosylation at a highly conserved N-glycosylation site within the Fc region. Stable clones can be validated for the expression of antibodies that specifically bind to hPRL. Positive clones can be amplified in chemically defined serum-free culture medium for antibody production in a bioreactor. Antibodies secreted into the medium can be purified by conventional methods. For example, the medium can be conveniently applied to a Protein A or G Sepharose FF column equilibrated with a suitable buffer such as sodium phosphate (pH 7.0). The column is washed to remove nonspecific binding components. The bound antibody is eluted, for example, by a pH gradient, and the antibody fraction is detected, for example, by reverse-phase chromatography and then pooled. The antibody can be concentrated and / or filtered sterile using common techniques. Soluble aggregates and polymers can be effectively removed by common techniques including size exclusion, hydrophobic interactions, ion exchange, or hydroxyapatite chromatography. The product can be immediately frozen at -80°C or lyophilized, for example.

[0087] The antibody or antigen-binding fragment can be incorporated into a pharmaceutical composition comprising the antibody of the present invention and one or more pharmaceutically acceptable carriers, diluents, or excipients, which can be prepared by methods well known in the art.

[0088] antibody manipulation Parental mouse monoclonal anti-hPRL antibodies were produced by immunizing mice with hPRL and then generating and screening hybridomas using a well-characterized method. The CDRs of the parental antibodies were sequenced, and humanized anti-hPRL antibodies were optimized using a framework library approach. For the framework library, four human VH framework germline genes (IGHV1-46, IGHV1-69, IGHV1-3, IGHV1-46*01 / 4m) containing anti-hPRL CDRs and four human VL framework genes (IGKV7-3, IGKV4-1, IGKV1-39, IGKV7-3*01 / 4m) were synthesized and cloned into HC and LC human IgG1 expression vectors. All 16 HC and LC combinations were transiently transfected into HEK 293 cells, and the supernatant was assayed by ELISA for binding to hPRL directly coated on plates. The purified antibodies were also screened for neutralization of hPRL activation by in vitro activity assays. Humanized antibodies with CDRs derived from parental mouse antibodies were selected for further development using the IGHV1-3 or IGHV1-46 HC human framework and the IGKV1-39 or IGKV4-1 human LC framework. The cDNA sequences of selected variable regions containing the IgG1 or IgG4 HC constant region (with a known hinge stability mutation) were cloned into GS expression vectors. The selected antibody candidates were compared by expression titer, purification yield, and neutralization activity, as well as by affinity for human, cynomolgus monkey, and mouse PRL, using in vitro activity assays. Overall, antibody 37, a humanized anti-hPRL antibody using the IGHV1-3 human HC framework and the IGKV1-39 human LC framework containing the human IgG4 HC constant region with a known hinge mutation, exhibited favorable properties compared to the other candidates.

[0089] Experimental example [Examples]

[0090] Antibody expression and purification The antibodies of the present invention can be biosynthesized, purified, and formulated for administration by known methods. Suitable host cells are commercially available from Lonza Bioscience. For example, a suitable host cell, e.g., HEK 293 or CHO, is transfected either transiently or stably with an expression system for antibody secretion, using a predetermined HC:LC vector ratio when using two vectors, or using a single vector system encoding both HC and LC. Suitable vectors for antibody expression and secretion from these commonly used host cells are well known.

[0091] After antibody expression and secretion, the culture medium is clarified to remove cells and purified using one of many commonly used methods. For example, the medium can be applied to a protein A or G column equilibrated with a buffer such as sodium phosphate (pH 7.0). The column is washed to remove nonspecific binding components. The bound antibody is eluted, for example, by a pH gradient (e.g., from 0.1 M sodium phosphate buffer 6.8 to 0.1 M sodium citrate buffer pH 2.5). The antibody fraction is detected by reverse-phase chromatography and subsequently pooled. Further purification is optional depending on the intended use. The antibody can be concentrated and / or filtered sterile using common methods. Substances other than the antibody, such as host cell and growth medium components, as well as soluble aggregates and polymers of the antibody, can be effectively reduced or removed by common methods including size exclusion, hydrophobic interactions, ion exchange, or hydroxyapatite chromatography. The product can be immediately frozen at -80°C or lyophilized, for example.

[0092] Exemplary antibody 37 was transiently expressed in HEK 293 cells after simultaneous transfection with separate HC expression DNA vectors and LC expression DNA vectors incorporating the DNA sequences of SEQ ID NO: 12 and SEQ ID NO: 13, respectively, or stably expressed in CHO cells after transfection with a single DNA vector incorporating the DNA sequences of both SEQ ID NO: 12 and SEQ ID NO: 13, which encode HC and LC, respectively. Culture media collected from either a 6-day transient HEK 293 culture or a 15-day CHO fuel-batch culture were clarified, and the resulting crude supernatant was purified by protein A chromatography. Antibody 37, bound to protein A resin, was eluted using a low pH buffer. The eluted antibody was further purified by size exclusion chromatography. The final purity of antibody 37 was evaluated using analytical size exclusion chromatography and LC / MS analysis. The purified antibody 37 was stored in phosphate-buffered saline (pH 7.4) at 4°C. [Examples]

[0093] In vitro binding affinity and kinetics The binding kinetics and affinity of antibody 37 to human, cynomolgus monkey, and mouse PRL were determined using biolayer interferometry on a ForteBio Octet-RED96 instrument. An anti-human IgG Fc capture sensor was used as the capture system. Antibody 37 was diluted to 5 μg / mL. Human, cynomolgus monkey, and mouse PRL were prepared at final concentrations of 100, 33, and 11 nM. Each analytical cycle included: (1) capturing the antibody on the sensor; (2) quenching the open binding site with 150 μg / mL control human gamma globulin; (3) immersing the sensor in PRL for 600 seconds; (4) returning to buffer for 600 seconds to monitor antigen dissociation; and (5) regenerating the sensor surface and repeating the cycle. Biomolecular interaction analysis was performed using ForteBio Data Analysis software with a 1:1 binding model to determine the association rate (on-rate, k). on M -1 s-1 (Units) and dissociation rate (off-speed, k off , s -1 Determine the unit. Equilibrium dissociation constant (K D ) is K D =k off / k on This is calculated from the relationship and is in moles.

[0094] Figure 1 shows the binding and dissociation dynamics of antibody 37 against hPRL. More specifically, Figure 1 shows curves representing the binding and dissociation of antibody 37 against 11 nM, 33 nM, and 100 nM hPRL, along with the best-fit curve. Antibody 37 exhibits rapid binding to hPRL, which is dependent on the antigen concentration. The best-fit curve for binding is used to determine the association rate (k on M -1 s -1 The (expressed in units) was determined. After binding, the sensor with immobilized antibody 37 and bound hPRL was moved into a buffer and the dissociation rate was monitored. Antibody 37 showed very slow dissociation over time, which indicates strong binding to hPRL (k off , s -1 (Represented by ). Binding curves for cynomolgus monkey PRL and mouse PRL are not shown. Antibody 37 shows similar binding kinetics and affinity for cynomolgus monkey PRL and hPRL. The interaction between antibody 37 and mouse PRL showed a limited response above the background, and therefore kinetic measurements could not be determined (ND). Thus, K for mouse PRL concentration D The values ​​are estimated to be higher than the maximum concentration tested (100 nM). Taken together, these results indicate that antibody 37 has high affinity for both hPRL and cynomolgus monkey PRL, but shows only limited cross-reactivity with mouse PRL.

[0095] Table 2 below lists the PRL binding kinetics and affinity of the antibody of the present invention, determined from a global fit of the curve shown in Figure 1 for hPRL.

[0096] [Table 2] [Examples]

[0097] Neutralization of hPRL activity in an in vitro CHO-hPRLR phospho-STAT5 cell signaling assay. Using CHO cells that stably express PRLR, we determined the ability of antibody 37 to inhibit PRL-inducible activity. In response to PRL, PRLR activates the JAK2 / STAT5 pathway. This activity can be measured using a fluorescence resonance energy transfer (FRET) assay designed to detect phosphorylated STAT5.

[0098] Cells were treated with hPRL, mouse PRL, cynomolgus monkey PRL, human growth hormone (GH), or human placental lactogen (PL) in or without antibody 37. For each test, antibody 37 was pre-incubated with PRL or other hormone at room temperature for 30 minutes. Prior to treatment, CHO-PRLR cells were plated in white, low-volume 384-well assay plates at a rate of 100,000 cells per well (8 μL). Subsequently, the plated cells were treated with 4 μl of antibody-PRL mixture added to each of the three wells. hPRL was added at a constant dose of 20 nM, and the dose range of antibody 37 was 0–150 nM. Culture medium only was used as the untreated control. Subsequently, the assay plates were sealed with a porous plate sealer and incubated at 37°C, 95% relative humidity, and 5% CO2 for 15 minutes. After incubation, cells were lysed by adding 3 μl of lysis buffer (Bioauxilium) containing a phosphatase inhibitor cocktail to each well. The plates were then resealed with a plate sealer and incubated at room temperature for 30 minutes with gentle shaking (400 rpm). After lysis, 5 μl of diluted detection antibody mixture (Bioauxilium) was added to each well. The assay plate was sealed and incubated overnight at room temperature in the dark. After incubation, the plate sealer was removed from the assay plate, and the TR-FRET fluorescence intensity signal was measured using a TECAN SPARK multimode plate reader at excitations of 620 nm and 665 nm. The results are shown in Table 3 below (Antibody 37 IC in in vitro CHO-hPRLR phospho-STAT5 cell signaling assay). 50 ).

[0099] The results of the hPRLR activity assay are shown in Figure 2, which demonstrates hPRLR activation by 20 nM hPRL upon addition of various concentrations of antibody 37. IC for neutralization of antibody 37 by hPRL and other agonists. 50 The measured values ​​are shown in Table 3.

[0100] [Table 3] [Examples]

[0101] Epitope mapping by cross-linked mass spectrometry The epitopes recognized on the hPRL antigen by antibody 37 were determined using a high-resolution method developed by CovalX. A 5 μM / 1.25 μM mixture of hPRL / antibody 37 was incubated with a deuterated crosslinking agent and then subjected to multi-enzyme cleavage. 1 μL of control solution and crosslinked solution were loaded into an UltiMate 3000-RSLC nanoliquid chromatography system connected in series with a Q-Exactive Plus Orbitrap mass spectrometer. The generated spectra were analyzed using XQuest and Stavrox software. Crosslinking analysis showed that the epitopes on hPRL recognized by antibody 37 were discontinuous and contained the following amino acid residues of SEQ ID NO: 11: T7, R10, Y58, R65, S76, T85, S117, and S125.

[0102] Array Overview The following array is referenced: HC is related to Sequence ID No. 1; LC is related to Sequence ID No. 2; HCVR is associated with sequence number 3; LCVR is related to sequence number 4; HCDR1 is associated with sequence number 5; HCDR2 is related to Sequence ID No. 6; HCDR3 is associated with sequence number 7; LCDR1 is related to sequence number 8; LCDR2 is related to sequence number 9; LCDR3 is related to Sequence ID 10; Human PRL amino acids 39-199 are related to SEQ ID NO: 11; The DNA encoding the HC in SEQ ID NO: 1 is related to SEQ ID NO: 12; The DNA encoding LC in SEQ ID NO: 2 is related to SEQ ID NO: 13.

[0103] More specifically, the following sequences are referenced: the HC of antibody 37 is SEQ ID NO: 1, the LC of antibody 37 is SEQ ID NO: 2, the HCVR of antibody 37 is SEQ ID NO: 3, the LCVR of antibody 37 is SEQ ID NO: 4, HCDR1 is SEQ ID NO: 5, HCDR2 is SEQ ID NO: 6, HCDR3 is SEQ ID NO: 7, LCDR1 is SEQ ID NO: 8, LCDR2 is SEQ ID NO: 9, LCDR3 is SEQ ID NO: 10, human PRL amino acids 39-199 are SEQ ID NO: 11, the DNA encoding the HC of SEQ ID NO: 12, and / or the DNA encoding the LC of SEQ ID NO: 2 is SEQ ID NO: 13.

[0104] The following sequence listings will also be submitted in the form of the corresponding WIPO standard ST.26.

[0105] Sequence List Sequence ID 1 QVQLVQSGAEVKKPGASVKVSCKASGYTFSNFWIEWVRQAPGQRLEWMGEIFPGTGSTYYTEKFKVRVTITRDTSASTAYMELSSLRSEDTAVYYCARRGYSDSWFAHWGQ GTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Sequence ID 2 DIQMTQSPSSLSASVGDRVTITCRASESVDMYGKSFMHWYQQKPGKAPKLLIYRASTLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNVEAPLTFGGGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence ID 3 QVQLVQSGAEVKKPGASVKVSCKASGYTFSNFWIEWVRQAPGQRLEWMGEIFPGTGSTYYTEKFKVRVTITRDTSASTAYMELSSLRSEDTAVYYCARRGYSDSWFAHWGQGTLVTVSS Sequence ID 4 DIQMTQSPSSLSASVGDRVTITCRASESVDMYGKSFMHWYQQKPGKAPKLLIYRASTLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNVEAPLTFGGGTKVEIK Sequence ID 5 YTFSNFWIE Sequence ID 6 EIFPGTGSTYYTEKFKV Sequence ID 7 RGYSDSWFAH Sequence ID 8 RASESVDMYGKSFMH Sequence ID 9 RASTLES Sequence ID 10 QQNVEAPLT Sequence ID 11 EFDKRYTHGRGFITKAINSCHTSSLATPEDKEQAQQMNQKDFLSLIVSILRSWNEPLYHLVTEVRGMQEAPEAILSKAVEIEEQTKRLLEGMELIVSQVHPETKENEIYPVWSGLPSLQMADEESRLSAYYNLLHCLRRDSHKIDNYLKLLKCRIIHNNNC Sequence ID 12 sequence number 13 GACATTCAGATGACACAGAGCCCTAGCAGCCTGAGCGCTAGCGTGGGCGACAGAGTGACCATCACCTGCAGAGCTAGCGAGAGCGTGGACATGTACGGCAAGAGCTTCATGCACTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACAGAG CTAGCACCCTGGAGAGGCGGCGTGCCTAGCAGATTCAGCGGCAGCGGCAGCGGCACCGACTTCCACCTGACCATCAGCAGCCTGCAGCCTGAGGACTTCGCCACCTACTGTCAGAACGTGGAGGCCCCTCTGACCTTCGGCGGCGCACCAAGGTGGAG ATCAAGAGAACCGTGGCGCCCTAGCGTGTTCATCTTCCCTCCTAGCGACGAGCAGCTGAAGAGCGGCACCGCTAGCGTGGTGTGCCTGCTGAACAACTTCTACCCTAGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCC AAGAGAGCGTGACCGAGCAAGACAGCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAAGGCCTGAGCAGCCCTGTGACCAAGAGCTTCAACAGAGGCGAGTGC

Claims

1. An antibody or its antigen-binding fragment that binds to human prolactin (hPRL).

2. The light chain (LC) includes one or more of the LC complementarity determining regions (CDRs) LCDR1, LCDR2, and / or LCDR3. LCDR1 has an amino acid sequence selected from SEQ ID NO: 8, LCDR2 has an amino acid sequence selected from SEQ ID NO: 9, and / or LCDR3 has an amino acid sequence selected from SEQ ID NO:

10. The antibody or its antigen-binding fragment according to claim 1.

3. It contains a heavy chain (HC) which includes one or more of HCDR1, HCDR2, and HCDR3, HCDR1 has an amino acid sequence selected from SEQ ID NO: 5, HCDR2 has an amino acid sequence selected from SEQ ID NO: 6, and / or HCDR3 has an amino acid sequence selected from SEQ ID NO:

7. The antibody or its antigen-binding fragment according to claim 1 or 2.

4. The antibody or antigen-binding fragment according to any one of the claims, comprising an LC variable region (LCVR) wherein the LCVR has an amino acid sequence selected from SEQ ID NO: 4, and / or comprising an HC variable region (HCVR) wherein the HCVR has an amino acid sequence selected from SEQ ID NO:

3.

5. An antibody or antigen-binding fragment thereof according to any one of the claims, which binds to an epitope having an amino acid sequence selected from SEQ ID NO:

11.

6. The antibody or antigen-binding fragment thereof according to any one of the claims, which is an antibody or antigen-binding fragment thereof of immunoglobulin G (IgG), for example, an antibody or antigen-binding fragment thereof of IgG of subclass IgG1, IgG2, IgG3 and / or IgG4.

7. An antibody or antigen-binding fragment thereof according to any one of the claims, which binds to PRL with an affinity of approximately 0.6 nM.

8. The hPRL-inducible activation of hPRLR is, for example, in an IC of approximately 3.1 nM. ５０ An antibody or antigen-binding fragment according to any of the claims, which neutralizes with in vitro efficacy.

9. (i) LCDR1 has an amino acid sequence selected from SEQ ID NO: 8, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to SEQ ID NO: 8, (ii) LCDR2 has an amino acid sequence selected from SEQ ID NO: 9, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to SEQ ID NO: 9, (iii)LCDR3 has an amino acid sequence selected from SEQ ID NO: 10, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to SEQ ID NO:

10. (iv) HCDR1 has an amino acid sequence selected from SEQ ID NO: 5, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to SEQ ID NO:

5. (v) HCDR2 has an amino acid sequence selected from SEQ ID NO: 6, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to SEQ ID NO: 6, and / or (vi) HCDR3 has an amino acid sequence selected from SEQ ID NO: 7, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to SEQ ID NO:

7. An antibody or its antigen-binding fragment according to any one of claims 1, 4 to 8.

10. (i) LCDR1 contains the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence that is at least 60% identical to SEQ ID NO: 8, (ii) LCDR2 contains the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence that is at least 60% identical to SEQ ID NO:

9. (iii)LCDR3 contains the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence that is at least 60% identical to SEQ ID NO: 10, (iv) HCDR1 contains the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence that is at least 60% identical to SEQ ID NO: 5, (v) HCDR2 contains the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence that is at least 60% identical to SEQ ID NO: 6, and / or (vi) HCDR3 contains the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence that is at least 60% identical to SEQ ID NO:

7. An antibody or its antigen-binding fragment according to any one of claims 1, 4 to 8.

11. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any of the above claims.

12. The pharmaceutical composition according to claim 11, comprising a pharmaceutically acceptable carrier, excipient and / or diluent.

13. A composition comprising an antibody or antigen-binding fragment according to any one of claims 1 to 10, for use in the treatment of a disease or condition modulated by hPRL, for example, for use in the treatment of pain, such as acute pain, neuropathic peripheral pain, chronic pain, and / or osteoarthritis.

14. Use of the composition according to claim 11 or 12 for the manufacture of a pharmaceutical product.

15. A method for treating a disease or condition modulated by PRL, for example, a pain, for example, acute pain, neuropathic peripheral pain, chronic pain, or osteoarthritis, comprising the step of administering an effective amount of the antibody or antigen-binding fragment described in any one of claims 1 to 10 to a patient.

Images