Novel Fab Dimer

Abstract

The present invention relates to novel dimers composed of a first Fab monomer and a second Fab monomer, each Fab monomer comprising a VH domain and a VL domain, where two of such VH or VL domains are covalently linked by a disulfide bond between additional non-naturally occurring cysteine ​​residues at their respective N-termini.

Description

TECHNICAL FIELD

[0001] The present invention relates to a novel dimer composed of a first Fab monomer and a second Fab monomer, each Fab monomer comprises a VH region and a VL region, wherein two of said VH regions or VL regions are covalently linked via a disulfide bond between an additional non-naturally occurring cysteine residue at their respective N-terminus. BACKGROUND ART

[0002] Carbonic anhydrases are a family of enzymes that catalyze the reversible hydration of carbon dioxide into bicarbonate ions and protons (hydrogen ions), and are therefore involved in maintaining pH homeostasis in the body (Badger et al., Annu Rev Plant Physiol Plant Mol Bio (1994), 45: 369-392). In the absence of a catalyst, this reaction proceeds quite slowly. Since most carbonic anhydrases contain a zinc ion at their active site, they are classified as metalloenzymes. The carbonic anhydrase family includes several members. There are at least five distinct carbonic anhydrase subfamilies (α, β, γ, δ and ε). These subfamilies do not share significant amino acid sequence similarity and are considered in most cases to be an example of convergent evolution. α-carbonic anhydrases (CA) are found in mammals. Members of this subfamily can be distinguished with respect to their kinetics, tissue expression, and intracellular localization (Kivela et. al., World J Gastroenterol (2005), 11(2): 155-163).

[0003] α-CA enzymes are divided into four broad subgroups: cytoplasmic CAs (CA-I, CA-II, CA-III, CA-VII, and CA-XIII), mitochondrial CAs (CA-VA and CA-VB), secretory CAs (CAVI), and membrane-bound CAs (CA-IV, CA-IX, CA-XII, CA-XIV, and CA-XV) (Breton et al. (2001), JOP 2 (4 Suppl):159-64). In addition, there are three "catalyst" CA isoforms (CA-VIII, CA-X, and CA-XI), whose functions are still unknown. Several further isoforms exist for all of these CAs.

[0004] CA-II, CA-IX, and CA-XII are associated with tumorigenetic processes and may serve as histological and prognostic biomarkers for various tumors (Nordfors et al. (2010), BMC cancer; 10:148). CA-II is the most widely expressed member of the α-CA gene family and is present in virtually every human tissue and organ. It is known as one of the most catalytically efficient enzymes. It is present to some extent in malignant cells and, interestingly, has recently been found to be ectopically expressed in endothelial cells of tumor neovascularization. CA-IX, a transmembrane enzyme, was first recognized as a novel tumor-associated antigen expressed not only in normal gastrointestinal tissue but also in several types of human cancers. Functionally, CA-IX is associated with cell adhesion, differentiation, proliferation, and carcinogenesis processes, and its enzymatic activity is comparable to that of CA-II. Another transmembrane CA isozyme, CA-XII, was first discovered in normal kidney tissue and renal cell carcinoma. Further studies have shown that it is expressed in several other tumors (Ulmasov et al., PNAS (2000), 97(26): 1412-1417); however, it is also expressed in some normal organs such as the colon and uterus. X-ray crystallography of human CA-XII reveals that it is a bitopic dimeric protein, with its short intracellular C-terminus located opposite the active site domain, and the latter (active site domain) face toward the extracellular space.

[0005] The high expression of CA-II, CA-IX, and CA-XII in tumors, particularly those in a hypoxic state, further suggests that these enzymes may functionally be involved in the invasive process facilitated by the acidification of the extracellular space. Supporting this hypothesis, CA inhibitors have been shown in vitro to reduce the invasiveness and proliferation of cancer cells (Manokaran et al. (2008), J Biomed Nanotechnol., 4(4):491-498). In particular, CA-IX and CA-XII appear to be regulated by similar mechanisms, as the transcription of these isozymes is induced in hypoxic tumors via a hypoxia-inducible factor-1α (HIF-1α)-mediated pathway (Chiche et al. (2009)). Furthermore, CA-XII expression has been shown to be highly correlated with estrogen receptor alpha (ERα) in mammary tumors (Barnett et al. (2008), Cancer Res 68:3505-3515). To elaborate on the importance of CA in cancer progression, rapidly growing tumor cells rapidly overgrow as oxygen diffusion from the nearest blood vessels (100-150 μm) is impaired. As a result, tumor cells receive low levels of oxygen, leading to localized hypoxic centers and tissue necrosis. Hypoxia creates selective pressure in cells to adapt to the stressful state, resulting in the expression of approximately 50 additional proteins, including enzymes involved in pH homeostasis (Potter et al. (2004), Gell Cycle, 3:164-167). As described above, the latter is achieved, at least in part, by complex coordination among selected carbonic anhydrase (CA) isozymes, particularly between CA-II, CA-IX, and CA-XII. A direct link between CA XII and cancer has been demonstrated by Proescholdt et al. (2005), Neuro Onco 7:465-475. This study shows that CA XII expression is upregulated in endogenous and metastatic brain tumors compared to normal brain tissue.Furthermore, Ilie et al. (2011), In J Cancer, 128(7): 1614-23 and Hynninen et al. (2006), Histopathology, 49:594-602 demonstrated overexpression of CA XII in resectable non-small cell lung cancer and ovarian cancer-derived tissues, respectively. Hsieh et al. (2010), Eur J Gell Biol, 89:598-606 revealed that CA XII is associated with tumor cell line invasion and metastasis both in vivo and in vitro.

[0006] Furthermore, CA inhibitors, particularly those of CA-II and CA-XII, lower intraocular pressure and are therefore used to treat ocular hypertension (Al-Barrag et al. (2009), Clinical Ophthalomology 3:357-362). CA inhibitors have also been shown to be useful in treating glaucoma (Haapasalo et al. (2008), Neuro Oncology 3:357-362 and Vullo et al. 2005).

[0007] Thus, CA, particularly CA-XII, is known to contribute to hypoxia, cancer, and eye diseases, and is therefore an important target for therapeutic intervention or diagnosis (Thiry et al 2008; Vulo et al 2005; and Haapasalo et al. (2008), Neuro Oncology 3:357-362). Systemic carbonic anhydrase inhibitors are known in the art, but they are associated with adverse side effects such as acid-base imbalances, hypersensitivity reactions, and fatal aplastic anemia (Gross et al. (1988), Am J Opthamol.; Naeser et al. (1986), Acta Opthalmol., 64:330-337; Mastropasqua et al. (1998), 212:318-321; and Passp et al. Br J Opthalmol. 1985; 69:572-575). Therefore, there is a need for further means and methods that can be employed in the development of further therapeutic and diagnostic means for the aforementioned diseases.

[0008] These and further difficulties need to be overcome. Therefore, the present invention addresses these needs and technical objectives by providing solutions as described herein and defined in the claims. [Overview of the project]

[0009] The present invention relates to a dimer composed of a first Fab monomer and a second Fab monomer, wherein each Fab monomer includes a VH region and a VL region, where (i) The VL region of the first Fab monomer and the VL region of the second Fab monomer are covalently linked by a disulfide bond between an additional cysteine ​​residue that is not naturally present, located at the N-terminus of the VL region of the first Fab monomer and the N-terminus of the VL region of the second Fab monomer; (ii) The VH region of the first Fab monomer and the VH region of the second Fab monomer are covalently linked by a disulfide bond between an additional cysteine ​​residue that is not naturally present, located at the N-terminus of the VH region of the first Fab monomer and the N-terminus of the VH region of the second Fab monomer; (iii) The VL region of the first Fab monomer and the VH region of the second Fab monomer are covalently linked by a disulfide bond between an additional cysteine ​​residue that is not naturally present, located at the N-terminus of the VL region of the first Fab monomer and the N-terminus of the VH region of the second Fab monomer; or (iv) The VH region of the first Fab monomer and the VL region of the second Fab monomer are covalently linked by disulfide bonds between additional cysteine ​​residues that do not exist in nature, located at the N-terminus of the VH region of the first Fab monomer and the N-terminus of the VL region of the second Fab monomer.

[0010] As found in connection with the present invention, Fab monomers are dimerized via cysteine ​​bonds (disulfide bonds) between additional cysteine ​​residues that are not naturally present in the N-terminus of the VL and / or VH regions of the Fab monomer. Therefore, according to the present invention, the presence of additional cysteine ​​residues that are not naturally present in the N-terminus of the VL and / or VH regions of the Fab monomer enables the formation of cysteine ​​bonds (disulfide bonds) between Fab monomers, thereby constructing Fab dimers as described and provided herein. Surprisingly, as found in connection with the present invention and as shown herein, these Fab dimers containing cysteine ​​bonds (disulfide bonds) exhibit increased binding affinity and / or binding activity to target structures (e.g., antigens) in an acidic environment compared to each Fab monomer (directed to the same target structure (e.g., antigen)) that does not contain such cysteine ​​bonds (disulfide bonds) or additional cysteine ​​residues that are not naturally present in the N-terminus of the VL and / or VH regions. According to the present invention, this increased binding affinity and / or affinity to a target structure (e.g., an antigen) in an acidic environment may be particularly important when the target structure (also referred to herein as the target; e.g., an antigen) bound by the Fab dimer is present in cancer or is suitable for the treatment or diagnosis of cancer due to the acidic microenvironment within the tumor (see, for example, Estrella et al., Microenviron Immunol (2013), 73(5): 1524-1535; Boedtkjer et al., Annual Rev Physiol (2020), 82: 103-126; Pillai et al., Cancer Metastasis Rev (2019), 38: 205-222; Ji et al., Cancer Metastasis Rev (2019): 38: 103-112; Gatenby et al., Nature Rev Cancer (2004): 891-899).

[0011] From the above, it was surprising that such dimers could bind to a target, such as a target protein; because it was expected that dimerization via the N-terminus of any of the following regions—two VH regions, two VL regions, a VH region and a VL region, or a VL region and a VH region—would not be able to bind to the target, for example, due to steric hindrance in the variable region that holds the CDR.

[0012] In fact, dimerization very close to the N-terminus of the variable region that provides antigen binding, preferably direct dimerization at the N-terminus, was expected to have an unfavorable effect on antigen binding because it could sterically hinder the antigen-binding site (paratope) from binding to the epitope of the target protein. However, as shown in the examples, the dimer of the present invention, in which two VH regions are dimerized as described herein, binds to the target. Therefore, it is quite reasonable that dimerization at the N-terminus of two VL regions, or between a VH region and a VL region, or between a VL region and a VH region, as described herein, as well as dimerization at the N-terminus of two VH regions, also enables binding to the target.

[0013] In fact, in other antibody formats, the VH region can be fused with the VL region by classical N-terminal-C-terminal fusion, for example, the N-terminus of the VL region can be fused with the C-terminus of the VH region, and vice versa. However, N-terminal / N-terminal fusion is not performed. Similarly, the introduction of disulfide bonds may be a suitable means, for example, to stabilize or dimerize the antibody or its fragments, but to the best of the inventor's knowledge, disulfide bonds are not placed very close to the N-terminus of the variable region, preferably at the N-terminus of the variable region. Rather, cysteine ​​residues are introduced far away from the variable region. [Invention 1001] A dimer composed of a first Fab monomer and a second Fab monomer, where each Fab monomer includes a VH region and a VL region, (i) The VL region of the first Fab monomer and the VL region of the second Fab monomer are covalently linked by a disulfide bond between an additional cysteine ​​residue that is not naturally present, located at the N-terminus of the VL region of the first Fab monomer and the N-terminus of the VL region of the second Fab monomer; (ii) The VH region of the first Fab monomer and the VH region of the second Fab monomer are covalently linked by a disulfide bond between an additional cysteine ​​residue that is not naturally present, located at the N-terminus of the VH region of the first Fab monomer and the N-terminus of the VH region of the second Fab monomer; (iii) The VL region of the first Fab monomer and the VH region of the second Fab monomer are covalently linked by a disulfide bond between an additional cysteine ​​residue that is not naturally present, located at the N-terminus of the VL region of the first Fab monomer and the N-terminus of the VH region of the second Fab monomer; or (iv) The VH region of the first Fab monomer and the VL region of the second Fab monomer are covalently linked by a disulfide bond between an additional cysteine ​​residue that does not exist in nature, located at the N-terminus of the VH region of the first Fab monomer and the N-terminus of the VL region of the second Fab monomer. Dimer. [Invention 1002] A dimer of the present invention 1001, wherein a naturally unpresent cysteine ​​residue is located at amino acid position 1 or amino acid position 2 in the N-terminus of the VL or VH region, where the amino acids are counted from the N-terminus of the VL or VH region, and position 1 is the first amino acid at the N-terminus. [Invention 1003] A dimer of the present invention 1001 or 1002, wherein the first Fab monomer and the second Fab monomer are the same or different. [Invention 1004] A dimer of the present invention, wherein the first Fab monomer and the second Fab monomer are directed toward the same target. [Invention 1005] A dimer according to any of invention 1001 to 1003, wherein the first Fab monomer is directional to a first target, and the second Fab monomer is directional to a second target, and the first target and the second target are different. [Invention 1006] A dimer of any of the present invention, wherein the first Fab monomer comprises VH-CDR1 shown in SEQ ID NO: 1, VH-CDR2 shown in SEQ ID NO: 2, VH-CDR3 shown in SEQ ID NO: 3, VL-CDR1 shown in SEQ ID NO: 4, VL-CDR2 shown in SEQ ID NO: 5, and VL-CDR3 shown in SEQ ID NO: 6. [Invention 1007] Any of the dimers of the present invention, wherein the VL region of the first Fab monomer and the VL region of the second Fab monomer each contain a secretory sequence indicated by SEQ ID NO: 16 at their N-terminus. [Invention 1008] Any of the dimers of the present invention, wherein the VH region of the first Fab monomer and the VH region of the second Fab monomer each contain a secretory sequence indicated by SEQ ID NO: 16 at their N-terminus. [Invention 1009] Any of the dimers of the present invention, wherein the VL region of the first Fab monomer and the VH region of the second Fab monomer each contain a secretory sequence indicated by SEQ ID NO: 16 at their N-terminus. [Invention 1010] Any of the dimers of the present invention, wherein the VH region of the first Fab monomer and the VL region of the second Fab monomer each contain a secretory sequence indicated by SEQ ID NO: 16 at their N-terminus. [Invention 1011] A dimer of the present invention 1007, wherein the VH region of the first Fab monomer and the VH region of the second Fab monomer each contain a secretory sequence indicated by SEQ ID NO: 15 at their N-terminus. [Invention 1012] A dimer of the present invention 1008, wherein the VL region of the first Fab monomer and the VL region of the second Fab monomer each contain a secretory sequence indicated by SEQ ID NO: 15 at their N-terminus. [Invention 1013] A dimer of the present invention 1009, wherein the VH region of the first Fab v and the VL region of the second Fab monomer contain a secretory sequence indicated by SEQ ID NO: 15 at their N-terminus. [Invention 1014] A dimer of the present invention 1010, wherein the VL region of the first Fab monomer and the VH region of the second Fab monomer each contain a secretory sequence indicated by SEQ ID NO: 15 at their N-terminus. [Invention 1015] A dimer of any of the present inventions, wherein the first Fab monomer comprises a VH region indicated by SEQ ID NO: 7 and a VL region indicated by SEQ ID NO: 8. [Invention 1016] A dimer of any of the present inventions, wherein the first Fab monomer comprises a VH region encoded by the nucleotide sequence shown in SEQ ID NO: 9 and a VL region encoded by the nucleotide sequence shown in SEQ ID NO: 10. [Invention 1017] A dimer of any of the present inventions, wherein the first Fab monomer comprises a VH region shown in SEQ ID NO: 11 and a VL region shown in SEQ ID NO: 12. [Invention 1018] A dimer of any of the present inventions, wherein the first Fab monomer comprises a VH region encoded by the nucleotide sequence shown in SEQ ID NO: 13 and a VL region encoded by the nucleotide sequence shown in SEQ ID NO: 14. [Invention 1019] (a) CHO cells expressing the first and second Fab monomers as defined in any of invention 1001 to 1018 are cultured until they reach a stationary phase; (b) optionally adding an oxidizing agent to the CHO cell culture; and (c) Obtaining the dimer A dimer of any of the above-described dimers of the present invention obtained by the present invention. [Invention 1020] The first and / or second Fab monomers are (a) labeling group; (b) toxins; or (c) Antitumor drugs A dimer of any of the present inventions bonded to the above. [Invention 1021] A composition comprising any dimer of invention 1001 to 1020. [Invention 1022] A composition of the present invention 1021, further comprising a Fab monomer as defined in any of the present inventions 1001 to 1020. [Invention 1023] A dimer of any of Invention 1001 to 1020 or a composition of Invention 1021 or 1022 for use as a pharmaceutical. [Invention 1024] A dimer of any of Invention 1001 to 1020 or a composition of Invention 1021 or 1022 for use in a method for treating or suppressing hypoxia, solid tumors, or eye diseases. [Invention 1025] A dimer or composition of the present invention 1024, wherein the hypoxia is selected from tumor hypoxia, nerve hypoxia, cerebral hypoxia, stenosis, and ischemia. [Invention 1026] A dimer or composition of the present invention 1024, wherein the solid tumor is selected from sarcoma, glioma, carcinoma, mesothelioma, lymphoma, renal tumor, lung tumor, mammary gland tumor, cervical tumor, ovarian tumor, colorectal tumor, liver tumor, prostate tumor, pancreatic tumor, and head and neck tumor. [Invention 1027] A dimer or composition of the present invention 1024, wherein the aforementioned eye disease is selected from ocular hypertension, glaucoma, macular degeneration, age-related macular degeneration, uveitis, retinitis, X-linked retinoschisis, and hypertensive retinopathy. [Modes for carrying out the invention]

[0014] In general, in relation to the present invention, the additional cysteine ​​residue at the N-terminus of the VL and / or VH regions of the Fab monomer described herein, which is not naturally occurring, may be located at any position within each VL or VH region, closer to the N-terminus than to the C-terminus. In one aspect of the present invention, it may be within the first 10 N-terminal amino acids of each VL or VH region (the first amino acid of each VL or VH region is counted as N-terminal position 1), more preferably within the first 3 N-terminal amino acids, and most preferably within the first 2 N-terminal amino acids. That is, in this aspect of the present invention, the cysteine ​​residue at the N-terminus of the VL or VH region, which is not naturally occurring, is located at amino acid position 1 or amino acid position 2; where the amino acids are counted from the N-terminus of each VL or VH region, with position 1 being the first N-terminal amino acid.

[0015] As used herein and as readily apparent to those skilled in the art, “Fab” (or “Fab molecule”) is an antibody fragment, such as those described herein, and is widely known in the art. It may be a binding substance comprising a variable light chain (VL chain) or VL region and a variable heavy chain (VH chain) or VH region, which together form a binding region or motif that enables the binding of Fab to a target structure such as an antigen or epitope. The VH chain or region and the VL chain or region of “Fab” may further comprise a constant region or a portion of a constant region. As used herein and as reasonably applicable to those skilled in the art, the term “Fab” may also include F(ab')2, Fab', Fv, scFv, Fd, dAb, and other antibody fragments that retain antigen-binding function. Typically, such fragments would contain an antigen-binding domain and would have the same properties as the Fab described herein.

[0016] In relation to the present invention, as used herein, "additional" in relation to "additional cysteine ​​residues not naturally occurring" at the N-terminus of the VL and / or VH regions of the Fab monomer described herein may mean: (i) a cysteine ​​residue is present at the N-terminus of each VL and / or VH region of the Fab monomer in addition to the amino acids constituting the respective reference (i.e., natural) sequence (thus extending the total length of the amino acid sequence of the reference VL or VH region by one amino acid); or (ii) one of the amino acids constituting the respective reference (i.e., natural) sequence of each VL and / or VH region is replaced with a cysteine ​​residue (thus remaining unchanged in total length of the amino acid sequence of the reference VL or VH region, but adding one cysteine ​​residue compared to the reference sequence). In preferred embodiments of the present invention, "additional" means the addition of a cysteine ​​residue to another amino acid constituting the respective reference (i.e., natural) sequence of each VL and / or VH region, in accordance with (i) above.

[0017] Similarly, in relation to the present invention, as used herein, "naturally absent" in relation to the "naturally absent additional cysteine ​​residues" at the N-terminus of the VL and / or VH regions of the Fab monomer described herein may mean that the additional cysteine ​​residues are absent at each of these positions or at all of them in the amino acid sequence of the respective reference VL or VH regions, or are absent at each of these positions or at all of them.

[0018] According to the present invention, the first Fab monomer and the second Fab monomer, in which the VL and / or VH regions are covalently linked by disulfide bonds between additional cysteine ​​residues that do not naturally exist at the N-terminus of each VL and / or VH region, may be the same or different. In either case, the first Fab monomer and the second Fab monomer, in which the VL and / or VH regions are covalently linked by disulfide bonds between additional cysteine ​​residues that do not naturally exist at the N-terminus of each VL and / or VH region, may be directional to the same target or to different targets (e.g., antigens). In one embodiment, according to the present invention, the first Fab monomer and the second Fab monomer, in which the VL and / or VH regions are covalently linked by disulfide bonds between additional cysteine ​​residues that do not naturally exist at the N-terminus of each VL and / or VH region, may be the same, and both Fab monomers are directional to the same target. In another aspect of the present invention, the first and second Fab monomers, which may be different and are directed toward the same target, are covalently linked by disulfide bonds between additional cysteine ​​residues that do not naturally exist at the N-terminus of each VL and / or VH region. In yet another aspect of the present invention, the first and second Fab monomers, which may be different and are directed toward different targets, are covalently linked by disulfide bonds between additional cysteine ​​residues that do not naturally exist at the N-terminus of each VL and / or VH region. In the latter aspect of the present invention, the first Fab monomer is directed toward a first target and the second Fab monomer is directed toward a second target, and the first and second targets are different. In this case, the dimer described and provided herein would be a bispecific Fab dimer because it is directed toward two different targets.

[0019] According to the present invention, the target directed by the Fab dimer of the present invention may be any target to which tagging, marking, neutralization, or other binding is desired. In some aspects of the present invention, such targets may be antigens present in subjects suffering from hypoxia or eye disease, antigens present in or on cancerous or neoplastic cells, or antigens present in subjects suffering from cancer, particularly solid tumors. Such targets may be antigens, or any other structures having a peptide moiety, a glycoside moiety, and / or other moieties to which an antibody or its fragment (e.g., a Fab or F(ab')2 fragment) can bind. In specific aspects of the present invention, the target directed by the dimer of the present invention is α-carbonic anhydrase XII (CA-XII).

[0020] Therefore, in specific embodiments of the present invention, the dimer described and provided herein binds to CA-XII (preferably specifically). In this regard, according to the present invention, the dimer described and provided herein can bind to CA-XII (preferably specifically) in one or both, preferably both, of the first and second Fab monomers described herein.

[0021] The term “specifically recognize” (as used herein in the same way as “specifically bind,” “direction to,” or “react with”) means that, according to the present invention, a recognition molecule can specifically interact with and / or bind to at least two, preferably at least three, more preferably at least four amino acids of an epitope as defined herein. Such binding can be explained by the specificity of the “lock and key principle.” Thus, the term “specifically” in this context means that the recognition molecule binds to a given target epitope but does not essentially bind to other proteins. The term “other proteins” includes all proteins, including proteins that are closely related to or homologous to the epitope directed by the recognition molecule. However, the term “other proteins” does not include the recognition molecule cross-reacting with epitopes from a different species than the one to which the recognition molecule was produced.

[0022] As used herein, the term “essentially non-binding” means that the epitope recognition molecules of the present invention do not bind to other proteins, i.e., they exhibit less than 30%, preferably less than 20%, more preferably less than 10%, and particularly preferably less than 9, 8, 7, 6, or 5% cross-reactivity with other proteins.

[0023] Specific binding is thought to occur when a specific motif in the amino acid sequence of the binding domain and the antigen bind to each other, not only as a result of their primary, secondary, or tertiary structures, but also as a result of secondary modifications of those structures. Specific interaction between an antigen interaction site and a particular antigen may also result in simple binding between the site and the antigen. Furthermore, specific interaction between an antigen interaction site and a particular antigen may alternatively result in signal initiation, for example, due to the induction of conformational changes in the antigen or oligomerization of the antigen. A preferred example of a binding domain suitable for the present invention is an antibody. Typically, with a binding affinity of 10 -6 If the binding affinity is higher than M, the binding is considered "specific". Preferably, the binding affinity is about 10-11 ~10 -8 M(K D ), preferably about 10 -11 ~10 -9 If the binding is M, the binding is considered specific. If necessary, nonspecific binding can be reduced without substantially affecting specific binding by changing the binding conditions. Whether a recognition molecule reacts specifically as defined above can be easily tested, in particular, by comparing the reaction of the recognition molecule with the epitope with the reaction of that recognition molecule with other proteins(s).

[0024] In one embodiment of the present invention, CA-XII is human CA-XII. According to the present invention, the dimer described and provided will further bind to at least one epitope in the extracellular domain of CA-XII as described in WO2011 / 138279, or preferably to at least one epitope in the region of the discontinuous catalytic domain of CA-XII. The term “extracellular domain” according to the present invention is a well-known term in the art and, in relation to the present invention, refers to the portion of CA-XII that extends into the extracellular environment. Similarly, the term “catalytic domain” according to the present invention is a well-known term in the art and, in relation to the present invention, refers to the portion of CA-XII where the catalytic reaction from carbonic acid to bicarbonate ions and hydrogen ions occurs.

[0025] The terms “antigen” and “immunogen” are used herein interchangeably to refer to molecules or substances that elicit an immune response (preferably an antibody response) in animals, preferably non-human animals immunized thereby (i.e., the antigen is “immunogenic” in the animal), and that can typically be bound by an antibody or a portion thereof (e.g., a Fab monomer or dimer described and provided herein, or an F(ab')2 fragment or other Fab molecule described herein and known in the Art).

[0026] Furthermore, the term "epitope" refers to a site on an antigen to which a recognition molecule binds. Preferably, an epitope is a site on a molecule to which a recognition molecule, preferably an antibody, will be produced and / or to which the antibody will bind. For example, an epitope can be recognized by a recognition molecule, particularly preferably by an antibody that determines the epitope. A "linear epitope" is an epitope that constitutes a recognized epitope with a primary sequence of amino acids. A linear epitope typically contains at least three, more commonly at least five, for example, about eight to about ten amino acids in a unique sequence.

[0027] A "structural epitope," in contrast to a linear epitope, is an epitope in which the primary sequence of amino acids constituting the epitope is not the sole definitive component of the recognized epitope (for example, an epitope whose primary sequence of amino acids is not necessarily recognized by the antibody determining the epitope). Typically, structural epitopes contain an increased number of amino acids compared to linear epitopes. In relation to the recognition of structural epitopes, the recognizing molecule recognizes the three-dimensional structure of the antigen, preferably a peptide or protein or a fragment thereof. For example, when a protein molecule folds to form a three-dimensional structure, the specific amino acids and / or polypeptide backbone constituting the structural epitope are juxtaposed, allowing the antibody to recognize the epitope. Methods for determining the three-dimensional structure of an epitope include, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy, site-directed spin labeling, and electron paramagnetic resonance spectroscopy.

[0028] As used herein, “antibody” is a protein consisting of one or more polypeptides (including one or more binding domains, preferably antigen-binding domains) substantially or partially encoded by an immunoglobulin gene or a fragment of an immunoglobulin gene. The term “immunoglobulin” (Ig) is used herein interchangeably with “antibody.” Widely recognized immunoglobulin genes include not only the numerous immunoglobulin variable region genes, but also the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes.

[0029] In particular, the “antibody” as used herein is typically a tetrameric glycosylated protein composed of two light chains (L chains) of approximately 25 kDa each and two heavy chains (H chains) of approximately 50 kDa each. Antibodies can have two types of light chains, called lambda and kappa. Depending on the amino acid sequence of the constant domain of the heavy chain, immunoglobulins can be assigned to five major classes: A, D, E, G, and M, some of which can be further classified into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2 (in the case of humans). IgM antibodies consist of five basic heterotetrameric units and an additional polypeptide called a J chain, containing 10 antigen-binding sites; IgA antibodies, on the other hand, consist of 2 to 5 basic quad-chain units, which can polymerize with the J chain to form a multivalent aggregate. In the case of IgG, the quad-chain unit is generally about 150,000 daltons.

[0030] Each light chain contains an N-terminal variable (V) domain (VL) and a constant (C) domain (CL). Each heavy chain contains an N-terminal V domain (VH), three or four C domains (CH), and a hinge region. The constant domain is not directly involved in the binding of the antibody to the antigen.

[0031] When the VH and VL domains pair together, a single antigen-binding site is formed. The CH domain closest to the VH is designated CH1. Each L chain is attached to the H chain by one covalent disulfide bond, while the two H chains are attached to each other by one or more disulfide bonds depending on the H chain isotype. The VH and VL domains consist of four relatively conserved regions of sequence called framework regions (FR1, FR2, FR3, and FR4), which form a scaffold for three regions of hypervariable sequence (complementarity-determining regions; CDRs). The CDRs contain most of the residues involved in the specific interaction between the antibody and the antigen. The CDRs are called CDR1, CDR2, and CDR3. Thus, the CDR components on the heavy chain are called H1, H2, and H3, while the CDR components on the light chain are called L1, L2, and L3. The term "variable" refers to the portion of the immunoglobulin domain (i.e., "variable domains") that exhibits variability in the immunoglobulin sequence and is involved in determining the specificity and binding affinity of a particular antibody. Variability is not uniformly distributed throughout the antibody's variable domain; it is concentrated in the respective subdomains of the heavy and light chain variable regions. These subdomains are called "hypervariable" regions or "complementarity-determining regions" (CDRs). The more conserved (i.e., non-hypervariable) portions of the variable domain are called "framework" regions (FRMs). Naturally occurring heavy and light chain variable domains each contain four FRM regions, primarily in a β-sheet configuration, and these FRM regions are connected by three hypervariable regions; the hypervariable regions form loops connecting the β-sheet structures and, in some cases, form parts of the β-sheet structures. The hypervariable regions of each chain are held together in close proximity by the FRMs and, together with the hypervariable region of the other chain, contribute to the formation of the antigen-binding site (Kabat et al., see below). The constant domain does not directly participate in antigen binding, but it exhibits various effector functions, such as antibody-dependent, cell-mediated cytotoxicity, and complement activation.

[0032] The terms "CDR" and its plural form "CDRs" refer to complementarity-determining regions (CDRs), three of which constitute the binding properties of the light chain variable region (CDRL1, CDRL2, and CDRL3; also referred to herein as VL-CDR, VL-CDR2, and VL-CDR3), and three which constitute the binding properties of the heavy chain variable region (CDRH1, CDRH2, and CDRH3; also referred to herein as VH-CDR, VH-CDR2, and VH-CDR3). CDRs contribute to the functional activity of antibody molecules and are separated by amino acid sequences that constitute the scaffold or framework region. The precise definition of CDR boundaries and lengths varies depending on the various classification and numbering systems. Therefore, CDRs may be referred to by Kabat, Chothia, contact, or other boundary definitions, including the numbering systems described herein. Despite the differing boundaries, each of these systems contains some overlap in the portions that constitute the so-called "hypervariable regions" within the variable sequences. Therefore, the definitions of CDRs following these systems may differ in length and boundary region with respect to adjacent framework regions. See, for example, Kabat, Chothia, and / or MacCallum (Kabat et al., loc. cit.; Chothia et al., J MoI Biol (1987), 196: 901; and MacCallum et al., J MoI Biol (1996), 262: 732). However, numbering following the so-called Kabat system is preferred.

[0033] In specific embodiments of the present invention, the first and second Fab monomers may include specific VL and VH regions, each region containing one or more specific CDR regions. For example, in one specific embodiment of the present invention, the first Fab monomer includes (A) VH-CDR1 shown at SEQ ID NO: 1, VH-CDR2 shown at SEQ ID NO: 2, VH-CDR3 shown at SEQ ID NO: 3, VL-CDR1 shown at SEQ ID NO: 4, VL-CDR2 shown at SEQ ID NO: 5 and / or (preferably, and) VL-CDR3 shown at SEQ ID NO: 6, or (B) any one or all of the sequences of (A), where at least one, and up to five, preferably up to four, more preferably up to three, more preferably up to two, and more preferably exactly one amino acid is substituted, deleted, or added (preferably substituted) compared to each amino acid sequence SEQ ID NO: 1 to 6. In specific embodiments of the present invention, the first Fab monomer includes VH-CDR1 shown as SEQ ID NO: 1, VH-CDR2 shown as SEQ ID NO: 2, VH-CDR3 shown as SEQ ID NO: 3, VL-CDR1 shown as SEQ ID NO: 4, VL-CDR2 shown as SEQ ID NO: 5, and VL-CDR3 shown as SEQ ID NO: 6. In another specific aspect of the present invention, the first Fab monomer comprises VH-CDR1 shown in SEQ ID NO: 1, VH-CDR2 shown in SEQ ID NO: 2, VH-CDR3 shown in SEQ ID NO: 3, VL-CDR1 shown in SEQ ID NO: 4, VL-CDR2 shown in SEQ ID NO: 5, and / or (preferably, and) VL-CDR3 shown in SEQ ID NO: 6, wherein in any one or all of the sequences of SEQ ID NO: 1 to 6, at least one and up to five, preferably up to four, more preferably up to three, more preferably up to two, and more preferably exactly one amino acid is substituted, and at least one or (preferably) all of the substitutions are conserved substitutions or highly conserved substitutions.

[0034] As used herein, “conservative” substitutions mean those listed as “exemplary substitutions” in Table I. As used herein, “highly conservative” substitutions mean those listed as “preferred substitutions” in Table I.

[0035] (Table I) Amino acid substitutions TIFF0007927055000001.tif101128

[0036] Unless otherwise specified herein, the term “position” as used in accordance with the present invention means the position of an amino acid within the amino acid sequence shown herein. Unless otherwise specified herein, the term “corresponding” in this context also includes the fact that the position is not determined solely by the number of preceding nucleotides / amino acids.

[0037] As used herein, the terms “amino acid” or “amino acid residue” typically refer to amino acids with definitions widely recognized in the art, such as alanine (Ala or A); arginine (Arg or R); asparagine (Asn or N); aspartic acid (Asp or D); cysteine ​​(Cys or C); glutamine (Gln or Q); glutamic acid (Glu or E); glycine (Gly or G); histidine (His or H); isoleucine (He or I); leucine (Leu or L); lysine (Lys or K); methionine (Met or M); phenylalanine (Phe or F); proline (Pro or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp or W); tyrosine (Tyr or Y); and valine (Val or V), although modified amino acids, synthetic amino acids, or rare amino acids may be used as desired. Generally, amino acids can be grouped based on the presence of the following side chains: nonpolar side chains (e.g., Ala, Cys, He, Leu, Met, Phe, Pro, Val); negatively charged side chains (e.g., Asp, Glu); positively charged side chains (e.g., Arg, His, Lys); or uncharged polar side chains (e.g., Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr, Trp, and Tyr).

[0038] According to the present invention, the VL and / or VH regions of each first and second Fab monomer may also include a secretory sequence at their N-terminus. As is commonly known in the art, such a secretory sequence may be suitable for causing the synthesized Fab monomer to be secreted from Fab-producing cells. For example, in connection with the present invention, such a secretory sequence at the N-terminus of the VL and / or VH regions of the first and / or second Fab monomer may include or consist of the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO: 16, or may include or consist of the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO: 16, where at least one, and up to five, preferably up to four, more preferably up to three, more preferably up to two, and more preferably exactly one amino acid is substituted, deleted, or added (preferably substituted) compared to each amino acid sequence SEQ ID NO: 15 or SEQ ID NO: 16. In specific embodiments of the present invention, such secretory sequences at the N-terminus of the VL and / or VH regions of the first and / or second Fab monomer may include or consist of the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO: 16. In another specific embodiment of the present invention, such secretory sequences at the N-terminus of the VL and / or VH regions of the first and / or second Fab monomer may include or consist of the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO: 16, wherein at least one, and up to five, preferably up to four, more preferably up to three, more preferably up to two, and more preferably exactly one amino acid is substituted compared to the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO: 16, and at least one or (preferably) all of the substitutions are conserved substitutions or highly conserved substitutions as defined herein.

[0039] In this regard, in a specific embodiment of the present invention, the VL region of the first Fab monomer and the VL region of the second Fab monomer include a secretory sequence indicated by SEQ ID NO: 16 at their N-terminus. In another specific embodiment of the present invention, the VH region of the first Fab monomer and the VH region of the second Fab monomer include a secretory sequence indicated by SEQ ID NO: 16 at their N-terminus. In another specific embodiment of the present invention, the VL region of the first Fab monomer and the VH region of the second Fab monomer include a secretory sequence indicated by SEQ ID NO: 16 at their N-terminus. In another specific embodiment of the present invention, the VH region of the first Fab monomer and the VL region of the second Fab monomer include a secretory sequence indicated by SEQ ID NO: 16 at their N-terminus.

[0040] In this regard, in another specific embodiment of the present invention, the VH region of the first Fab monomer and the VH region of the second Fab monomer include a secretory sequence at their N-terminus indicated by SEQ ID NO: 15. In another specific embodiment of the present invention, the VL region of the first Fab monomer and the VL region of the second Fab monomer include a secretory sequence at their N-terminus indicated by SEQ ID NO: 15. In another specific embodiment of the present invention, the VH region of the first Fab monomer and the VL region of the second Fab monomer include a secretory sequence at their N-terminus indicated by SEQ ID NO: 15. In another specific embodiment of the present invention, the VL region of the first Fab monomer and the VH region of the second Fab monomer include a secretory sequence at their N-terminus indicated by SEQ ID NO: 15.

[0041] In a more specific embodiment of the present invention, the VL region of the first Fab monomer and the VL region of the second Fab monomer include a secretory sequence indicated by SEQ ID NO: 16 at their N-terminus, and the VH region of the first Fab monomer and the VH region of the second Fab monomer include a secretory sequence indicated by SEQ ID NO: 15 at their N-terminus. In another more specific embodiment of the present invention, the VH region of the first Fab monomer and the VH region of the second Fab monomer include a secretory sequence indicated by SEQ ID NO: 16 at their N-terminus, and the VL region of the first Fab monomer and the VL region of the second Fab monomer include a secretory sequence indicated by SEQ ID NO: 15 at their N-terminus. In another more specific embodiment of the present invention, the VL region of the first Fab monomer and the VH region of the second Fab monomer include a secretory sequence indicated by SEQ ID NO: 16 at their N-terminus, and the VH region of the first Fab monomer and the VL region of the second Fab monomer include a secretory sequence indicated by SEQ ID NO: 15 at their N-terminus. In another more specific embodiment of the present invention, the VH region of the first Fab monomer and the VL region of the second Fab monomer include a secretory sequence indicated by SEQ ID NO: 16 at their N-terminus, and the VL region of the first Fab monomer and the VH region of the second Fab monomer include a secretory sequence indicated by SEQ ID NO: 15 at their N-terminus.

[0042] In a very specific embodiment of the present invention, the first and / or second Fab monomer comprises a VH region shown in SEQ ID NO: 7 and / or (preferably, and) a VL region shown in SEQ ID NO: 8. In another specific embodiment of the present invention, the first and / or second Fab monomer comprises a VH region shown in SEQ ID NO: 7 and / or (preferably, and) a VL region shown in SEQ ID NO: 8, wherein at least one, and up to five, preferably up to four, more preferably up to three, more preferably up to two, and more preferably exactly one amino acid is substituted, deleted, or added (preferably substituted) compared to the respective amino acid sequence SEQ ID NO: 7 or SEQ ID NO: 8. In relation to the latter, in one aspect of the present invention, the first and / or second Fab monomer comprises a VH region shown in SEQ ID NO: 7 and / or (preferably, and) a VL region shown in SEQ ID NO: 8, wherein at least one and up to five, preferably up to four, more preferably up to three, more preferably up to two, and more preferably exactly one amino acid is substituted compared to the respective amino acid sequence SEQ ID NO: 7 or SEQ ID NO: 8, and at least one or (preferably) all of such substitutions are conserved substitutions or highly conserved substitutions as defined herein.

[0043] In another very specific embodiment of the present invention, the first and / or second Fab monomer comprises a VH region encoded by the nucleotide sequence shown in SEQ ID NO: 9 and / or (preferably, and) a VL region encoded by the nucleotide sequence shown in SEQ ID NO: 10, or the first and / or second Fab monomer comprises a VH region encoded by the nucleotide sequence shown in SEQ ID NO: 9 and / or (preferably, and) a VL region encoded by the nucleotide sequence shown in SEQ ID NO: 10, wherein 1 to 30, preferably 1 to 21, more preferably 1 to 12, and more preferably 1 to 6 nucleotides are substituted, added or deleted (preferably substituted) compared to the respective nucleic acid sequences of SEQ ID NO: 9 or SEQ ID NO: 10. In the latter case, 1 to 30 nucleotides, preferably 1 to 21, more preferably 1 to 12, and more preferably 1 to 6 nucleotides are substituted in each nucleic acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10, and such substitutions are preferably conserved substitutions, highly conserved substitutions, or more preferably silent substitutions.

[0044] As used herein, a “silent” substitution or mutation means a base substitution within a nucleic acid sequence that does not alter the individual amino acid sequence encoded by the nucleic acid sequence. A “conservative” substitution means the substitutions listed as “exemplary substitutions” in Table I. As used herein, a “highly conservative” substitution means the substitutions shown in the “preferred substitutions” column of Table I.

[0045] Unless otherwise defined, the terms “nucleic acid” or “nucleic acid molecule” as used herein are synonymous with “oligonucleotide,” “nucleic acid chain,” etc., and mean, for example, a polymer containing one, two, or more nucleotides, whether single-stranded or double-stranded.

[0046] Similarly, the terms “polynucleotide,” “nucleic acid,” and “nucleic acid molecule” as used herein should be interpreted synonymously. Generally, a nucleic acid molecule may include, among other things, DNA molecules, RNA molecules, oligonucleotide thiophosphates, substituted ribo-oligonucleotides, or PNA molecules. Furthermore, the term “nucleic acid molecule” may refer to DNA or RNA, or hybrids thereof, or modifications thereof known in the art (see, for example, US 5525711, US 471 1955, US 5792608, or EP 302175). Polynucleotide sequences may be single-stranded or double-stranded, linear or cyclic, natural or synthetic, and there are no size limitations. For example, a polynucleotide sequence may be genomic DNA, cDNA, mitochondrial DNA, mRNA, antisense RNA, ribozyme RNA, or DNA encoding such RNA, or a chimeroplast (Gamper, Nucleic Acids Research, 2000, 28, 4332-4339). The polynucleotide sequence may be in the form of a vector, plasmid, or viral DNA or RNA. This specification also describes nucleic acid molecules complementary to the above nucleic acid molecules, and nucleic acid molecules that can hybridize to the nucleic acid molecules described herein. The nucleic acid molecules described herein may also be fragments of those nucleic acid molecules in connection with the present invention. In particular, such fragments are functional fragments. An example of such a functional fragment is a nucleic acid molecule that can function as a primer.

[0047] In a further specific embodiment of the present invention, the first and / or second Fab monomer comprises a VH region shown in SEQ ID NO: 11 and / or (preferably, and) a VL region shown in SEQ ID NO: 12. In another specific embodiment of the present invention, the first and / or second Fab monomer comprises a VH region shown in SEQ ID NO: 11 and / or (preferably, and) a VL region shown in SEQ ID NO: 12, wherein at least one, and up to five, preferably up to four, more preferably up to three, more preferably up to two, and more preferably exactly one amino acid is substituted, deleted, or added (preferably substituted) compared to the respective amino acid sequence SEQ ID NO: 11 or SEQ ID NO: 12. In relation to the latter, in one aspect of the present invention, the first and / or second Fab monomer comprises a VH region shown in SEQ ID NO: 11 and / or (preferably, and) a VL region shown in SEQ ID NO: 12, wherein at least one and up to five, preferably up to four, more preferably up to three, more preferably up to two, and more preferably exactly one amino acid is substituted compared to the respective amino acid sequence SEQ ID NO: 11 or SEQ ID NO: 12, and at least one or (preferably) all of such substitutions are conserved substitutions or highly conserved substitutions as defined herein.

[0048] In a further specific embodiment of the present invention, the first and / or second Fab monomer comprises a VH region encoded by the nucleotide sequence shown in SEQ ID NO: 13 and a VL region encoded by the nucleotide sequence shown in SEQ ID NO: 14, wherein the first and / or second Fab monomer comprises a VH region encoded by the nucleotide sequence shown in SEQ ID NO: 13 and / or (preferably, and) a VL region encoded by the nucleotide sequence shown in SEQ ID NO: 14, wherein 1 to 30, preferably 1 to 21, more preferably 1 to 12, and more preferably 1 to 6 nucleotides are substituted, added, or deleted (preferably substituted) compared to the respective nucleic acid sequences of SEQ ID NO: 13 or SEQ ID NO: 14. In the latter case, compared to the respective nucleic acid sequences of SEQ ID NO: 13 or SEQ ID NO: 14, 1 to 30, preferably 1 to 21, more preferably 1 to 12, and more preferably 1 to 6 nucleotides are substituted, and such substitutions are preferably conserved substitutions, highly conserved substitutions, or more preferably silent substitutions.

[0049] The present invention further, (a) Culturing suitable cells expressing the first and second Fab monomers as defined herein (e.g., CHO cells or HEK293 cells) until they reach a stationary phase; (b) Optionally, add an oxidizing agent to the cell culture (e.g., O2, 1 mM H2O2, and / or 1 mM MnO4) - ) adding; and (c) Obtaining the dimer This relates to the dimers described and provided herein, obtained by the means described herein.

[0050] As used herein, “stationary phase” may mean the absence of any further substantial net increase in cell number (e.g., by the balance between dividing and dying cells) (e.g., by about 2% or more, preferably about 1% or more over 2 hours), preferably no further net increase in cell number.

[0051] Furthermore, according to the present invention, the first and / or second Fab monomers can be coupled to other compounds, for example, for diagnostic or therapeutic purposes. Such compounds may include, for example, labeling groups, toxins, or antitumor agents (e.g., fluorochromes, enzymes (such as peroxidases), radionuclides, rizin, ozogamicin, emtansine, monomethyl auristatin E, α-amanitin). Such coupling may be carried out chemically after the expression of an antibody or antigen at the binding site, or the coupling product may be engineered to be incorporated into the antibody or antigen of the present invention at the DNA level. Subsequently, the DNA is expressed in a suitable host system, the expressed protein is recovered, and if necessary, restored, as described later herein. Coupling may be carried out via linkers known in the art. In particular, various linkers that release toxins or antitumor agents under acidic or reducing conditions, or upon exposure to specific proteases, can be employed in conjunction with this technique.

[0052] In certain contexts, it may be desirable for labeling groups, toxins, or antitumor drugs to be coupled by spacer arms of varying lengths to reduce potential steric hindrance.

[0053] The present invention further relates to a compound comprising a dimer described and provided herein. According to the present invention, such a compound comprising a dimer described and provided herein may additionally comprise one or more Fab monomers (non-dimerized form) described and provided herein. In one aspect of the present invention, such a compound may be for use as a pharmaceutical. For example, a compound comprising a dimer described and provided herein may be a pharmaceutical composition.

[0054] Unless otherwise specified, when “dimer” is referred to herein, it means the Fab dimer described herein. Similarly, when “monomer” is referred to herein, it means the Fab monomer described herein.

[0055] In general, in connection with the present invention, the formulations described and provided herein may comprise one or more dimers described herein and one or more monomers described herein. For example, in the compositions described and provided herein, the ratio of dimer to monomer may be in the range of about 100:0, about 99:1, about 90:10, about 80:20, about 70:30, about 60:40, about 50:50, about 40:60, about 30:70, about 20:80, about 10:90, or about 1:99. As a specific example, the ratio of dimer to monomer may be about 40:60.

[0056] The (pharmaceutical) composition is preferably administered to mammals such as livestock and pets. Most preferably, it is administered to humans. The pharmaceutical composition described herein can be administered to a subject in an appropriate dose. Pharmaceutical compositions for use in accordance with the present invention can be conventionally formulated with one or more physiological carriers or excipients according to methods found in the art; see, for example, Ansel et al., "Pharmaceutical Dosage Forms and Drug Delivery Systems", 7th edition, Lippincott Williams & Wilkins Publishers, 1999. The (pharmaceutical) composition can be administered orally, parenterally, for example, subcutaneously, intravenously, intramuscularly, intraperitoneally, intrathecally, percutaneously, transmucosally, subdurally, topically or externally via iontophoresis, sublingually, by inhalation spray, aerosol, or rectally, as a dosage unit formulation, optionally containing conventionally pharmaceutically acceptable additives. For oral administration, the (pharmaceutical) compositions of the present invention may take the form of, for example, tablets or capsules, which are prepared by conventional means using pharmaceutically acceptable additives such as binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, hydroxypropyl methylcellulose), fillers (e.g., lactose, microcrystalline cellulose, calcium hydrogen phosphate), lubricants (e.g., magnesium stearate, talc, silica), disintegrants (e.g., potato starch, sodium starch glycolate), or wetting agents (e.g., sodium lauryl sulfate). The (pharmaceutical) compositions may be administered to a patient together with a physiologically acceptable carrier as described herein. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle administered together with the therapeutic agent. Such pharmaceutical carriers may be sterile liquids, such as water, and oils, such as petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, or sesame oil. Water is a preferred carrier when the (pharmaceutical) composition is administered intravenously. Physiological saline, as well as aqueous solutions of glucose and glycerol, can also be used as liquid carriers, particularly for injection solutions.Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium ions, dried skim milk, glycerol, propylene glycol, water, and ethanol. The composition may also contain small amounts of emulsifiers or pH buffers, if necessary. Such compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, or sustained-release formulations. The composition can be formulated as suppositories using conventional binders and carriers such as triglycerides. Oral formulations may contain standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions contain a therapeutically effective amount of the aforementioned compound, preferably in a purified form, along with an appropriate amount of carrier to provide a suitable dosage form for the patient. The formulation must be suitable for the method of administration.

[0057] The pharmaceutical composition of the present invention may be administered as the sole active agent, or it may be administered in combination with other agents, preferably agents known in the art to be suitable for treating the disease in question.

[0058] According to the present invention, dimers described and provided herein, compositions comprising such dimers (and one or more Fab monomers (non-dimerized)) described and provided herein, may also be used in methods for treating or inhibiting hypoxia, solid tumors, or eye diseases.

[0059] In relation to the present invention, for example, hypoxia may be selected from tumor hypoxia, neurological hypoxia, cerebral hypoxia, stenosis, and ischemia. In this context, the term "hypoxia" refers to a pathological condition in which the entire body (systemic hypoxia) or a part of the body (tissue hypoxia) is deprived of sufficient oxygen supply. For example, a mismatch between the amount of oxygen supplied at the cellular level and its demand can lead to a hypoxic state. Hypoxia in which oxygen supply is completely deprived is called anoxia and is included in the broader term hypoxia.

[0060] The term “solid tumor” in this invention defines an abnormal mass of tissue that does not typically contain cysts or fluid. Solid tumors may be benign (not cancerous) or malignant (often referred to as cancerous in the art). Various types of solid tumors are named according to the types of cells that form them. In connection with this invention, for example, solid tumors may be selected from sarcomas, gliomas, carcinomas, mesotheliomas, lymphomas, renal tumors, lung tumors, mammary gland tumors, cervical tumors, ovarian tumors, colorectal tumors, liver tumors, prostate tumors, pancreatic tumors, and head and neck tumors. In one aspect of this invention, the solid tumor is a glioma or a lung tumor. In a specific aspect of this invention, the solid tumor is a glioma.

[0061] As used herein, the term “ocular disease” refers to a pathological condition or injury of the eye, including its adnexa. Ocular diseases may include, for example, opacity or clouding of the natural lens of the eye, swelling of the macula or central retina, small accumulation of hyaline bodies (vitreous humor) beneath the retina, damage to the optic nerve, degeneration of macular cells, decreased visual acuity or inflammation of the conjunctiva and cornea of ​​the eye, and the formation of scar tissue. In connection with the present invention, for example, ocular diseases may be selected from ocular hypertension, glaucoma, macular degeneration, age-related macular degeneration, uveitis, retinitis, X-linked retinoschisis, and hypertensive retinopathy.

[0062] The embodiments characterizing the present invention are described herein, shown in the drawings, illustrated in the examples, and reflected in the claims.

[0063] The present invention is also characterized by the following: 1. A dimer composed of a first Fab monomer and a second Fab monomer, where each Fab monomer includes a VH region and a VL region, (i) The VL region of the first Fab monomer and the VL region of the second Fab monomer are covalently linked by a disulfide bond between an additional cysteine ​​residue that is not naturally present, located at the N-terminus of the VL region of the first Fab monomer and the N-terminus of the VL region of the second Fab monomer; (ii) The VH region of the first Fab monomer and the VH region of the second Fab monomer are covalently linked by a disulfide bond between an additional cysteine ​​residue that is not naturally present, located at the N-terminus of the VH region of the first Fab monomer and the N-terminus of the VH region of the second Fab monomer; (iii) The VL region of the first Fab monomer and the VH region of the second Fab monomer are covalently linked by a disulfide bond between an additional cysteine ​​residue that is not naturally present, located at the N-terminus of the VL region of the first Fab monomer and the N-terminus of the VH region of the second Fab monomer; or (iv) The VH region of the first Fab monomer and the VL region of the second Fab monomer are covalently linked by a disulfide bond between an additional cysteine ​​residue that does not exist in nature, located at the N-terminus of the VH region of the first Fab monomer and the N-terminus of the VL region of the second Fab monomer. Dimer. 2. A dimer of item 1, in which a naturally unpresent cysteine ​​residue is located at amino acid position 1 or amino acid position 2 in the N-terminus of the VL or VH region, where the amino acids are counted from the N-terminus of the VL or VH region, respectively, with position 1 being the first amino acid at the N-terminus. 3. A dimer of item 1 or 2, wherein the first Fab monomer and the second Fab monomer are the same or different. 4. A dimer of any of the above items, wherein the first Fab monomer and the second Fab monomer are directed toward the same target. 5. A dimer according to any of items 1 to 3, wherein the first Fab monomer is directed toward a first target, and the second Fab monomer is directed toward a second target, and the first target and the second target are different. 6. The first Fab monomer is a dimer of any of the above items, comprising VH-CDR1 shown in SEQ ID NO: 1, VH-CDR2 shown in SEQ ID NO: 2, VH-CDR3 shown in SEQ ID NO: 3, VL-CDR1 shown in SEQ ID NO: 4, VL-CDR2 shown in SEQ ID NO: 5, and VL-CDR3 shown in SEQ ID NO: 6. 7. A dimer of any of the above items, wherein the VL region of the first Fab monomer and the VL region of the second Fab monomer contain a secretory sequence indicated by SEQ ID NO: 16 at their N-terminus. 8. A dimer of any of the above items, wherein the VH region of the first Fab monomer and the VH region of the second Fab monomer contain a secretory sequence indicated by SEQ ID NO: 16 at their N-terminus. 9. A dimer of any of the above items, wherein the VL region of the first Fab monomer and the VH region of the second Fab monomer contain a secretory sequence indicated by SEQ ID NO: 16 at their N-terminus. 10. A dimer of any of the above items, wherein the VH region of the first Fab monomer and the VL region of the second Fab monomer contain a secretory sequence indicated by SEQ ID NO: 16 at their N-terminus. 11. The first Fab monomer is a dimer of any of the above items, comprising a VH region shown in SEQ ID NO: 7 and a VL region shown in SEQ ID NO: 8. 12. The first Fab monomer is a dimer of any of the above items, comprising a VH region shown in SEQ ID NO: 11 and a VL region shown in SEQ ID NO: 12. 13. (a) Culturing CHO cells expressing the first and second Fab monomers as defined in any one of claims 1 to 12 until they reach a stationary phase; (b) optionally adding an oxidizing agent to the CHO cell culture; and (c) Obtaining the dimer A dimer of any of the above items obtained by the above method. 14. A composition comprising any of the dimers listed in items 1 through 13. 15. A composition according to item 14, further comprising a Fab monomer as defined in any of items 1 to 13.

[0064] It should be noted that the singular forms “a,” “an,” and “the” used herein include plural referents unless otherwise indicated by the context. Therefore, for example, a reference to “a reagent” includes one or more of such reagents, and a reference to “the method” includes equivalent steps and methods known to those skilled in the art that can be used to modify or substitute the method described herein.

[0065] Unless otherwise indicated, the term “at least” preceding a set of elements should be understood to refer to all elements of that set. Those skilled in the art will recognize, or can verify by routine experimentation, many equivalents corresponding to specific embodiments of the invention described herein. Such equivalents shall be incorporated herein.

[0066] As used herein, the terms "and / or" include the meanings of "and," "or," and "all or any other combination of the elements linked by the aforementioned terms."

[0067] Throughout this specification and the subsequent claims, unless otherwise indicated by context, the word “comprise,” and variations such as “comprises” and “comprising,” are understood to mean encompassing the integer or step or group of integers or steps described, but not to mean excluding other integers or steps or groups of integers or steps. As used herein, the term “comprising” may be replaced with the terms “containing” or “including,” and sometimes with the term “having.”

[0068] As used herein, "consisting of" excludes any element, step, or component not explicitly stated in the claims. As used herein, "consisting essentially of" does not exclude any material or step that does not materially affect the basic and novel properties of the claims.

[0069] In each of the cases herein, the terms “include,” “essentially consist of,” and “consist of” may be replaced with any of the other two terms.

[0070] The present invention is not limited to the specific methodologies, protocols, reagents, etc., described herein, and should therefore be understood to be subject to change. The terms used herein are for the sole purpose of describing specific aspects and are not intended to limit the scope of the present invention as defined solely by the claims.

[0071] All publications and patents cited throughout this specification (including all patents, patent applications, scientific publications, manufacturer specifications, instructions, etc.) are incorporated herein by reference in their entirety, regardless of whether they are referenced above or below. Nothing in this specification should be construed as acknowledging that the present invention has no prior rights to such disclosures for the sake of prior art. This specification shall prevail only in the event that any material incorporated by reference is inconsistent or contradictory to this specification. [Brief explanation of the drawing]

[0072] drawing The drawing is shown below.

[0073] (Figure 1-1) Figure 1: Vector structure. HitbasisHygro_ch6A10_LC and HitbasisNeo_ch6A10_HC vectors. Translation of HitbasisHygro_ch6A10_LC: TIFF0007927055000002.tif31135 (Bold = Predicted signal (secretion) sequence). (Figure 1-2) See the explanation for Figure 1-1. (Figure 2) Figure 2: SDS-PAGE. Reduced SDS-PAGE analysis of the 6A10 Fab dimer revealed characteristic bands of approximately 25-30 kDa corresponding to the light and heavy chains of the 6A10 Fab fragment. Under non-reducing conditions, bands of approximately 50 kDa and 100 kDa corresponding to the precursor monomer and homodimer forms were detected. (Figure 3) Figure 3: "Intact Test" using LC-ESI-TOF. Further analysis of the monomer and dimer forms of the precursor is performed by LC-ESI-TOF. Homodimers are formed by an additional cysteine ​​disulfide bond at the N-terminus of the light chain. Furthermore, different monomer forms with and without the additional cysteine ​​or glutathione are detected in varying proportions. (Figure 4) Figure 4: Binding of monomers and dimers to CA12-positive human A549 lung cancer cells. Flow cytometry showed no significant difference in the binding behavior of monomers and dimers at pH 7.4. 50,000 A549 cells were incubated with serial dilutions of monomers or dimers (6A10). After washing, the cells were incubated with Cy5-labeled secondary antibodies (donkey anti-human IgG light and heavy chains). Secondary antibody binding was analyzed using BD FACSCanto and DIVA software. RU = relative luminescence. (Figure 5-1) Figure 5: Flow cytometry. Flow cytometry revealed that in the acidic environment (pH 5.5) typical of solid tumors, monomer binding behavior dramatically decreases, but dimer (610A) binding behavior does not decrease. (Figure 5-2) See the explanation for Figure 5-1. (Figure 5-3) See the explanation for Figure 5-1. (Figure 5-4) See the explanation for Figure 5-1. (Figure 5-5) See the explanation for Figure 5-1. (Figure 6) Figure 6: The amino acid sequences of Fab6A10 VH with the IgG secretion sequence and Fab6A10 VL with the IgL secretion sequence are shown. (Figure 7) Figure 7: The amino acid sequence of Fab6A10 VH after sequencing of the N-terminus and C-terminus, and the amino acid sequence of Fab6A10 VL after sequencing of the N-terminus and C-terminus.

[0074] This application also provides the following sequences referenced herein, and a sequence protocol for them has also been created, which is also part of this application. However, in the event of any discrepancy between the following sequences and the sequences listed in the sequence listing, the following sequences shall prevail. SEQ ID NO: 1 - VH-CDR1 of Fab6A10: GFSLTTYSVS SEQ ID NO: 2 - VH-CDR2 of Fab6A10: RMWYDGDTV SEQ ID NO: 3 - VH-CDR3 of Fab6A10: DFGYFDGSSPFDY SEQ ID NO: 4 - VL-CDR1 of Fab6A10: RASQGISTSIH SEQ ID NO: 5 - VL-CDR2 of Fab6A10: FASQSIS SEQ ID NO: 6 - VL-CDR3 of Fab6A10: QQTYSLPYTF SEQ ID NO: 7 - VH of Fab6A10: TIFF0007927055000003.tif21159 (CDR is in bold and in a box, regular parts are underlined) SEQ ID NO: 8 - VL of Fab6A10: TIFF0007927055000004.tif21160 (CDR is in bold and in a box, regular parts are underlined) SEQ ID NO: 9 - Nucleic acid of Fab6A10 VH: TIFF0007927055000005.tif60160SEQ ID NO: 10 - Nucleic acid of Fab6A10 VL: TIFF0007927055000006.tif60160SEQ ID NO: 11 - VH of Fab6A10 with IgG secretion sequence: TIFF0007927055000007.tif27160 (Secretion sequence is italicized and bold, CDR is bold and in a box, constant state is underlined) SEQ ID NO: 12 - VL of Fab6A10 with IgL secretion sequence: TIFF0007927055000008.tif21160 (Secretion sequence is italicized and bold, CDR is bold and in a box, constant state is underlined) SEQ ID NO: 13 - Nucleic acid of VH Fab6A10 with IgG secretion sequence: TIFF0007927055000009.tif65160SEQ ID NO: 14 - Nucleic acid of VL Fab6A10 with IgL secretion sequence: TIFF0007927055000010.tif65160SEQ ID NO: 15 - IgG secretion sequence: TIFF0007927055000011.tif4128SEQ ID NO: 16 - IgL secretion sequence: TIFF0007927055000012.tif4128SEQ ID NO: 17 - N-terminal fragment of VL of Fab6A10: TIFF0007927055000013.tif4128SEQ ID NO: 18 - N-terminal fragment of VL of Fab6A10: TIFF0007927055000014.tif4128SEQ ID NO: 19 - N-terminal fragment of VH of Fab6A10: TIFF0007927055000015.tif4128SEQ ID NO: 20 - C-terminal fragment of VL of Fab6A10: TIFF0007927055000016.tif3128SEQ ID NO: 21 - C-terminal fragment of VH of Fab6A10: TIFF0007927055000017.tif3128

[0075] The present invention is further illustrated by the following embodiments. However, the embodiments and specific aspects described herein should not be construed as limiting the present invention to such specific aspects. [Examples]

[0076] Creation of a CHO cell line that produces 6A10 Fab fragments Cell line clones expressing 6A10Fab, derived from Chinese hamster ovaries (CHO), were established using recombinant DNA technology. The construction of the production cell line was carried out by Fraunhofer ITEM, PhB, Braunschweig, under the responsibility of Helmholtz Zentrum Muenchen. The host suspension cell line CHO HIT was developed from CHO K1 (ECACC, No. 85051005). The 6A10Fab used had the sequences shown in Figures 6 and 7, respectively. Figure 6 shows the sequence of the 6A10Fab monomer before treatment with host cells. Figure 7 shows the sequence obtained after sequencing of the 6A10Fab dimer.

[0077] The vectors HitbasisHygro_ch6A10_LC and HitbasisNeo_ch6A10_HC (see Figure 1) were prepared for transfection into CHO HIT cells. For this purpose, the coding sequences of the 6A10Fab light and heavy chains were amplified from the plasmids pUC57-ch6A10 LC and pUC57-ch6A10 HC using PCR. The cDNA of the 6A10Fab light chain was inserted into HITbasisHygro. The cDNA of the 6A10Fab heavy chain was inserted into HITbasisNeo.

[0078] The structure of the corresponding vector is shown in Figure 1. The accuracy and completeness of the plasmid were demonstrated by combining the results from restriction enzyme analysis and partial sequencing.

[0079] After transforming competent Escherichia coli (E. coli) TopTen cells with the expression vector, the cells were plated on LB agar containing ampicillin. Resistant colonies were selected, and plasmid DNA was isolated.

[0080] To prepare the host cell line, one vial of the CHO HIT host cell line was thawed, seeded in "host cell growth medium" (ProCHO5 medium containing 4 mM GlutaMAX), and expanded for transfection.

[0081] Transfection of host cells with both expression vectors (together or sequentially) was performed by electroporation using the AMAXA nucleofection system (LONZA) according to the manufacturer's manual. Transfected cells were incubated at 37°C.

[0082] Two days after transfection, the cell pool was transferred to either "Selective Medium 1" or "Selective Medium 2" (proCHO medium containing 4 mM GlutaMAX and antibiotics) and subcultured until viability was restored and cell proliferation began. For pools showing acceptable proliferation, titer was determined by SDS-PAGE densitometry and cell-specific productivity was estimated. For single-cell cloning, the cell pool "MP P6-A4" was selected.

[0083] Clones were isolated using image-assisted cloning with limiting dilution combined with image-based documentation. All proliferated clones were then expanded and cultured for later freezing in five vials. Clones exhibiting the best product concentration, as measured by PAIA assay, were expanded to shaken flask levels (9 clones). Clones "P1D20," "P1E20," "P1H06," and "P5E17" were selected as preferred production cell lines and used for expression stability testing. Based on data obtained during expression stability testing, clones "P1E20" and "P5E17" were considered phenotypically stable. Ultimately, cell clone "P1E20" was selected for USP method development based on quantity and quality.

[0084] N-terminus and C-terminus sequencing MALDI-TOF-MS sequencing of the N-terminus and C-terminus is a method for confirming the amino acid sequences at the N-terminus and C-terminus of a protein. A reduced sample is spotted onto a polished steel target using 1,5-diaminonaphthalene and measured using positive ion mode MALDI-TOF-MS. Fragmentation along the amino acid backbone of the peptide yields fragments specific to the N-terminus and C-terminus amino acid sequences of the protein. The theoretical amino acid sequence is verified by comparing the molecular weight of the experimentally measured fragments with that of the theoretically predicted fragments.

[0085] The identity of 6A10Fab-CHX-A''-DTPA was confirmed by N-terminal and C-terminal sequencing using MALDI-ISD-MS. Sequencing detected additional arginine (R) and cysteine ​​(C) amino acids at the N-terminus of the light chain; see Table 2. No such additions or modifications were observed in the heavy chain.

[0086] (Table 2) N-terminus and C-terminus of 6A10Fab chain (reference and batch) TIFF0007927055000018.tif62161

[0087] While not bound by theory, the predicted signal sequence selected for optimal translation and secretion of the light chain was not correctly cleaved by the signal sequence peptidase (SSP) enzyme. Consequently, the VL regions of the first Fab6A10 monomer and the second Fab6A10 monomer are covalently linked by disulfide bonds between additional cysteine ​​residues that are not naturally present, located at the N-terminus of the VL region of the first Fab6A10 monomer and the N-terminus of the VL region of the second Fab6A10 monomer.

[0088] As shown in Table 2 above, the first Fab6A10 monomer contains an additional cysteine ​​residue (Cys) at the N-terminus of the VL region (underlined in SEQ ID NO: 18 above). This cysteine ​​residue can form a disulfide bridge with the additional cysteine ​​residue (Cys) present at the N-terminus of the VL region of the second Fab6A10 monomer. As a result, a Cys-Cys bridge is formed between the VL regions of the first Fab6A10 monomer and the VL region of the second Fab6A10 monomer, thereby yielding the Fab6A10 dimer.

[0089] Of course, even if disulfide bonds are formed as a result of improper cleavage by signal sequence peptidase (SSP) enzymes, such cysteine ​​residues forming disulfide bonds may, but do not necessarily, be introduced via signal / secretion sequences at the N-terminus of the variable region. In fact, as is well known in the art, such cysteine ​​residues can also be introduced by corresponding engineering of the nucleotide sequences encoding such variable regions.

[0090] From the observation that Fab dimers can construct dimers via unique disulfide bonds not seen before, the following can be reasonably inferred: (i) The VL region of the first Fab monomer and the VL region of the second Fab monomer may be covalently bonded by a disulfide bond between an additional cysteine ​​residue that is not naturally present, located at the N-terminus of the VL region of the first Fab monomer and the N-terminus of the VL region of the second Fab monomer; (ii) The VH region of the first Fab monomer and the VH region of the second Fab monomer may be covalently bonded by a disulfide bond between an additional cysteine ​​residue that is not naturally present, located at the N-terminus of the VH region of the first Fab monomer and the N-terminus of the VH region of the second Fab monomer; (iii) The VL region of the first Fab monomer and the VH region of the second Fab monomer may be covalently bonded by a disulfide bond between an additional cysteine ​​residue that is not naturally present, located at the N-terminus of the VL region of the first Fab monomer and the N-terminus of the VH region of the second Fab monomer; or (iv) The VH region of the first Fab monomer and the VL region of the second Fab monomer are covalently linked by disulfide bonds between additional cysteine ​​residues that do not exist in nature, located at the N-terminus of the VH region of the first Fab monomer and the N-terminus of the VL region of the second Fab monomer.

[0091] Binding of monomers and dimers to CA12-positive human A549 lung cancer cells 50,000 A549 cells were incubated with serial dilutions of Fab6A10 monomers or Fab6A10 dimers. Fab6A10 has the following CDRs: VH-CDR1 shown in SEQ ID NO: 1, VH-CDR2 shown in SEQ ID NO: 2, VH-CDR3 shown in SEQ ID NO: 3, VL-CDR1 shown in SEQ ID NO: 4, VL-CDR2 shown in SEQ ID NO: 5, and VL-CDR3 shown in SEQ ID NO: 6. In the Fab6A10 dimer used in this example, the VL region of the first Fab6A10 monomer and the VL region of the second Fab6A10 monomer are covalently linked by a disulfide bond between additional cysteine ​​residues that are not naturally present, located at the N-terminus of the VL region of the first Fab6A10 monomer and the N-terminus of the VL region of the second Fab6A10 monomer.

[0092] As shown in Table 2 above, the first Fab6A10 monomer contains an additional cysteine ​​residue (Cys) at the N-terminus of the VL region (underlined in SEQ ID NO: 18 above). This cysteine ​​residue can form a disulfide bridge with the additional cysteine ​​residue (Cys) present at the N-terminus of the VL region of the second Fab6A10 monomer. As a result, a Cys-Cys bridge is formed between the VL regions of the first Fab6A10 monomer and the VL region of the second Fab6A10 monomer, thereby yielding the Fab6A10 dimer.

[0093] The 6A10Fab used has the sequences shown in Figures 6 and 7, respectively. Figure 6 shows the sequence of the 6A10Fab monomer before treatment with host cells. Figure 7 shows the sequence obtained after sequencing of the 6A10Fab dimer.

[0094] After washing, cells were incubated with Cy5-labeled secondary antibodies (donkey anti-human IgG light and heavy chains). Secondary antibody binding was analyzed using BD FACSCanto and DIVA software. RU = relative luminescence.

[0095] Figure 4 shows that the binding of Fab6A10 dimers to CA12-positive A549 cells is comparable to that of monomers at pH 7.4.

[0096] Flow cytometry CA12-positive A549 cells were incubated with varying amounts of Fab6A10 dimer or monomer at the indicated pH, followed by incubation with a suitable Cy5-labeled secondary antibody at a neutral pH (approximately 7.2).

[0097] Fab6A10 has the following CDRs: VH-CDR1 shown in SEQ ID NO: 1, VH-CDR2 shown in SEQ ID NO: 2, VH-CDR3 shown in SEQ ID NO: 3, VL-CDR1 shown in SEQ ID NO: 4, VL-CDR2 shown in SEQ ID NO: 5, and VL-CDR3 shown in SEQ ID NO: 6. In the Fab6A10 dimer used in this example, the VL region of the first Fab6A10 monomer and the VL region of the second Fab6A10 monomer are covalently linked by a disulfide bond between additional cysteine ​​residues that do not exist in nature, located at the N-terminus of the VL region of the first Fab6A10 monomer and the N-terminus of the VL region of the second Fab6A10 monomer.

[0098] As shown in Table 2 above, the first Fab6A10 monomer contains an additional cysteine ​​residue (Cys) at the N-terminus of the VL region (underlined in SEQ ID NO: 18 above). This cysteine ​​residue can form a disulfide bridge with the additional cysteine ​​residue (Cys) present at the N-terminus of the VL region of the second Fab6A10 monomer. As a result, a Cys-Cys bridge is formed between the VL regions of the first Fab6A10 monomer and the VL region of the second Fab6A10 monomer, thereby yielding the Fab6A10 dimer.

[0099] The 6A10Fab used had the sequences shown in Figures 6 and 7, respectively. Figure 6 shows the sequence of the 6A10Fab monomer before treatment with host cells. Figure 7 shows the sequence obtained after sequencing of the 6A10Fab dimer. Binding of this dimer or monomer was measured by flow cytometry.

[0100] Figure 5 shows that Fab6A10 dimer binding is considerably superior to monomer binding, for example, at low pH levels found in tumor environments.

Claims

1. A dimer composed of a first Fab monomer and a second Fab monomer, wherein each Fab monomer contains a VH region and a VL region. (i) The VL region of the first Fab monomer and the VL region of the second Fab monomer are covalently linked by a disulfide bond between an additional cysteine ​​residue that is not naturally present, located at the N-terminus of the VL region of the first Fab monomer and the N-terminus of the VL region of the second Fab monomer; (ii) The VH region of the first Fab monomer and the VH region of the second Fab monomer are covalently linked by a disulfide bond between an additional cysteine ​​residue that is not naturally present, located at the N-terminus of the VH region of the first Fab monomer and the N-terminus of the VH region of the second Fab monomer; (iii) The VL region of the first Fab monomer and the VH region of the second Fab monomer are covalently linked by a disulfide bond between an additional cysteine ​​residue that is not naturally occurring, located at the N-terminus of the VL region of the first Fab monomer and the N-terminus of the VH region of the second Fab monomer; or (iv) The VH region of the first Fab monomer and the VL region of the second Fab monomer are covalently linked by a disulfide bond between an additional cysteine ​​residue that does not exist in nature, located at the N-terminus of the VH region of the first Fab monomer and the N-terminus of the VL region of the second Fab monomer. A naturally occurring cysteine ​​residue is located at the N-terminus of either the VL or VH region, at amino acid position 1 or amino acid position 2. The amino acids are counted from the N-terminus of either the VL or VH region, with position 1 being the first amino acid of the VL or VH region, and position 2 being the second amino acid of the VL or VH region. The first Fab monomer and the second Fab monomer are either the same or different. The first Fab monomer and the second Fab monomer are directed toward the same target, or the first Fab monomer is directed toward the first target and the second Fab monomer is directed toward the second target, and the first target and the second target are different. The aforementioned dimer.

2. The VL region of the first Fab monomer and the VL region of the second Fab monomer are covalently bonded by a disulfide bond between an additional cysteine ​​residue that does not exist in nature, located at the N-terminus of the VL region of the first Fab monomer and the N-terminus of the VL region of the second Fab monomer. The dimer according to claim 1, wherein a cysteine ​​residue, which does not naturally exist, is located at amino acid position 2 in the N-terminus of the VL region, and amino acids are counted from the N-terminus of the VL region, with position 2 being the second amino acid of the VL region.

3. The dimer according to claim 1, wherein the target is α-carbonic anhydrase XII (CA-XII).

4. The dimer according to claim 1, wherein the first Fab monomer and / or the second Fab monomer comprises VH-CDR1 shown in SEQ ID NO: 1, VH-CDR2 shown in SEQ ID NO: 2, VH-CDR3 shown in SEQ ID NO: 3, VL-CDR1 shown in SEQ ID NO: 4, VL-CDR2 shown in SEQ ID NO: 5, and VL-CDR3 shown in SEQ ID NO:

6.

5. The dimer according to claim 1, wherein the target is human CA-XII, and the first Fab monomer and / or second Fab monomer comprises VH-CDR1 shown in SEQ ID NO: 1, VH-CDR2 shown in SEQ ID NO: 2, VH-CDR3 shown in SEQ ID NO: 3, VL-CDR1 shown in SEQ ID NO: 4, VL-CDR2 shown in SEQ ID NO: 5, and VL-CDR3 shown in SEQ ID NO:

6.

6. i) The VL region of the first Fab monomer and the VL region of the second Fab monomer each contain a secretory sequence at their N-terminus indicated by SEQ ID NO: 16; ii) The VH region of the first Fab monomer and the VH region of the second Fab monomer each contain a secretory sequence at their N-terminus indicated by SEQ ID NO: 16; iii) The VL region of the first Fab monomer and the VH region of the second Fab monomer contain the secretory sequence indicated by SEQ ID NO: 16 at their N-terminus; or iv) The VH region of the first Fab monomer and the VL region of the second Fab monomer contain the secretory sequence indicated by SEQ ID NO: 16 at their N-terminus, The dimer according to claim 1.

7. i) The VL region of the first Fab monomer and the VL region of the second Fab monomer contain a secretory sequence indicated by SEQ ID NO: 16 at their N-terminus, and the VH region of the first Fab monomer and the VH region of the second Fab monomer contain a secretory sequence indicated by SEQ ID NO: 15 at their N-terminus; ii) The VH region of the first Fab monomer and the VH region of the second Fab monomer contain a secretory sequence indicated by SEQ ID NO: 16 at their N-terminus, and the VL region of the first Fab monomer and the VL region of the second Fab monomer contain a secretory sequence indicated by SEQ ID NO: 15 at their N-terminus; iii) The VL region of the first Fab monomer and the VH region of the second Fab monomer contain a secretory sequence indicated by SEQ ID NO: 16 at their N-terminus, and the VH region of the first Fab monomer and the VL region of the second Fab monomer contain a secretory sequence indicated by SEQ ID NO: 15 at their N-terminus; or vi) The dimer according to claim 1, wherein the VH region of the first Fab monomer and the VL region of the second Fab monomer each contain a secretory sequence indicated by SEQ ID NO: 16 at their N-terminus, and the VL region of the first Fab monomer and the VH region of the second Fab monomer each contain a secretory sequence indicated by SEQ ID NO: 15 at their N-terminus.

8. The dimer according to claim 1, wherein the first Fab monomer and / or the second Fab monomer include a VH region indicated by SEQ ID NO: 7 and a VL region indicated by SEQ ID NO:

8.

9. The dimer according to claim 1, wherein the first Fab monomer and / or the second Fab monomer comprises a VH region encoded by the nucleotide sequence shown in SEQ ID NO: 9 and a VL region encoded by the nucleotide sequence shown in SEQ ID NO:

10.

10. The dimer according to claim 1, wherein the first Fab monomer and / or the second Fab monomer include a VH region indicated by SEQ ID NO: 11 and a VL region indicated by SEQ ID NO:

12.

11. The dimer according to claim 1, wherein the first Fab monomer and / or the second Fab monomer comprises a VH region encoded by the nucleotide sequence shown in SEQ ID NO: 13 and a VL region encoded by the nucleotide sequence shown in SEQ ID NO:

14.

12. The dimer according to claim 1, obtained by (A) or (B) below: （A） (a) Culturing CHO cells expressing the first and second Fab monomers as defined in any one of claims 1 to 11 until they reach a stationary phase; (b) Adding an oxidizing agent to the CHO cell culture; and (c) obtaining the dimer, or (B) (a) Culturing CHO cells expressing the first and second Fab monomers as defined in any one of claims 1 to 11 until they reach a stationary phase; and (b) Obtain the dimer.

13. The first and / or second Fab monomers are (a) Label group; (b) Toxins; or (c) Antitumor drugs The dimer according to claim 1, which is bonded to the dimer.

14. A composition comprising the dimer described in claim 1.

15. The composition according to claim 14, further comprising the first and second Fab monomers defined in claim 1.

16. (i) the dimer described in claim 1, or (ii) the dimer described in claim 1 and the first and second Fab monomers as defined in claim 1.

17. A pharmaceutical composition for treating or inhibiting hypoxia, solid tumors, or eye diseases, comprising (i) the dimer described in claim 1, or (ii) the dimer described in claim 1 and first and second Fab monomers as defined in claim 1, wherein the target directed by the dimer and the first and second Fab monomers is CA-XII.

18. i) The hypoxia is selected from tumor hypoxia, nerve hypoxia, cerebral hypoxia, stenosis, and ischemia; ii) The solid tumor is selected from sarcoma, glioma, carcinoma, mesothelioma, lymphoma, renal tumor, lung tumor, mammary gland tumor, cervical tumor, ovarian tumor, colorectal tumor, liver tumor, prostate tumor, pancreatic tumor, and head and neck tumor; or iii) The pharmaceutical composition according to claim 17, wherein the eye disease is selected from ocular hypertension, glaucoma, macular degeneration, age-related macular degeneration, uveitis, retinitis, X-linked retinal schizophrenia, and hypertensive retinopathy.

Images