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 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. [Background technology]

[0002] Carbonic anhydrases are a family of enzymes that catalyze the reversible hydration of carbonic acid to 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 occurs fairly slowly. Most carbonic anhydrases contain a zinc ion in their active site, so they are classified as metalloenzymes. The family of carbonic anhydrases includes several members. There are at least five different carbonic anhydrase subfamilies (α, β, γ, δ, and ε). These subfamilies lack significant amino acid sequence similarity and are often considered to be an example of convergent evolution. α-carbonic anhydrase (CA) is found in mammals. Members of this subfamily can be distinguished with respect to their kinetics, tissue expression, and subcellular localization (Kivela et. al., World J Gastroenterol (2005), 11(2): 155-163).

[0003] The 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), secreted 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). Besides, there exist three "catalytic" CA isoforms (CA-VIII, CA-X, and CA-XI) whose functions are still unknown. Several further isoforms exist for all these CAs.

[0004] CA-II, CA-IX and CA-XII are associated with tumorigenesis processes, and they may be 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 to be one of the most catalytically efficient enzymes. It is present to some extent in malignant cells and, interestingly, has recently been shown to be ectopically expressed in endothelial cells of tumor neovasculature. CA-IX, a transmembrane enzyme, was first recognized as a novel tumor-associated antigen expressed not only in normal gastrointestinal tissues but also in several types of human cancers. CA-IX is functionally associated with cell adhesion, differentiation, proliferation and carcinogenesis processes, and its enzymatic activity is comparable to that of CA II. CA-XII, another transmembrane CA isozyme, was first found in normal kidney tissues and renal cell carcinoma. Further studies showed 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, the latter face of which faces towards the extracellular space.

[0005] The high expression of CA-II, CA-IX and CA-XII in tumors, especially in hypoxic tumors, further suggests that these enzymes may be functionally involved in the invasion process promoted by acidification of the extracellular space. In support of this hypothesis, it has been shown in vitro that CA inhibitors can reduce the invasive potential and proliferation of cancer cells (Manokaran et al. (2008), J Biomed Nanotechnol., 4(4):491-498). Notably, CA IX and CA XII appear to be regulated by a similar mechanism; since the transcription of these isozymes is induced in hypoxic tumors through a hypoxia-inducible factor-1α (HIF-1α)-mediated pathway (Chiche et al. (2009)). Moreover, expression of CA-XII has been shown to be highly correlated with estrogen receptor alpha (ERα) in breast tumors (Barnett et al. (2008), Cancer Res 68:3505-3515). Further elucidating the importance of CA in cancer progression, rapidly proliferating tumor cells overgrow so rapidly that 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 stress conditions, resulting in the expression of about 50 additional proteins, including enzymes involved in pH homeostasis (Potter et al. (2004), Gell Cycle, 3:164-167). As explained above, the latter is achieved, at least in part, by a complex coordination between selected carbonic anhydrase (CA) isozymes, particularly between CA-II, CA-IX, and CA-XII. A direct link between CA XII and cancer is demonstrated by Proescholdt et al. (2005), Neuro Onco 7:465-475, where it is shown that expression of CA XII is upregulated in intrinsic and metastatic brain tumors compared to normal brain tissue.Furthermore, Ilie et al. (2011), In J Cancer, 128(7): 1614-23 and Hinninen et al. (2006), Histopathology, 49:594-602 demonstrated overexpression of CA XII in tissues from resectable non-small cell lung cancer and ovarian cancer, respectively. Hsieh et al. (2010), Eur J Gell Biol, 89:598-606 revealed that CA XII is associated with invasion and metastasis of tumor cell lines in vivo and in vitro.

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

[0007] Thus, CAs, especially CA-XII, are known to contribute to hypoxia, cancer, and eye diseases and therefore represent important targets for therapeutic treatment 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 imbalance, 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). Thus, there is a need for additional tools and methods that can be employed to develop additional therapeutic and diagnostic tools for the above diseases.

[0008] There is a need to overcome these and further difficulties. Accordingly, the present invention addresses these needs and technical objectives and provides a solution as described herein and defined in the claims. Summary of the Invention

[0009] The present invention relates to a dimer composed of a first Fab monomer and a second Fab monomer, each Fab monomer comprising a VH domain and a VL domain, wherein (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 additional non-naturally occurring cysteine ​​residues 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 additional non-naturally occurring cysteine ​​residues 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 additional non-naturally occurring cysteine ​​residues 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 additional non-naturally occurring cysteine ​​residues 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 the context of the present invention, Fab monomers are dimerized via cysteine ​​bonds (disulfide bonds) between the additional non-naturally occurring cysteine ​​residues at the N-terminus of the VL and / or VH regions of the Fab monomers. Thus, according to the present invention, the presence of the additional non-naturally occurring cysteine ​​residue at the N-terminus of the VL and / or VH regions of the Fab monomers allows the formation of cysteine ​​bonds (disulfide bonds) between the Fab monomers to construct Fab dimers as described and provided herein. Surprisingly, as found in the context of the present invention and as shown herein, these Fab dimers containing cysteine ​​bonds (disulfide bonds) show increased binding affinity and / or avidity to a target structure (e.g., antigen) in an acidic environment compared to the respective Fab monomers (directed to the same target structure (e.g., antigen)) that do not contain such cysteine ​​bonds (disulfide bonds) or do not contain the additional non-naturally occurring cysteine ​​residue at the N-terminus of the VL and / or VH regions. According to the present invention, this increased binding affinity and / or avidity for a target structure (e.g., an antigen) in an acidic environment may be particularly important when the target structure (also referred to herein as a target; e.g., an antigen) bound by the Fab dimer is present in a cancer or is suitable for the treatment or diagnosis of cancer due to the acidic microenvironment within the tumor (see, e.g., 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] In view of the above, it was surprising that such dimers are capable of binding to a target, e.g. a target protein; since dimerization via the N-terminus of either the two VH regions, the two VL regions, the VH region and the VL region, or the VL region and the VH region would not be expected to bind to the target, e.g. due to steric hindrance of the variable regions bearing the CDRs.

[0012] In fact, dimerization close to the N-terminus of the variable region that provides antigen binding, preferably directly at the N-terminus, was expected to have a detrimental effect on antigen binding, since the antigen binding site (paratope) may be sterically hindered 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 not only the dimerization of two VH regions at the N-terminus, but also the dimerization of two VL regions, or VH region and VL region, or VL region and VH region at the N-terminus as described herein, allows binding to the target.

[0013] Indeed, in other antibody formats, the VH region can be fused to the VL region by classical N-terminal-C-terminal fusion, e.g., the N-terminus of the VL region can be fused to the C-terminus of the VH region, or vice versa. However, no N-terminal / N-terminal fusions have been performed. Similarly, the introduction of disulfide bonds can be a suitable means, e.g., to stabilize or dimerize antibodies or fragments thereof, but to the inventor's knowledge, no disulfide bonds are located in close proximity to, and preferably at, the N-terminus of the variable region. Instead, cysteine ​​residues are introduced far from the variable region. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Generally, in the context of the present invention, the additional non-naturally occurring cysteine ​​residue at the N-terminus of the VL and / or VH region of a Fab monomer described herein may be located at any position within the respective VL or VH region closer to the N-terminus than the C-terminus. In one aspect of the invention, it may be within the first 10 N-terminal amino acids of the respective VL or VH region (the first amino acid of the respective VL or VH region being counted as position 1 of the N-terminus), 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 invention, the non-naturally occurring cysteine ​​residue at the N-terminus of the VL or VH region is located at amino acid position 1 or amino acid position 2; where the amino acids are counted from the N-terminus of the VL or VH region, respectively, and position 1 is the first amino acid at the N-terminus.

[0015] As used herein and as readily understood by one of skill in the art, a "Fab" (or "Fab molecule") is a fragment of an antibody, e.g., as described herein, and is widely known in the art. It may be a binding agent that includes 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 allows the Fab to bind to a target structure, such as an antigen or epitope. The VH and VL chains or regions of the "Fab" may further include a constant region or a portion of a constant region. As used herein and as reasonably applied by one of skill 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 will include an antigen-binding domain and have the same properties as the Fabs described herein.

[0016] In the context of the present invention, as used herein, "additional" in the context of an "additional non-naturally occurring cysteine ​​residue" at the N-terminus of the VL and / or VH region of a Fab monomer described herein can mean: (i) the presence of a cysteine ​​residue at the N-terminus of each of the VL and / or VH regions of a Fab monomer in addition to the amino acids constituting the respective canonical (i.e. native) sequence of each VL and / or VH region (thus extending the overall length of the amino acid sequence of the respective canonical VL or VH region by one amino acid), or (ii) the replacement of one of the amino acids constituting the respective canonical (i.e. native) sequence of each VL and / or VH region with a cysteine ​​residue (thus leaving the overall length of the amino acid sequence of the respective canonical VL or VH region unchanged but adding one cysteine ​​residue compared to the canonical sequence). In a preferred embodiment of the present invention, "additional" refers to the addition of a cysteine ​​residue to the other amino acids constituting the respective canonical (i.e. native) sequence of each VL and / or VH region in accordance with (i) above.

[0017] Similarly, in the context of the present invention, as used herein, "non-naturally occurring" in the context of an "additional non-naturally occurring cysteine ​​residue" at the N-terminus of the VL and / or VH region of a Fab monomer described herein can mean that the additional cysteine ​​residue is not present at each or all of these positions in the amino acid sequence of the respective reference VL or VH region, or is not present at each or all of these positions.

[0018] According to the invention, the first and second Fab monomers, whose VL and / or VH regions are covalently linked by a disulfide bond between the additional non-naturally occurring cysteine ​​residues at the N-terminus of the respective VL and / or VH regions, may be the same or different. In either case, the first and second Fab monomers, whose VL and / or VH regions are covalently linked by a disulfide bond between the additional non-naturally occurring cysteine ​​residues at the N-terminus of the respective VL and / or VH regions, may be directed against the same target or against different targets (e.g. antigens). In one aspect, according to the invention, the first and second Fab monomers, whose VL and / or VH regions are covalently linked by a disulfide bond between the additional non-naturally occurring cysteine ​​residues at the N-terminus of the respective VL and / or VH regions, may be the same, and both Fab monomers are directed against the same target. In another embodiment of the invention, the first and second Fab monomers, whose VL and / or VH regions are covalently linked by a disulfide bond between the additional non-naturally occurring cysteine ​​residues at the N-terminus of the respective VL and / or VH regions, may be different and directed against the same target. In yet another embodiment of the invention, the first and second Fab monomers, whose VL and / or VH regions are covalently linked by a disulfide bond between the additional non-naturally occurring cysteine ​​residues at the N-terminus of the respective VL and / or VH regions, may be different and directed against different targets. In the latter embodiment of the invention, the first Fab monomer is directed against a first target and the second Fab monomer is directed against a second target, the first and second targets being different. In this case, the dimers described and provided herein would be bispecific Fab dimers, since they are directed against two different targets.

[0019] According to the present invention, the target directed by the Fab dimer of the present invention can be any target for which tagging, marking, neutralization, or other binding is desired. In some embodiments of the present invention, such target can be an antigen present in a subject suffering from hypoxia or eye disease, an antigen present in or on a cancerous or neoplastic cell, or an antigen present in a subject suffering from cancer, particularly a solid tumor. Such target can be an antigen or any other structure having a peptide moiety, glycosidic moiety, and / or other moiety to which an antibody or a fragment thereof (e.g., Fab or F(ab')2 fragment) can bind. In a specific embodiment of the present invention, the target directed by the dimer of the present invention is α-carbonic anhydrase XII (CA-XII).

[0020] Thus, in a specific embodiment of the invention, the dimers described and provided herein bind (preferably specifically bind) to CA-XII. In this regard, according to the invention, the dimers described and provided herein are capable of binding (preferably specifically bind) to CA-XII via one or both, preferably both, of the first and second Fab monomers described herein.

[0021] The term "specifically recognize" (used herein similarly to "specifically bind", "directed to" or "react with") means, according to the present invention, that the recognition molecule is able to specifically interact and / or bind to at least two, preferably at least three, more preferably at least four amino acids of an epitope 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 any protein, including proteins closely related to or homologous to the epitope against which the recognition molecule is directed. However, the term "other proteins" does not include that the recognition molecule cross-reacts with epitopes from species other than the species against which the recognition molecule was generated.

[0022] The term "essentially does not bind" as used herein means that the epitope recognition molecule of the present invention does not bind to other proteins, i.e., exhibits 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 believed to result from the binding of specific motifs in the amino acid sequence of the binding domain and the antigen to each other, not only as a result of their primary, secondary or tertiary structure, but also as a result of secondary modifications of said structure. The specific interaction of the antigen interaction site with its specific antigen may result in a simple binding of said site with said antigen. Furthermore, the specific interaction of the antigen interaction site with its specific antigen may alternatively result in the initiation of a signal, for example due to the induction of a conformational change of the antigen, oligomerization of the antigen, etc. A preferred example of a binding domain according to the present invention is an antibody. Usually, the binding affinity is greater than 10 -6 Binding is considered "specific" if the binding affinity is greater than M. Preferably, the binding affinity is about 10-11 ~10 -8 M(K D ), preferably about 10 -11 ~10 -9 If M, the binding is considered specific. If necessary, the binding conditions can be changed to reduce non-specific binding without substantially affecting specific binding. Whether a recognition molecule reacts specifically as defined above can be easily tested, inter alia, by comparing the reaction of the recognition molecule with the epitope with the reaction of the recognition molecule with other protein(s).

[0024] In one embodiment of the invention, CA-XII is human CA-XII. According to the invention, furthermore, the described and provided dimers will 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 invention is a term well known in the art and in the context of the present invention relates to the part of CA-XII that extends to the extracellular environment. The term "catalytic domain" according to the invention is also a term well known in the art and in the context of the present invention relates to the part of CA-XII where the catalytic reaction of carbonic acid to bicarbonate and hydrogen ions takes place.

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

[0026] The term "epitope" also refers to a site on an antigen to which a recognition molecule binds. Preferably, an epitope is a site on a recognition molecule, preferably a molecule to which an antibody will be produced and / or to which an antibody will bind. For example, an epitope can be recognized by a recognition molecule, particularly preferably an antibody that defines the epitope. A "linear epitope" is an epitope in which a primary sequence of amino acids constitutes the epitope being recognized. A linear epitope typically contains at least 3, more usually at least 5, for example about 8 to about 10 amino acids in a unique sequence.

[0027] A "conformational epitope" is an epitope in which, in contrast to a linear epitope, the primary sequence of amino acids that constitute the epitope is not the only determining component of the recognized epitope (e.g., an epitope in which the primary sequence of amino acids is not necessarily recognized by the antibody that defines the epitope). Typically, a conformational epitope contains an increased number of amino acids compared to a linear epitope. For the recognition of a conformational epitope, the recognition 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, certain amino acids and / or polypeptide backbones that constitute the conformational epitope are juxtaposed, allowing the antibody to recognize the epitope. Methods for determining the conformation of an epitope include, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy, site-specific spin labeling, and electron paramagnetic resonance spectroscopy.

[0028] As used herein, an "antibody" is a protein composed of one or more polypeptides (containing one or more binding domains, preferably an antigen-binding domain) substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The term "immunoglobulin" (Ig) is used interchangeably herein with "antibody." Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes.

[0029] In particular, an "antibody" as used herein is a tetrameric glycosylated protein typically composed of two light chains (L chains) of about 25 kDa each and two heavy chains (H chains) of about 50 kDa each. There can be two types of light chains in an antibody, 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 humans). IgM antibodies are composed of five basic heterotetrameric units and an additional polypeptide called the J chain, which contains 10 antigen-binding sites; whereas IgA antibodies are composed of two to five basic four-chain units, which can polymerize to form multivalent aggregates with the J chain. In the case of IgG, the four-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 domains are not directly involved in binding the antibody to the antigen.

[0031] When VH and VL are paired together, a single antigen-binding site is formed. The CH domain closest to VH is designated CH1. Each L chain is linked to the H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the isotype of the H chain. The VH and VL domains are composed of four regions of relatively conserved 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 of the antibody with the antigen. The CDRs are termed 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 portions of an immunoglobulin domain (i.e., the "variable domain(s)") that exhibit variability in the immunoglobulin sequence and are responsible for determining the specificity and binding affinity of a particular antibody. The variability is not uniformly distributed throughout the variable domain of an antibody; it is concentrated in subdomains of each of the heavy and light chain variable regions. These subdomains are called "hypervariable" or "complementarity determining regions" (CDRs). The more conserved (i.e., non-hypervariable) portions of the variable domains are called "framework" regions (FRMs). Naturally occurring heavy and light chain variable domains each contain four FRM regions that predominantly adopt a β-sheet configuration, and these FRM regions are connected by three hypervariable regions; the hypervariable regions form loops that connect, and in some cases form part of, the β-sheet structure. The hypervariable regions of each chain are held together in close proximity by the FRMs and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site (Kabat et al., infra). The constant domains are not directly involved in antigen binding, but exhibit various effector functions, such as antibody-dependent, cell-mediated cytotoxicity, and complement activation.

[0032] The term "CDR" and its plural "CDRs" refer to the 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 of 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). The CDRs contribute to the functional activity of the antibody molecule and are separated by amino acid sequences that constitute the scaffolding or framework regions. The exact definition of the boundaries and lengths of the CDRs varies according to the various classification and numbering systems. Thus, CDRs may be referred to by Kabat, Chothia, contact or other boundary definitions, including the numbering systems described herein. Despite the different boundaries, each of these systems includes some overlap in the portions of the variable sequences that constitute the so-called "hypervariable regions". Therefore, the definition of CDR according to these systems may differ in length and border regions with respect to the 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, the numbering according to the so-called Kabat system is preferred.

[0033] In a specific embodiment of the invention, the first and second Fab monomers can comprise specific VL and VH regions, each of which comprises one or more specific CDR regions. For example, in one specific embodiment of the invention, the first Fab monomer comprises (A) a VH-CDR1 as set forth in SEQ ID NO: 1, a VH-CDR2 as set forth in SEQ ID NO: 2, a VH-CDR3 as set forth in SEQ ID NO: 3, a VL-CDR1 as set forth in SEQ ID NO: 4, a VL-CDR2 as set forth in SEQ ID NO: 5 and / or (preferably) a VL-CDR3 as set forth in SEQ ID NO: 6, or (B) any one or all of the sequences of (A), 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 has been substituted, deleted or added (preferably substituted) compared to the respective amino acid sequence SEQ ID NO: 1-6. In a specific embodiment of the invention, the first Fab monomer comprises a VH-CDR1 as set forth in SEQ ID NO:1, a VH-CDR2 as set forth in SEQ ID NO:2, a VH-CDR3 as set forth in SEQ ID NO:3, a VL-CDR1 as set forth in SEQ ID NO:4, a VL-CDR2 as set forth in SEQ ID NO:5 and a VL-CDR3 as set forth in SEQ ID NO:6. In another specific embodiment of the invention, the first Fab monomer comprises a VH-CDR1 as depicted in SEQ ID NO:1, a VH-CDR2 as depicted in SEQ ID NO:2, a VH-CDR3 as depicted in SEQ ID NO:3, a VL-CDR1 as depicted in SEQ ID NO:4, a VL-CDR2 as depicted in SEQ ID NO:5 and / or (preferably) a VL-CDR3 as depicted in SEQ ID NO:6, wherein at least one, and up to five, preferably up to four, more preferably up to three, more preferably up to two, more preferably exactly one amino acid substitution in any one or all of SEQ ID NOs:1-6, respectively, and wherein at least one and (preferably) all of said substitutions are conservative or highly conservative.

[0034] As used herein, a "conservative" substitution refers to a substitution listed under "exemplary substitutions" in Table I herein. As used herein, a "highly conservative" substitution refers to a substitution set forth under the heading "preferred substitutions" in Table I herein.

[0035] Table I: Amino acid substitutions TIFF2024525722000001.tif101128

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

[0037] The term "amino acid" or "amino acid residue" as used herein typically refers to an amino acid having a definition generally recognized in the art, e.g., an amino acid selected from the group consisting of 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, synthetic, or rare amino acids may be used if desired. In general, amino acids can be grouped as having 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 of the first and second Fab monomers may also comprise a secretory sequence at their N-terminus. As is generally known in the art, such a secretory sequence may be suitable for allowing the synthesized Fab monomer to be secreted from the Fab-producing cell. For example, in the context of the present invention, such a secretory sequence at the N-terminus of the VL and / or VH region of the first and / or second Fab monomer may comprise or consist of the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO: 16, or may comprise or consist of the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO: 16, in which at least one and up to five, preferably up to four, more preferably up to three, more preferably up to two, more preferably exactly one amino acid has been substituted, deleted or added (preferably substituted) compared to the respective amino acid sequence SEQ ID NO: 15 or SEQ ID NO: 16. In a specific embodiment of the invention, such secretory sequence at the N-terminus of the VL and / or VH region of the first and / or second Fab monomer may comprise or consist of the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO: 16. In another specific embodiment of the invention, such secretory sequence at the N-terminus of the VL and / or VH region of the first and / or second Fab monomer may comprise 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, more preferably exactly one amino acid substitution has been made compared to the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO: 16, and wherein at least one or (preferably) all of said substitutions are conservative or highly conservative substitutions as defined herein.

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

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

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

[0042] In a very specific embodiment of the invention, the first and / or the second Fab monomer comprises a VH region as depicted in SEQ ID NO:7 and / or (preferably) a VL region as depicted in SEQ ID NO:8. In another specific embodiment of the invention, the first and / or the second Fab monomer comprises a VH region as depicted in SEQ ID NO:7 and / or (preferably) a VL region as depicted in SEQ ID NO:8, wherein at least 1, and up to 5, preferably up to 4, more preferably up to 3, more preferably up to 2, more preferably exactly 1 amino acid has been 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 invention, the first and / or second Fab monomer comprises a VH region as depicted in SEQ ID NO:7 and / or (preferably) a VL region as depicted 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, more preferably exactly one amino acid substitution compared to the respective amino acid sequence SEQ ID NO:7 or SEQ ID NO:8, and wherein at least one or (preferably) all of said substitutions are conservative or highly conservative substitutions as defined herein.

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

[0044] As used herein, a "silent" substitution or mutation refers to a base substitution in a nucleic acid sequence that does not change the respective amino acid sequence encoded by the nucleic acid sequence. A "conservative" substitution refers to a substitution listed in Table I under the heading "exemplary substitutions." As used herein, a "highly conservative" substitution refers to a substitution shown in Table I under the heading "preferred substitutions."

[0045] As used herein, unless otherwise defined, the terms "nucleic acid" or "nucleic acid molecule" are used interchangeably with "oligonucleotide," "nucleic acid strand," and the like, and refer to, for example, a single-stranded or double-stranded polymer containing one, two, or more nucleotides.

[0046] Similarly, the terms "polynucleotide", "nucleic acid" and "nucleic acid molecule" used herein should be interpreted synonymously. In general, nucleic acid molecules can include, inter alia, DNA molecules, RNA molecules, oligonucleotide thiophosphates, substituted ribo-oligonucleotides or PNA molecules. Furthermore, the term "nucleic acid molecule" can refer to DNA or RNA, or hybrids thereof, or modifications thereof known in the art (for examples of modifications, see, for example, US 5525711, US 471 1955, US 5792608 or EP 302175). Polynucleotide sequences can be single-stranded or double-stranded, linear or circular, natural or synthetic, and have no size limitations. For example, polynucleotide sequences can be genomic DNA, cDNA, mitochondrial DNA, mRNA, antisense RNA, ribozyme RNA, or DNA encoding such RNA, or chimeroplasts (Gamper, Nucleic Acids Research, 2000, 28, 4332-4339). Said polynucleotide sequence may be in the form of vector, plasmid, or viral DNA or RNA.Also described herein are 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 said nucleic acid molecules in the context of 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 invention, the first and / or second Fab monomer comprises a VH region as depicted in SEQ ID NO:11 and / or (preferably) a VL region as depicted in SEQ ID NO:12. In another specific embodiment of the invention, the first and / or second Fab monomer comprises a VH region as depicted in SEQ ID NO:11 and / or (preferably) a VL region as depicted in SEQ ID NO:12, wherein at least 1, and up to 5, preferably up to 4, more preferably up to 3, more preferably up to 2, more preferably exactly 1 amino acid has been substituted, deleted or added (preferably substituted) compared to the respective amino acid sequence SEQ ID NO:11 or SEQ ID NO:12. In the latter context, in one aspect of the invention, the first and / or second Fab monomer comprises a VH region as depicted in SEQ ID NO:11 and / or (preferably) a VL region as depicted in SEQ ID NO:12, in which at least one, and up to five, preferably up to four, more preferably up to three, more preferably up to two, more preferably exactly one amino acid substitution compared to the respective amino acid sequence SEQ ID NO:11 or SEQ ID NO:12, and in which at least one or (preferably) all of said substitutions are conservative or highly conservative substitutions as defined herein.

[0048] In a further specific embodiment of the 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. In a further specific embodiment of the 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) 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, more preferably 1 to 6 nucleotides have been substituted, added or deleted (preferably substituted) compared to the nucleic acid sequence of SEQ ID NO:13 or SEQ ID NO:14, respectively. In the latter case, 1 to 30, preferably 1 to 21, more preferably 1 to 12, more preferably 1 to 6 nucleotides are substituted compared to the nucleic acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14, respectively, and such substitutions are preferably conservative substitutions, highly conservative substitutions or more preferably silent substitutions.

[0049] The present invention further comprises: (a) culturing suitable cells (e.g., CHO cells or HEK293 cells) expressing a first and second Fab monomer as defined herein until they reach stationary phase; (b) Optionally, the cell culture is supplemented with an oxidizing agent (e.g., O2, 1 mM H2O2, and / or 1 mM MnO4 - ) addition; and (c) obtaining the dimer. The present invention relates to a dimer as described and provided herein, which is obtained by

[0050] As used herein, "stationary phase" may mean no further substantial (e.g., no more than about 2%, preferably no more than about 1% over a 2 hour period) net increase in cell number (e.g., due to a balance between dividing and dying cells), 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, e.g., for diagnostic or therapeutic purposes. Such compounds can include, for example, labeling groups, toxins, or antitumor drugs (e.g., fluorochromes, enzymes (such as peroxidases), radionuclides, rizin, ozogamicin, emtansine, monomethylauristatin E, α-amanitin). Such coupling can be performed chemically after expression of the antibody or antigen to the binding site, or the coupling product can be engineered into the antibody or antigen of the present invention at the DNA level. The DNA is then expressed in a suitable host system, and the expressed protein is recovered and, if necessary, renatured, as described later in this specification. Coupling can be performed via linkers known in the art. In particular, a variety of linkers that release the toxin or antitumor drug under acidic or reducing conditions or upon exposure to specific proteases can be employed with this technology.

[0052] In certain aspects, it may be desirable to attach the labeling group, toxin, or anti-tumor drug by spacer arms of various lengths to reduce potential steric hindrance.

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

[0054] Unless otherwise specified, reference herein to a "dimer" refers to a Fab dimer as described herein. Similarly, reference herein to a "monomer" refers to a Fab monomer as described herein.

[0055] Generally, in the context of the present invention, the formulations described and provided herein may include one or more dimers as described herein and one or more monomers as 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 a mammal, such as a livestock or pet. Most preferably, it is administered to a human. The pharmaceutical composition described herein can be administered to a subject in an appropriate dose. The pharmaceutical composition for use according to 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 then be administered orally, parenterally, for example, subcutaneously, intravenously, intramuscularly, intraperitoneally, intrathecally, transdermally, transmucosally, subdurally, topically or externally via iontophoresis, sublingually, by inhalation spray, aerosol, or rectally, etc., as a dosage unit formulation, optionally including conventional pharmaceutical acceptable additives. For oral administration, the (pharmaceutical) composition of the invention can take the form of, for example, tablets or capsules, which are prepared by conventional means with pharma- ceutical acceptable additives, such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone, hydroxypropylmethylcellulose), 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) composition can be administered to a patient with a physiologically acceptable carrier, as described herein. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, e.g., oils of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the (pharmaceutical) composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium ion, dried skim milk, glycerol, propylene glycol, water, ethanol, etc. The composition can also contain small amounts of emulsifiers or pH buffers, if necessary. Such compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, etc. The composition can be formulated as a suppository, using conventional binders and carriers, such as triglycerides. Oral formulations can include standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin. Such compositions will contain a therapeutically effective amount of the compound described above, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the method of administration.

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

[0058] In accordance with the present invention, the dimers described and provided herein, compositions comprising such dimers (and one or more Fab monomers (non-dimeric forms)) described and provided herein may also be for use in methods for treating or preventing hypoxia, solid tumors, or eye diseases.

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

[0060] The term "solid tumor" according to the present invention defines an abnormal mass of tissue that does not usually contain cysts or fluid. Solid tumors can be benign (not cancerous) or malignant (often referred to in the art as cancer). Various types of solid tumors are named according to the type of cells that form them. In the context of the present invention, for example, the solid tumor can be selected from sarcoma, glioma, carcinoma, mesothelioma, lymphoma, renal tumor, lung tumor, breast tumor, cervical tumor, ovarian tumor, colorectal tumor, liver tumor, prostate tumor, pancreatic tumor and head and neck tumor. In one embodiment of the present invention, the solid tumor is a glioma or a lung tumor. In a specific embodiment of the present invention, the solid tumor is a glioma.

[0061] The term "ocular disease" as used herein refers to a pathological condition or injury of the eye, including its adnexa. Ocular diseases may involve, for example, clouding or opacification of the natural lens of the eye, swelling of the macula or swelling of the central retina, for example due to leakage and accumulation of fluid, small accumulations of hyaline bodies (vitreous bodies) under the retina, damage to the optic nerve, degeneration of cells in the macula, loss of vision or inflammation of the conjunctiva and cornea of ​​the eye, and formation of scar tissue. In the context of 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 aspects that characterize the 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 items. 1. A dimer composed of a first Fab monomer and a second Fab monomer, each Fab monomer comprising a VH domain and a VL domain, wherein: (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 additional non-naturally occurring cysteine ​​residues 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 additional non-naturally occurring cysteine ​​residues 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 additional non-naturally occurring cysteine ​​residues 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 additional non-naturally occurring cysteine ​​residues 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. 2. A dimer of item 1, wherein the non-naturally occurring cysteine ​​residue at the N-terminus of the VL or VH region is at amino acid position 1 or amino acid position 2, where the amino acids are counted from the N-terminus of the VL or VH region, respectively, and position 1 is the first amino acid at the N-terminus. 3. 3. The dimer of item 1 or 2, wherein said first Fab monomer and said second Fab monomer are the same or different. 4. The dimer of any of the preceding items, wherein the first Fab monomer and the second Fab monomer are directed against the same target. 5. 4. The dimer of any of items 1 to 3, wherein the first Fab monomer is directed against a first target and the second Fab monomer is directed against a second target, wherein the first target and the second target are different. 6. The dimer of any of the preceding items, wherein the first Fab monomer comprises a VH-CDR1 as set forth in SEQ ID NO: 1, a VH-CDR2 as set forth in SEQ ID NO: 2, a VH-CDR3 as set forth in SEQ ID NO: 3, a VL-CDR1 as set forth in SEQ ID NO: 4, a VL-CDR2 as set forth in SEQ ID NO: 5, and a VL-CDR3 as set forth in SEQ ID NO: 6. 7. 16. The dimer of any of the preceding items, wherein the VL region of the first Fab monomer and the VL region of the second Fab monomer comprise at their N-terminus the secretory sequence shown in SEQ ID NO:16. 8. 16. The dimer of any of the preceding items, wherein the VH region of the first Fab monomer and the VH region of the second Fab monomer comprise at their N-terminus the secretory sequence shown in SEQ ID NO:16. 9. 16. The dimer of any of the preceding items, wherein the VL region of the first Fab monomer and the VH region of the second Fab monomer comprise at their N-terminus the secretory sequence shown in SEQ ID NO:16. 10. 16. The dimer of any of the preceding items, wherein the VH region of the first Fab monomer and the VL region of the second Fab monomer comprise at their N-terminus the secretory sequence shown in SEQ ID NO:16. 11. The dimer of any of the preceding items, wherein the first Fab monomer comprises a VH region as depicted in SEQ ID NO:7 and a VL region as depicted in SEQ ID NO:8. 12. The dimer of any of the preceding items, wherein the first Fab monomer comprises a VH region as depicted in SEQ ID NO:11 and a VL region as depicted 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 stationary phase; (b) optionally adding an oxidizing agent to the CHO cell culture; and (c) obtaining the dimer. 2. A dimer according to any of the preceding claims, obtained by 14. A composition comprising the dimer of any one of items 1 to 13. 15. 15. The composition of item 14, further comprising a Fab monomer as defined in any one of items 1 to 13.

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

[0065] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0066] As used herein, the term "and / or" includes the meaning of "and" as well as "or" and "all or any other combination of the elements connected by said term."

[0067] Throughout this specification and the claims which follow, unless the context dictates otherwise, the word "comprise" and variations such as "comprises" and "comprising" are understood to mean the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of other integers or steps or group of integers or steps. As used herein, the term "comprising" can be interchanged with the terms "containing" or "including," and sometimes can also be interchanged with the term "having" as used herein.

[0068] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0069] In each instance herein, any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with either of the other two terms.

[0070] It is to be understood that this invention is not limited to the particular methodology, protocols, reagents, etc. described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0071] All publications and patents cited throughout the text of this specification (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether referenced supra or infra, are hereby incorporated by reference in their entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent that the material incorporated by reference contradicts or is inconsistent with the present specification, the present specification shall supersede such material. [Brief description of the drawings]

[0072] drawing The drawings show:

[0073] (Figure 1-1) Figure 1: Vector structure. HitbasisHygro_ch6A10_LC and HitbasisNeo_ch6A10_HC vectors. Translation of HitbasisHygro_ch6A10_LC: TIFF2024525722000002.tif31135 (bold = predicted signal (secretion) sequence). (Figure 1-2) See the explanation for Figure 1-1. (Figure 2)Figure 2: SDS-PAGE. Reducing SDS-PAGE analysis of the 6A10 Fab dimer showed characteristic bands at approximately 25-30 kDa corresponding to the light and heavy chains of the 6A10 Fab fragment. Under non-reducing conditions, bands at approximately 50 and 100 kDa corresponding to the precursor monomeric and homodimeric forms are detected. (Figure 3)Figure 3: "Intact test" by LC-ESI-TOF. Further analysis of the monomer and dimeric forms of the precursor is performed by LC-ESI-TOF. Homodimers are formed by disulfide bonding of an additional cysteine ​​at the N-terminus of the light chain. Furthermore, different forms of the monomer with and without an additional cysteine ​​or glutathione are detected in varying ratios. (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 the monomer and dimer at pH 7.4. 50.000 A549 cells were incubated with serial dilutions of monomer or dimer (6A10). After washing, cells were incubated with Cy5-labeled secondary antibody (donkey anti-human IgG light and heavy chains). Secondary antibody binding was analyzed using a BD FACSCanto and DIVA software. RU = relative light units. (Figure 5-1)Figure 5: Flow cytometry. Flow cytometry revealed that the binding behavior of the monomer, but not that of the dimer (610A), was dramatically reduced in the acidic environment (pH 5.5) typical of solid tumors. (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. (FIG. 6) FIG. 6: Shows the amino acid sequence of VH of Fab6A10 with the IgG secretory sequence, and the amino acid sequence of VL of Fab6A10 with the IgL secretory sequence. (Figure 7) Figure 7: Shows the amino acid sequence of VH of Fab6A10 after N-terminal and C-terminal sequencing, and the amino acid sequence of VL of Fab6A10 after N-terminal and C-terminal sequencing.

[0074] This application also provides the following sequences referred to in this application, for which sequence protocols have been prepared and are also part of this application, provided that in the event of any discrepancy between the following sequences and those shown 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: TIFF2024525722000003.tif21159 (CDRs are in bold and boxed, constant regions are underlined) SEQ ID NO: 8 - VL of Fab6A10: TIFF2024525722000004.tif21160 (CDRs are in bold and boxes, constant regions are underlined) SEQ ID NO: 9 - Nucleic acid of VH of Fab6A10: TIFF2024525722000005.tif60160SEQ ID NO: 10 - Nucleic acid of VL of Fab6A10: TIFF2024525722000006.tif60160SEQ ID NO: 11 - VH of Fab6A10 with IgG secretory sequence: TIFF2024525722000007.tif27160 (secretory sequence is in bold italics, CDRs are in bold and boxed, constant regions are underlined) SEQ ID NO: 12 - VL of Fab6A10 with IgL secretory sequence: TIFF2024525722000008.tif21160 (secretory sequence is in bold italics, CDRs are in bold and boxed, constant regions are underlined) SEQ ID NO: 13 - Nucleic acid of VH Fab6A10 with IgG secretory sequence: TIFF2024525722000009.tif65160SEQ ID NO: 14 - Nucleic acid of VL Fab6A10 with IgL secretory sequence: TIFF2024525722000010.tif65160SEQ ID NO: 15 - IgG secretion sequence: TIFF2024525722000011.tif4128SEQ ID NO: 16 - IgL secretion sequence: TIFF2024525722000012.tif4128SEQ ID NO: 17 - N-terminal fragment of VL of Fab6A10: TIFF2024525722000013.tif4128SEQ ID NO: 18 - N-terminal fragment of VL of Fab6A10: TIFF2024525722000014.tif4128SEQ ID NO: 19 - N-terminal fragment of VH of Fab6A10: TIFF2024525722000015.tif4128SEQ ID NO: 20 - C-terminal fragment of VL of Fab6A10: TIFF2024525722000016.tif3128SEQ ID NO: 21 - C-terminal fragment of VH of Fab6A10: TIFF2024525722000017.tif3128

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

[0076] Construction of CHO cell line producing 6A10 Fab fragment A Chinese Hamster Ovary (CHO) derived cell line clone expressing 6A10 Fab was 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 München. The host suspension cell line CHO HIT was developed from CHO K1 (ECACC, No. 85051005). The 6A10 Fab used has the sequence shown in Figure 6 and Figure 7, respectively. Figure 6 shows the sequence of the 6A10 Fab monomer before processing by the host cell. Figure 7 shows the sequence obtained after sequencing of the 6A10 Fab dimer.

[0077] The vectors HitbasisHygro_ch6A10_LC and HitbasisNeo_ch6A10_HC (see Figure 1) were generated for transfection into CHO HIT cells. For this purpose, the coding sequences of the 6A10 Fab light and heavy chains were amplified from the plasmids pUC57-ch6A10 LC and pUC57-ch6A10 HC using PCR techniques. The cDNA of the 6A10 Fab light chain was inserted into HITbasisHygro. The cDNA of the 6A10 Fab heavy chain was inserted into HITbasisNeo.

[0078] The structure of the corresponding vector is shown in Figure 1. The combined results from restriction enzyme analysis and partial sequencing verified the accuracy and integrity of the plasmid.

[0079] After the expression vector was transformed into competent E. coli TopTen cells, the cells were plated on LB agar medium 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 into "host cell growth medium" (ProCHO5 medium with 4mM GlutaMAX) and expanded for transfection.

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

[0082] Two days after transfection, cell pools were transferred to "selection medium 1" or "selection medium 2" (proCHO medium containing 4 mM GlutaMAX and antibiotics) and subcultured until viability was restored and cells began to grow. For pools that showed acceptable growth, titers were determined by densitometric evaluation of SDS-PAGE to estimate cell-specific productivity. Cell pool "MP P6-A4" was selected for single cell cloning.

[0083] Clones were isolated by image-assisted cloning using limiting dilution combined with image-based documentation. All expanded clones were then expanded for later freezing in 5 vials each. Clones showing the best product concentration as measured by PAIA assay were expanded to shake flask level (9 clones). Clones “P1D20”, “P1E20”, “P1H06”, and “P5E17” were selected as the preferred production cell lines and used for expression stability testing. Based on the data obtained during expression stability testing, clones “P1E20” and “P5E17” were considered phenotypically sufficiently stable. Finally, cell clone “P1E20” was selected for USP method development based on quantity and quality.

[0084] N- and C-terminal sequencing N- and C-terminal sequencing by MALDI-TOF-MS is a method for confirming the amino acid sequence at the N- and C-terminus of a protein. Reduced samples are spotted onto a polished steel target with 1,5-diaminonaphthalene and measured by MALDI-TOF-MS in positive ion mode. Fragmentation along the amino acid backbone of the peptide produces fragments specific to the amino acid sequence at the N- and C-terminus of the protein. The theoretical amino acid sequence is verified by comparing the molecular masses of the experimentally measured fragments with those theoretically predicted.

[0085] The identity of 6A10Fab-CHX-A''-DTPA was confirmed by N- 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- and C-termini of 6A10 Fab chain (reference and batch) TIFF2024525722000018.tif62161

[0087] Without wishing to be bound by theory, the predicted signal sequence selected for optimal translation and secretion of the light chain was not properly cleaved by the signal sequence peptidase (SSP) enzyme. As a result, 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 non-naturally occurring cysteine ​​residues 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 can be seen 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), which can form a disulfide bridge with an additional cysteine ​​residue (Cys) present at the N-terminus of the VL region of the second Fab6A10 monomer, resulting in a Cys-Cys bridge between the VL region of the first Fab6A10 monomer and the VL region of the second Fab6A10 monomer, thereby obtaining a Fab6A10 dimer.

[0089] Of course, such disulfide bond-forming cysteine ​​residues may, but do not necessarily have to, be introduced via a signal / secretion sequence at the N-terminus of the variable region, even though such a disulfide bond would likely not be cleaved correctly by a signal sequence peptidase (SSP) enzyme as a result. Indeed, such cysteine ​​residues can also be introduced by corresponding engineering of the nucleotide sequence encoding such variable region, as is well known in the art.

[0090] From the observation that Fab dimers can assemble via a previously unseen unique disulfide bond, one can reasonably infer that: (i) the VL region of the first Fab monomer and the VL region of the second Fab monomer may be covalently linked by a disulfide bond between additional non-naturally occurring cysteine ​​residues 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 linked by a disulfide bond between additional non-naturally occurring cysteine ​​residues 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 linked by a disulfide bond between additional non-naturally occurring cysteine ​​residues 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 additional non-naturally occurring cysteine ​​residues 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 monomer or Fab6A10 dimer. Fab6A10 has the following CDRs: VH-CDR1 as shown in SEQ ID NO: 1, VH-CDR2 as shown in SEQ ID NO: 2, VH-CDR3 as shown in SEQ ID NO: 3, VL-CDR1 as shown in SEQ ID NO: 4, VL-CDR2 as shown in SEQ ID NO: 5, and VL-CDR3 as 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 non-naturally occurring cysteine ​​residues 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 can be seen 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), which can form a disulfide bridge with an additional cysteine ​​residue (Cys) present at the N-terminus of the VL region of the second Fab6A10 monomer, resulting in a Cys-Cys bridge between the VL region of the first Fab6A10 monomer and the VL region of the second Fab6A10 monomer, thereby obtaining a Fab6A10 dimer.

[0093] The 6A10 Fab used has the sequence shown in Figure 6 and Figure 7, respectively. Figure 6 shows the sequence of the 6A10 Fab monomer before processing by the host cell. Figure 7 shows the sequence obtained after sequencing the 6A10 Fab 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 a BD FACSCanto and DIVA software. RU = relative light units.

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

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

[0097] Fab6A10 has the following CDRs: VH-CDR1 as shown in SEQ ID NO: 1, VH-CDR2 as shown in SEQ ID NO: 2, VH-CDR3 as shown in SEQ ID NO: 3, VL-CDR1 as shown in SEQ ID NO: 4, VL-CDR2 as shown in SEQ ID NO: 5, and VL-CDR3 as 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 non-naturally occurring cysteine ​​residues 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 can be seen 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), which can form a disulfide bridge with an additional cysteine ​​residue (Cys) present at the N-terminus of the VL region of the second Fab6A10 monomer, resulting in a Cys-Cys bridge between the VL region of the first Fab6A10 monomer and the VL region of the second Fab6A10 monomer, thereby obtaining a Fab6A10 dimer.

[0099] The 6A10Fab used has the sequence shown in Figure 6 and Figure 7, respectively. Figure 6 shows the sequence of the 6A10 Fab monomer before processing by the host cell. Figure 7 shows the sequence obtained after sequencing the 6A10Fab dimer. Binding of the dimer or monomer was measured by flow cytometry.

[0100] FIG. 5 shows that binding of the Fab6A10 dimer is significantly superior to that of the monomer at low pH, such as that found in the tumor environment.

Claims

**Claim 1** A dimer composed of a first Fab monomer and a second Fab monomer, wherein each Fab monomer comprises a VH region and a VL region, wherein (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 additional cysteine residues that are not naturally present 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 additional cysteine residues that are not naturally present 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 additional cysteine residues that are not naturally present 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 additional cysteine residues that are not naturally present 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, the dimer. **Claim 2** The dimer according to claim 1, wherein the cysteine residue that is not naturally present at the N-terminus of the VL region or the VH region is at amino acid position 1 or amino acid position 2, where the amino acid is counted from the N-terminus of the respective VL region or VH region, and position 1 is the first amino acid at the N-terminus. **Claim 3** The dimer according to claim 1, wherein the first Fab monomer and the second Fab monomer are the same or different. **Claim 4** The dimer according to claim 1, wherein the first Fab monomer and the second Fab monomer are directed against the same target, or the first Fab monomer is directed against a first target and the second Fab monomer is directed against a second target, and the first target and the second target are different. **Claim 5** The dimer according to claim 1, 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. **Claim 6**: (i) The VL region of the first Fab monomer and the VL region of the second Fab monomer each contain a secretion sequence shown in SEQ ID NO: 16 at their N-terminus; (ii) The VH region of the first Fab monomer and the VH region of the second Fab monomer each contain a secretion sequence shown in SEQ ID NO: 16 at their N-terminus; (iii) The VL region of the first Fab monomer and the VH region of the second Fab monomer each contain a secretion sequence shown in 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 each contain a secretion sequence shown in SEQ ID NO: 16 at their N-terminus, the dimer according to claim 1. **Claim 7**: (i) The VL region of the first Fab monomer and the VL region of the second Fab monomer each contain a secretion sequence shown in 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 each contain a secretion sequence shown in 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 each contain a secretion sequence shown in 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 each contain a secretion sequence shown in 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 each contain a secretion sequence shown in 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 each contain a secretion sequence shown in SEQ ID NO: 15 at their N-terminus; or vi) The VH region of the first Fab monomer and the VL region of the second Fab monomer contain a secretion sequence shown in SEQ ID NO: 16 at their N-terminals, and the VL region of the first Fab monomer and the VH region of the second Fab monomer contain a secretion sequence shown in SEQ ID NO: 15 at their N-terminals. The dimer according to claim 1.

8. The dimer according to claim 1, wherein the first Fab monomer contains a VH region shown in SEQ ID NO: 7 and a VL region shown in SEQ ID NO:

8.

9. The dimer according to claim 1, wherein the first Fab monomer contains 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 contains a VH region shown in SEQ ID NO: 11 and a VL region shown in SEQ ID NO:

12.

11. The dimer according to claim 1, wherein the first Fab monomer contains 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. (a) Culturing CHO cells expressing the first and second Fab monomers defined in any one of claims 1 to 11 until reaching the stationary phase; (b) Optionally, adding an oxidizing agent to the CHO cell culture; and (c) Obtaining the dimer The dimer according to claim 1, obtained by the above method.

13. The first and / or second Fab monomer is (a) a labeling group; (b) a toxin; or (c) an antitumor drug The dimer according to claim 1, which is bound to the above.

14. A composition comprising the dimer according to claim 1.

15. The composition according to claim 14, further comprising the Fab monomer defined in claim 1.

16. (i) The pharmaceutical composition comprising the dimer according to claim 1, or (ii) the dimer according to claim 1 and the Fab monomer defined in claim 1.

17. A pharmaceutical composition for treating or suppressing hypoxia, solid tumors, or eye diseases, comprising (i) the dimer according to claim 1, or (ii) the dimer according to claim 1 and the Fab monomer defined in claim 1.

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, kidney tumor, lung tumor, breast tumor, cervical tumor, ovarian tumor, colorectal tumor, liver tumor, prostate tumor, pancreatic tumor, and head and neck tumor; or iii) The eye disease is selected from glaucoma, cataract, macular degeneration, age-related macular degeneration, uveitis, retinitis, X-linked retinoschisis, and hypertensive retinopathy, the pharmaceutical composition according to claim 17.

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