Modified Fc-region to enhance the functional affinity of antibodies and antigen-binding fragments thereof

Abstract

The present invention involves identifying key residues within mouse IgG3 antibodies (MABs) responsible for intermolecular cooperativity and transferring them to IgG1 antibodies to enhance their functional affinity and direct cell killing.

Description

Technical Field

[0001] The invention involves identifying key residues within mouse IgG3 antibodies (MABs) responsible for intermolecular cooperativity and transferring them to IgG1 antibodies to enhance their functional affinity and direct cell killing. Background Art

[0002] Mouse (m)IgG3 is highly protective against bacterial infection and is the only isotype of mIgG that forms non-covalent oligomers, strongly affecting its biological activity (Abdelmoula et al., 1989) and increasing its functional affinity for multivalent antigens. The tendency of mIgG3 to oligomerize was initially identified by Grey et al. (Grey, Hirst, and Cohn, 1971), who showed that binding to multivalent antigens promoted interactions between mIgG3 molecules, resulting in increased functional affinity for antigens (Greenspan, Monafo, and Davie, 1987; Greenspan and Cooper, 1993), a feature that was termed "intermolecular cooperativity" (Greenspan and Cooper, 1993; Greenspan, Dacek, and Cooper, 1989; Greenspan and Cooper, 1992). This phenomenon was determined to be Fc dependent, as the IgG3 F(ab')2 fragment did not bind cooperatively to antigens (Greenspan, Monafo, and Davie, 1987). The mIgG3 isotype is associated with a tendency for intermolecular cooperativity towards repeated antigens, thereby rescuing low monovalent affinity through avidity-mediated enhanced binding (Greenspan and Cooper, 1993, 1992). Non-covalent interactions between adjacent mIgG3 Fc-regions are thought to underpin this enhanced functional affinity by prolonging target occupancy and reducing dissociation rates (Cooper et al., 1991; Greenspan, Dacek and Cooper, 1989)).

[0003] The tumor glycosome is a rich and underexplored source of cancer-specific targets for mAb development due to changes associated with the transformation process (Pinho and Reis, 2015). Glycan structures are present on both protein and glycolipid backbones and can be massively overexpressed in cancer due to altered expression of glycotransferases. Glycans have been shown to be key accessory molecules for cancer cell survival, proliferation, spread, and immune evasion; cross-linking these antigens can lead to direct cell death without the need for immune effector cells or complement (Dalziel et al., 2014; Rodríguez, Schetters, and van Kooyk, 2018; Rabu et al., 2012). Indeed, the inventors have generated a number of glycan-targeting mAbs with potential applications in cancer therapy due to their strong tumor differentiation relative to healthy tissue binding, high functional affinity, and ability to cure mice with metastatic colorectal cancer (Chua et al., 2015; Durrant et al., 2006; Noble et al., 2013), many other mAbs are in clinical development as passive or active immunotherapies, or reformatted for use in chimeric antigen receptor (CAR) T cells (Burris et al., 2010; Labrada et al., 2018; Hege et al., 2017), while Dinutuximab beta (an anti-GD2 mAb) is currently used for the treatment of neuroblastoma (Ladenstein et al., 2018).

[0004] The inventors previously described a group of Lewis a / c / x Lewis y and sialyl-di-Lewis aReactive cancer glycan-targeting mAbs (WO2017 / 033020A1; WO2015 / 063500A1; Chua et al., 2015; Noble et al., 2013). Interestingly, if the glycan-binding mAbs belong to the mIgG3 isotype, they exhibit direct cytotoxic effects on cancer cells expressing high density of targets independently of the presence of complement or immune effector cells, with the potential to initiate adaptive antitumor immune responses and long-term tumor control. This suggests that intermolecular synergy achieved by the mIgG3 isotype is responsible for the direct cell killing. This direct cytotoxicity has also been observed with other anti-glycan mAbs, typically involving mAb-induced homotypic cell adhesion, cytoskeletal rearrangements, subsequent cell swelling, membrane damage, and eventual cell death (Loo et al., 2007; Faraj et al., 2017; Roque-Navarro et al., 2008; Chua et al., 2015; Welt et al., 1987). In most cases, cell death is a non-classical form of apoptosis that may involve the generation of reactive oxygen species (ROS), most similar to tumor necrosis (Zheng et al., 2017; Hernandez et al., 2011). Importantly, similar to immunogenic or inflammatory cell death (ICD), the concurrent release of damage-associated molecular patterns (DAMPs) of inflammatory mediators has the potential to recruit innate immune cells to tumor sites, which can further increase mAb-mediated effector functions (Galluzzi et al., 2017). Therefore, these anti-glycan mAbs could be important tools to re-mobilize the full potential of the immune system in otherwise immunosuppressive settings.

[0005] For human antibody therapeutics, regions not responsible for target specificity are often replaced with their human equivalents to generate humanized antibody versions with clinical use. When we chimerize mouse mAbs with IgG1 (e.g., human IgG1, hIgG1), they lose direct cell killing and have reduced functional affinity because of the intermolecular cooperativity that is not present in human IgG. It has been previously suggested that mouse IgG3 antibodies, once bound to their targets, may bind together in a way that other antibody subclasses do not, to stabilize the binding and enhance the clinical efficacy of the antibody.

[0006] US2011 / 0123440 describes altered antibody Fc-regions and their uses. The altered Fc-regions have one or more amino acid substitutions. The patent claims any substitutions at many positions, but the exact examples are all different substitutions of our invention, which illustrate that these substitutions are not responsible for direct cell killing.

[0007] US2008 / 0089892 describes polypeptide Fc-region variants and compositions comprising these Fc-region variants, which have amino acid substitutions at positions 251, 256, 268, 280, 332, 378 and 440 to improve ADCC. The only similarity to the modifications outlined in this patent is position 378, but the inventors substituted serine instead of aspartic acid, and we show reduced ADCC in the present invention.

[0008] US2010 / 0184959 describes methods for providing Fc polypeptide variants with 2 or more amino acid substitutions and altered Fc ligand (e.g., FcγR or C1q) and / or effector function (e.g., ADCC) recognition. In contrast, the substitutions of the present invention are only associated with increased cell killing. The 26 and 23 amino acid motifs currently disclosed do include V305A, Q342R, and R344Q substitutions, while 15 amino acid motifs only include Q342R.

[0009] US2010 / 015133 describes a method for producing polypeptides by regulating polypeptide association. They claim protection of the CH3 constant region of an antibody at positions 356 / 439, 357 / 370 and 399 / 409, changing the charge of paired amino acids from negative to positive, or vice versa. In contrast, this patent describes changing amino acid 356 from aspartic acid (D) to glutamic acid (E) (without changing the charge), changing amino acid 357 from glutamic acid (E) to glutamine (Q) (changing the negative charge to neutral), and changing amino acid 370 from lysine (K) to threonine (changing the negative charge to neutral).

[0010] WO2018 / 146317 describes polypeptides and antibodies having an Fc-region and an antigen binding region, wherein the Fc-region has an Fc-Fc enhancing mutation and a C1q binding enhancing mutation, providing a polypeptide or antibody with increased CDC activity and / or agonistic activity. The Fc region is said to comprise (a) a substitution at a position selected from the group consisting of E430, E345 or S440Y or S440W substitutions, and (b) a substitution at one or more positions selected from the group consisting of: G236, S239, S267, H268, S324, K326, I332, E333 and P396, wherein these positions correspond to human IgG1. The polypeptide comprises at least one substitution from the group consisting of: E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y and S440W. The present invention does not include any of these substitutions.

[0011] WO2018 / 083126 describes polypeptides and antibodies comprising variant Fc-regions. When one or more polypeptides, antibodies or multiple antibodies bind to a target, antigen or multiple antigens on the cell surface, the Fc-region provides a stable Fc: Fc interaction, while also having reduced complement-dependent cytotoxicity (CDC), and may also reduce the activation of other effector functions due to one or more amino acid modifications in the Fc-region. The Fc region is said to include (i) a first mutation at E430, E345 or S440, provided that the mutation in S440 is S440Y or S440W; and (ii) a second mutation at K322 or P329. The first mutation is selected from the group consisting of: E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W and S440Y. The present invention does not include any of these substitutions.

[0012] WO2014 / 108198 discloses polypeptides, such as antibodies comprising variant Fc-regions having one or more amino acid modifications, which result in increased complement dependent cytotoxicity (CDC). The present invention does not include any of these substitutions.

[0013] WO2013 / 004842 describes polypeptides and related antibodies comprising variant Fc domains for stabilizing Fc:Fc interactions when one or more polypeptides, antibodies or antibodies bind to a target on the surface of a cell to improve effector functions, such as CDC responses. The present invention does not include any of these substitutions. Summary of the invention

[0014] The present invention provides polypeptides, antibodies, variants and antigen-binding fragments thereof having enhanced functional affinity compared to their parent polypeptides, antibodies, variants and antigen-binding fragments thereof. The present invention also provides polypeptides, antibodies, variants and antigen-binding fragments thereof having enhanced cell killing compared to their parent polypeptides, antibodies, variants and antigen-binding fragments thereof. Without being limited by theory, it is believed that such modified polypeptides, antibodies, variants and antigen-binding fragments thereof are capable of more stable binding interactions between the Fc-regions of two antibodies, thereby providing increased functional affinity of antibody-antigen binding. In addition, the inventors disclose a discontinuous region within the CH2 and CH3 domains of mIgG3, which confers increased functional affinity of the isotype to binding to repeating glycan antigens, as well as direct cytotoxicity to high binding cancer cell lines. The residues involved are transferred to the IgG1 subtype, resulting in an improved "i" IgG1, which increases anti-tumor activity in vitro and in vivo.

[0015] By cross-linking the chimeric mAbs with anti-human Ig antiserum or changing a key amino acid residue, we demonstrated that direct cell killing is directly related to functional affinity. In addition, we summarized the functional affinity of the novel mouse mAbs of the present invention by introducing intermolecular cooperativity in the chimeric constructs. No one has mapped the mIgG3 cooperativity, and the key amino acids that differ between IgG1 and mIgG3 have not been described before. Therefore, the inventors have not only advanced the development of our series of anti-glycolipid mAbs, but also developed a technology that can be used to improve the therapeutic index of any mAb.

[0016] Chimerization of mIgG3 monoclonal antibodies to the human IgG1 backbone was consistent with a significant reduction in direct cytotoxicity, leading the inventors to hypothesize that this was the result of reduced intermolecular synergy. Therefore, the inventors hoped to identify the key residues responsible for intermolecular synergy within mIgG3 and transfer them to IgG1 to summarize the observed direct cytotoxic activity of mIgG3, thereby generating a chimeric IgG1 with excellent clinical utility.

[0017] The inventors created IgG1 anti-glycan mAbs with increased functional affinity and direct cytotoxic activity by transferring selected mIgG3 constant region residues using our set or previously generated glycan-targeting mAbs. The mIgG3 contributing regions were determined by creating hybrid IgG1 constructs containing mIgG3 CH1, CH2, or CH3 domains. Candidate residues were identified by screening based on increased direct cytotoxicity and functional affinity when introduced into IgG1 (gain of function), and / or reduced direct cytotoxicity and functional affinity when replaced by corresponding IgG1 residues in mIgG3 (loss of function). Preliminary analysis determined that the contribution of mIgG3 CH1 to direct cytotoxic capacity was negligible, as the introduction of mIgG3CH1 into IgG1 did not result in a significant increase in cytotoxicity. In contrast, the introduction of human IgG1( h IgG1)CH 1 Introduction of mIgG 3 Similarly, there was no significant reduction in killing activity. Next, in the gain-of-function approach, the mIgG3 CH2 and CH3 domains were introduced into IgG1. 3 IgG1 showed a significant increase in cytotoxicity. 2 The introduction of IgG1 resulted in a small, but not significant, increase in killing activity. As confirmation of the contribution of these two domains, the opposite strategy was followed, whereby the loss of cytotoxic activity due to the introduction of either the IgG1 CH2 or CH3 domain into mIgG3 was assessed. This scenario resulted in IgG1 CH 3 mIgG 3The cytotoxicity of mIgG3 was significantly reduced, confirming previous gain-of-function results. Importantly, this strategy also identified a small contribution from mouse CH2, as mIgG3 containing human CH2 exhibited a significant reduction in cytotoxic activity.

[0018] According to one aspect of the present invention, a modified IgG1 antibody or an antigen-binding fragment thereof is provided, which comprises one or more residues of an immunoglobulin Fc-region and a binding region, wherein the one or more residues of the Fc-region are modified to the corresponding residues from a mouse IgG3 antibody and wherein the modified IgG1 antibody or the antigen-binding fragment thereof has an enhanced functional affinity compared to the corresponding IgG1 antibody or the antigen-binding fragment thereof comprising the wild-type Fc-region residues.

[0019] In some aspects of the present invention, a modified IgG1 antibody or antigen-binding fragment thereof is provided, wherein the functional affinity of the modified IgG1 antibody or antigen-binding fragment thereof is enhanced by at least about 10% compared to the corresponding IgG1 antibody or antigen-binding fragment thereof comprising wild-type Fc-region residues.

[0020] In some aspects of the invention, the functional affinity of the modified IgG1 antibody or antigen-binding fragment thereof is enhanced by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 100% compared to the corresponding IgG1 antibody or antigen-binding fragment thereof comprising wild-type Fc-region residues.

[0021] In some aspects of the invention, the modified IgG1 antibody or antigen-binding fragment thereof enhances direct cell killing compared to the corresponding IgG1 antibody or antigen-binding fragment thereof comprising wild-type Fc-region residues.

[0022] In some aspects of the invention, the modified IgG1 antibody or antigen-binding fragment thereof has at least about 10% enhancement in direct cell killing compared to a corresponding IgG1 antibody or antigen-binding fragment thereof comprising wild-type Fc-region residues.

[0023] In some aspects of the invention, the modified IgG1 antibody or antigen-binding fragment thereof has increased direct cell killing by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 100% compared to the corresponding IgG1 antibody or antigen-binding fragment thereof comprising wild-type Fc-region residues.

[0024] According to a first aspect of the present invention, there is provided a modified IgG1 antibody or antigen-binding fragment thereof, wherein one or more residues of the CH2 and / or CH3 domains are replaced with corresponding residues from mouse IgG3 CH2 and / or CH3 domains.

[0025] According to a second aspect of the present invention, there is provided a modified IgG1 antibody or an antigen-binding fragment thereof, wherein the CH2 and / or CH3 domains are replaced with mouse IgG3 CH2 and / or CH3 domains.

[0026] Further stripping of the combined CH2-CH3 region by subdomain analysis showed that the direct cytotoxicity of mIgG3 against IgG1 was fully restored by transferring a discontinuous section containing residues 286-306 and 339-378 (containing both CH2 and CH3 elements, a total of 26 mIgG3 residues). The 26 residues are N286T, K288W, K290Q, E294A, Y300F, V305A, A339P, Q342R, P343A, R344Q, E345T, L351I, S354P, D356E, E357Q, L358M, T359S, N361K, Q362K, K370T, G371N, Y373F, P374S, S375E, D376A, A378S. Due to the combined effects of direct interactions and conformational residues, which are required to increase functional affinity through intermolecular cooperativity, it is possible that the conformational residues create a permissive framework. The role of the charge distribution pattern, especially in CH2, cannot be excluded, as it has been shown to enhance mIgG3 binding to negatively charged multivalent antigens and is different from IgG1 (Klaus and Bereta 2018; Hovenden et al. 2013). Some of the residues involved in our approach are located within the larger consensus binding sites of many natural binding partners, such as protein A, protein G, rheumatoid factor, and neonatal FcRn (DeLano et al. 2000). However, none of our residues are directly involved in FcRn binding (Oganesyan et al. 2014).

[0027] According to a third aspect of the present invention, a modified IgG1 antibody or an antigen-binding fragment thereof is provided, which has modified one or more of the following residues in the Fc-region: N286, K288, K290, E294, Y300, V305, A339, Q342, P343, R344, E345, L351, S354, D356, E357, L358, T359, N361, Q362, K370, G371, Y373, P374, S375, D376, A378.

[0028] According to a fourth aspect of the present invention, a modified IgG1 antibody or an antigen-binding fragment thereof is provided, which has one or more of the following modifications to the Fc-region: N286T, K288W, K290Q, E294A, Y300F, V305A, A339P, Q342R, P343A, R344Q, E345T, L351I, S354P, D356E, E357Q, L358M, T359S, N361K, Q362K, K370T, G371N, Y373F, P374S, S375E, D376A, A378S.

[0029] In some aspects of the invention, the modified IgG1 antibody or antigen-binding fragment thereof comprises modifications at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 residues selected from positions N286, K288, K290, E294, Y300, V305, A339, Q342, P343, R344, E345, L351, S354, D356, E357, L358, T359, N361, Q362, K370, G371, Y373, P374, S375, D376, A378. In a preferred aspect of the invention, the modified IgG1 antibody or antigen-binding fragment thereof comprises modifications at all 26 residues.

[0030] In some aspects of the invention, the modified IgG1 antibody or antigen-binding fragment thereof comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 modifications selected from N286T, K288W, K290Q, E294A, Y300F, V305A, A339P, Q342R, P343A, R344Q, E345T, L351I, S354P, D356E, E357Q, L358M, T359S, N361K, Q362K, K370T, G371N, Y373F, P374S, S375E, D376A and A378S. In a preferred aspect of the invention, the modified IgG1 antibody or antigen-binding fragment thereof comprises all 26 modifications.

[0031] A large number of Fc engineering strategies have been described in the literature, mainly affecting mAb effector functions (ADCC and CDC) by altering FcγR or C1q binding, and affecting mAb half-life via FcRn ligation (Wang, Mathieu, and Brezski 2018; Carter, 2006). None of these describe the amino acid changes that form part of the present patent. Additionally, in many human mAb isotypes, crystal packing induces mAb oligomerization via Fc:Fc interactions (Saphire et al., 2001), forming the basis for the recently described hexameric mAb platform for improved complement activation (WO2014 / 006217A9; WO2018 / 083126A1; WO2018 / 146317A1, Ugurlar et al., 2018; de Jong et al., 2016; Diebolder et al., 2014). Effective hexamer arrangement and C1q binding depend on individual mutations (E345K or E430G), which are different from the amino acid changes that form the basis of this patent. In fact, the ability of the type II CD20 mAb 11B8 to induce programmed cell death (i.e., direct cytotoxicity) is not enhanced by these mutations that induce strong CDC capacity, indicating that this position is not involved in direct cell killing.

[0032] Most approved therapeutic mAbs are humanized or fully human IgG antibodies, as mouse or chimeric antibodies carry the risk of increasing adverse anti-mouse antibody (HAMA) reactions in patients (Schroff et al., 1985; Azinovic et al., 2006; Miotti et al., 1999; D'Arcy and Mannik 2001). The introduction of only 26 mIgG3s in IgG1 is unlikely to induce a HAMA response. However, they may generate MHCII binding epitopes that have the potential to drive a HAMA response in some patients. The Immune Epitope Database (IEDB) analyzed 2 hybrid 88 IgG1 containing 6 residues and found a cluster (Cluster 1, residues 294-315) containing several potential high-scoring epitopes. Restoring the three MIG3s at positions 294, 300, and 305 in Cluster 1 to IgG1 residues maintained functional affinity and direct cytotoxicity. Importantly, modifications to this 23-aa motif (N286, K288, K290, A339, Q342, P343, R344, E345, L351, S354, D356, E357, L358, T359, N361, Q362, K370, G371, Y373, P374, S375, D376, A378) when modified to the corresponding mouse IgG3 residues are (N286T, K374T, S374T, S374T, S374T, S374T, S374T, S374T, S374T, S374T, S374T, S374T, S374T, S374T, S374T, S374T, S374T, S374T, S374T, S374T, S374T, S374T, S374T 288W, K290Q, A339P, Q342R, P343A, R344Q, E345T, L351I, S354P, D356E, E357Q, L358M, T359S, N361K, Q362K, K370T, G371N, Y373F, P374S, S375E, D376A, A378S), induced direct cytotoxicity, cell aggregation, pore formation, and eventual cell lysis. Pore formation and eventual cell lysis share similar cell disintegration features with necroptosis but cannot be distinguished from necrosis or secondary necrosis (Vanden Berghe et al., 2010). The ultimate outcome of released DAMPs due to activated stress pathways or induction depends on the cellular context and the underlying signaling cascades, but in general it is possible to create an inflammatory environment that can further enhance immune effector function and / or stimulate adaptive immune responses through cross-presentation of released tumor antigens (Yatim, Cullen, and Albert 2017; Galluzzi et al., 2017).

[0033] According to a fifth aspect of the present invention, a modified IgG1 antibody or an antigen-binding fragment thereof is provided, wherein one or more of the following residues in the Fc-region are modified: N286, K288, K290, A339, Q342, P343, R344, E345, L351, S354, D356, E357, L358, T359, N361, Q362, K370, G371, Y373, P374, S375, D376, A378.

[0034] According to the sixth aspect of the present invention, a modified IgG1 antibody or an antigen-binding fragment thereof is provided, which has one or more of the following modifications to the Fc-region: N286T, K288W, K290Q, A339P, Q342R, P343A, R344Q, E345T, L351I, S354P, D356E, E357Q, L358M, T359S, N361K, Q362K, K370T, G371N, Y373F, P374S, S375E, D376A, A378S.

[0035] In some aspects of the invention, the modified IgG1 antibody or antigen-binding fragment thereof comprises modifications at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 residues selected from positions N286, K288, K290, A339, Q342, P343, R344, E345, L351, S354, D356, E357, L358, T359, N361, Q362, K370, G371, Y373, P374, S375, D376, A378. In a preferred aspect of the invention, the modified IgG1 antibody or antigen-binding fragment thereof comprises modifications at all 23 residues.

[0036] In some aspects of the invention, the modified IgG1 antibody or antigen-binding fragment thereof comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 modifications selected from N286T, K288W, K290Q, A339P, Q342R, P343A, R344Q, E345T, L351I, S354P, D356E, E357Q, L358M, T359S, N361K, Q362K, K370T, G371N, Y373F, P374S, S375E, D376A, A378S. In a preferred aspect of the invention, the modified IgG1 antibody or antigen-binding fragment thereof comprises all 23 modifications.

[0037] right Fig.9A and 9B The 88 antibodies in Figure 2 exemplify 23 modifications from this group.

[0038] By introducing 23 selected mIgG3 residues into the sialyl-di-Lewis a Targeting 129 mAb demonstrated further validation, sialyl-di-Lewis a The targeted 129 mAb has a more favorable normal tissue distribution while targeting a wide range of tumor tissues, especially more than 70% of pancreatic tumors and more than 30% of gastric and colorectal tumors, as well as more than 20% of ovarian and non-small cell lung cancer tumors. Interestingly, the parental 129 mAb produced by the hybridoma is an mIgG1 that lacks direct cytotoxicity. Therefore, the i129G1 mAb with significantly improved functional affinity compared to 129IgG1 was generated by a slower dissociation rate, consistent with the nanomolar direct cell killing ability against COLO205, suggesting that our approach may have broader applicability and is relevant to immunomodulatory mAbs that rely on avidity effects (Wang, Mathieu, & Brezski, 2018). The direct cell killing exerted by i129G1 behaved in a similar manner to i88G1, with the mAb inducing cell aggregation, followed by pore formation, and ultimately cell lysis. Introduction of 23 mIgG3 residues into the i129G1 mAb had mixed effects on effector function: although ADCC was significantly reduced, nanomolar EC was maintained. 50 , i129G1 had a significant increase in CDC activity. This mirrors the results obtained with i88G1, although the reduction in ADCC was stronger with i129G1, suggesting that the nature of the carbohydrate target also influences ADCC potency: whereas the 88mAb targets both glycoproteins and glycolipids, the 129mAb targets only glycoproteins. Notably, i129G1 showed effective tumor control, with i129G1 showing a significant reduction in tumor volume, significantly superior to 129IgG1, which did not show a significant tumor reduction. This suggests that the combined effector function of 129IgG1 is unable to control tumor growth in this model, further emphasizing the value of having direct cytotoxic capabilities.

[0039] In order to evaluate the individual contribution of each of the 23 selected mG3 residues, a single revertant strategy based on i88G1 was performed (Example 4). Each of the 23 mG3 residues in i88G1 was individually reverted to hG1 residues, and the resulting constructs were subjected to direct cell killing analysis, which confirmed that the above 23 residues can be further reduced to 15 residues without significantly losing direct cell killing ability. It is worth noting that the six mIgG3 residues in i88G1 can be reverted to IgG1 residues without significantly losing direct cell killing. This was further demonstrated using 129mAb, where the introduction of 15 mIgG3 residues maintained nanomolar direct cell killing of COLO205.

[0040] Immunogenicity can be a major barrier to successful treatment. Anti-drug antibodies may neutralize therapeutic function, affect pharmacokinetics, and may cause serious adverse reactions. T cell epitopes that pose a risk of immunogenicity can be relatively accurately assessed using in silico tools. Immunoinformatics algorithms that identify T cell epitopes can be used to assess and analyze whether immunotherapy will lead to unwanted immunogenicity. To evaluate the potential immunogenicity of our hybrid SD286-306+339-378 construct (resulting from the presence of 26 mIgG3 residues), we performed an in silico screening of the SD286-306+339-378 sequence for MHCII binding epitopes (Immune Epitope Database, IEDB). Class II-restricted T helper cells are more associated with humoral immune responses, and predicted binding clusters have been shown to be a strong indicator of T cell responses (Jawa et al., 2013). Two MHCII binding clusters (containing several high-scoring binding epitopes) were identified: Cluster 1 (residues 294-315) and Cluster 2 (residues 365-393) ( Figure 5 / Fig. 20 ). Restoring three mouse residues 294 (A to E), 300 (F to Y), and 305 (A to V) within cluster 1 to human residues generated an individually tolerable human sequence fragment. Similarly, reversal of two residues 351 (I to L) and 371 (N to G) within the 339378 region removed two MHCII binding cores.

[0041] Reversion of an immunogenic cluster identified in silico generated a lead candidate, improved “i”88G1, which possessed potent cell killing, pore-forming abilities, and well-characterized immune effector functions.

[0042] To demonstrate that the effect of introducing the selected residues is not limited to anti-glycan antibodies, trastuzumab (HER2) targeting the growth factor receptor The resulting iTrastuzumab had improved steady-state cell binding to moderately HER2-expressing cell lines, consistent with increased PI uptake. In addition, it had improved affinity compared to wild-type by a slower dissociation rate.

[0043] According to the seventh aspect of the present invention, a modified IgG1 antibody or an antigen-binding fragment thereof is provided, which has modified one or more of the following residues in the Fc-region: N286, K288, K290, Q342, P343, E345, L351, T359, N361, Q362, G371, P374, S375, D376, A378.

[0044] According to an eighth aspect of the present invention, a modified IgG1 antibody or an antigen-binding fragment thereof is provided, which has one or more of the following modifications to the Fc-region: N286T, K288W, K290Q, Q342R, P343A, E345T, L351I, T359S, N361K, Q362K, G371N, P374S, S375E, D376A, A378S.

[0045] right Fig. 9C and 9D Antibodies 129 and 88 in exemplify this set of 15 modifications.

[0046] In some aspects of the invention, the modified IgG1 antibody or antigen-binding fragment thereof comprises modifications at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 residues selected from positions N286, K288, K290, Q342, P343, E345, L351, T359, N361, Q362, G371, P374, S375, D376, A378. In a preferred aspect of the invention, the modified IgG1 antibody or antigen-binding fragment thereof comprises modifications at all 15 residues.

[0047] In some aspects of the invention, the modified IgG1 antibody or antigen-binding fragment thereof comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 modifications selected from N286T, K288W, K290Q, Q342R, P343A, E345T, L351I, T359S, N361K, Q362K, G371N, P374S, S375E, D376A, A378S. In a preferred aspect of the invention, the modified IgG1 antibody or antigen-binding fragment thereof comprises all 15 modifications.

[0048] According to a ninth aspect of the present invention, there is provided a modified IgG1 antibody or an antigen-binding fragment thereof, wherein one or more of the following residues in the Fc-region are modified: Q342, P343, E345, N361, Q362, P374, D376.

[0049] According to a tenth aspect of the present invention, there is provided a modified IgG1 antibody or antigen binding fragment thereof having one or more of the following modifications to the Fc-region: Q342R, P343A, E345T, N361K, Q362K, P374S, D376A.

[0050] right Fig.9F Antibody 88 in exemplifies this set of 7 modifications.

[0051] In some aspects of the invention, the modified IgG1 antibody or antigen-binding fragment thereof comprises modifications at 1, 2, 3, 4, 5, 6 or 7 residues selected from positions Q342, P343, E345, N361, Q362, P374, D376. In a preferred aspect of the invention, the modified IgG1 antibody or antigen-binding fragment thereof comprises modifications at all 7 residues.

[0052] In some aspects of the invention, the modified IgG1 antibody or antigen-binding fragment thereof comprises 1, 2, 3, 4, 5, 6 or 7 modifications selected from Q342R, P343A, E345T, N361K, Q362K, P374S, D376A. In a preferred aspect of the invention, the modified IgG1 antibody or antigen-binding fragment thereof comprises all 7 modifications.

[0053] In one aspect of the invention, a modified IgG1 antibody or antigen binding fragment thereof comprises one or more sequences disclosed in Figure 15. In one aspect of the invention, a modified IgG1 antibody or antigen binding fragment thereof comprising heavy and light chain sequences as disclosed in Figure 15 is provided.

[0054] In one aspect of the invention, a modified IgG1 antibody or antigen binding fragment thereof comprises one or more sequences disclosed in Figure 16. In one aspect of the invention, a modified IgG1 antibody or antigen binding fragment thereof comprising heavy and light chain sequences as disclosed in Figure 16 is provided.

[0055] In one aspect of the present invention, the modified IgG1 antibody or antigen-binding fragment thereof comprises Fig.17 In one aspect of the present invention, there is provided a method comprising: Fig.17 IgG1 antibodies or antigen-binding fragments thereof with modifications of the disclosed heavy and light chain sequences.

[0056] In one aspect of the present invention, the modified IgG1 antibody or antigen-binding fragment thereof comprises Fig.19 In one aspect of the present invention, there is provided a method comprising: Fig.19 IgG1 antibodies or antigen-binding fragments thereof with modifications of the disclosed heavy and light chain sequences.

[0057] In some aspects of the invention, a "corresponding IgG1 antibody or antigen-binding fragment thereof" refers to an unmodified (i.e., wild-type) antibody or antigen-binding fragment thereof that contains native residues and has not been changed to corresponding mouse IgG3 residues. In some aspects of the invention, the corresponding IgG1 antibody or antigen-binding fragment thereof binds to the same one or more targets (e.g., the same epitope) as the modified IgG1 antibody or antigen-binding fragment thereof. In some aspects of the invention, the modified and corresponding unmodified IgG1 antibody or antigen-binding fragment thereof contains at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6 of the same CDR sequences (i.e., VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, VLCDR3). In some aspects of the invention, the modified and corresponding unmodified IgG1 antibody or antigen-binding fragment thereof contains the same binding region sequence. In some aspects of the invention, the modified and corresponding unmodified IgG1 antibody or antigen-binding fragment thereof contains the same variable region sequence (e.g., the same variable heavy chain sequence and / or the same variable light chain sequence).

[0058] In some aspects of the invention, a "binding region" refers to the portion of an antibody or antigen-binding fragment thereof that is responsible for binding to a target antigen. For example, it may include a constant domain and a variable domain in each of the heavy and light chains. In some aspects of the invention, a binding region may refer to a complementary determining region (CDR) sequence (i.e., VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, VLCDR3).

[0059] The structure of an IgG1 antibody or antigen-binding fragment thereof is generally the sequence of an antibody heavy chain or light chain or a large portion thereof. The structure and location of immunoglobulin domains can be determined by referring to http: / / www.imgt.org / .

[0060] Throughout the specification, residue numbering refers to the standardized IMGT system for numbering antibody sequences, as disclosed in Lefranc MP, Giudicelli V, Ginestoux C, Jabado-Michaloud J, Folch G, Bellahcene F et al., IMGT, the international ImMunoGeneTics information system, Nucleic acids research, 2009: 37: D1006-12. Other suitable numbering systems are known to the skilled person. Other suitable numbering systems can be used to identify corresponding residues between modified IgG1 antibodies and their antigen-binding fragments or antigen-binding fragments thereof. Any numbering system that allows identification of corresponding residues is suitable for the present invention. The numbering system used herein does not limit the scope of the present invention, but is only used to identify relevant residues that can be modified. The term "corresponding residue" means a residue that is structurally or functionally in an equivalent position in two or more antibodies or antigen-binding fragments thereof being compared. In some cases, corresponding residues can be identified by sequence alignment. In some cases, corresponding residues can be identified by structural alignment.

[0061] In some aspects of the invention, an IgG1 antibody or antigen-binding fragment thereof comprising one or more residues of an immunoglobulin Fc-region comprises at least 10 amino acid residues of the Fc-region, at least 20 amino acids of the Fc-region, at least 30 amino acids of the Fc-region, at least 40 amino acids of the Fc-region, at least 50 amino acids of the Fc-region, at least 75 amino acids of the Fc-region, at least 100 amino acids of the Fc-region, at least 200 amino acids of the Fc-region, at least 300 amino acids of the Fc-region, at least 400 amino acids of the Fc-region, or at least 500 amino acids of the Fc-region. Preferably, an IgG1 antibody or antigen-binding fragment thereof comprises the entire Fc-region of an immunoglobulin.

[0062] In some aspects of the invention, one or more residues in the Fc-region are selected from the CH2 and / or CH3 domains. In some aspects, one or more residues are selected from the CH2 domain. In some aspects, one or more residues are selected from the CH3 domain.

[0063] In some aspects of the invention, the functional affinity of the modified IgG1 antibody or antigen-binding fragment thereof and the corresponding wild-type IgG1 antibody or antigen-binding fragment thereof can be determined by surface plasmon resonance (e.g., Biacore 3000 / T200, GE Healthcare), for example, by injecting increasing concentrations (0.3 nmol / L to 200 nmol / L) of the IgG1 antibody or antigen-binding fragment thereof into a CM5 chip containing an appropriate ligand, and fitting the data to an appropriate binding model using appropriate software (e.g., BIAevaluation4.1). The modified IgG1 antibody or antigen-binding fragment thereof can be subjected to corresponding experiments using the same conditions. In some aspects of the invention, when the surface plasmon resonance data indicates that the modified IgG1 antibody or antigen-binding fragment thereof binds more tightly to the ligand-coated CM5 chip, the modified IgG1 antibody or antigen-binding fragment thereof exhibits a greater functional affinity than the corresponding wild-type IgG1 antibody or antigen-binding fragment thereof. The ligand includes a control conjugate of glycan-HSA and an anti-glycan antibody.

[0064] The Biacore CM5 chip coated with anti-his antibody contains carboxymethyl dextran covalently linked to a gold surface. The molecules are covalently linked to the sensor surface through amines, thiols, aldehydes or carboxyl groups. Interactions involving small organic molecules (e.g., candidate drugs) to macromolecular assemblies or entire viruses can be studied. High binding capacity provides high response, which is beneficial for capture analysis and interactions involving small molecules. High surface stability can provide accuracy and precision and allow repeated analysis on the same surface. Other suitable chips are known to those skilled in the art, and the surface plasmon resonance protocol can be adjusted by standard techniques known in the art.

[0065] In some aspects of the invention, direct cell killing by the modified IgG1 antibody or antigen-binding fragment thereof and the corresponding wild-type IgG1 antibody or antigen-binding fragment thereof can be determined by propidium iodide (PI) uptake, for example, by incubating the IgG1 antibody or antigen-binding fragment thereof with COLO205 or HCT-15 cells (5 x 10 4) are incubated at 37°C for 2 hours, then 1 μg of PI is added for 30 minutes, the cells are resuspended in PBS, the cells are processed on a flow cytometer (e.g., Beckman Coulter FC 500 or MACSQuant 10), and the results are analyzed using appropriate software (e.g., WinMDI 2.9 or FlowJo v10). A corresponding experiment using the same conditions can be performed on the modified IgG1 antibody or antigen-binding fragment thereof. In some aspects of the invention, when the cells are incubated with the modified IgG1 antibody or antigen-binding fragment thereof, the modified IgG1 antibody or antigen-binding fragment thereof provides greater direct cell killing than the corresponding wild-type IgG1 antibody or antigen-binding fragment thereof when the PI uptake is greater.

[0066] The improved functional properties of the modified IgG1 antibody or its antigen-binding fragment are measured relative to the corresponding functional properties of the wild-type IgG1 antibody or its antigen-binding fragment (not comprising the modified residues in the Fc-region). Since the improved functional property is a relative measure, the accurate method for determining the functional affinity and / or direct cell killing or any other functional property of the claimed IgG1 antibody or its antigen-binding fragment does not affect the relative change of the functional property. Many suitable methods for measuring the functional properties of antibodies and their antigen-binding fragments are known to those skilled in the art. Any suitable method for measuring these functional properties can be used to measure the functional properties of the claimed modified IgG1 antibody and its antigen-binding fragment or its antigen-binding fragment, provided that the same method is applied to the corresponding wild-type IgG1 antibody or its antigen-binding fragment for fair comparison.

[0067] The generation of improved onco-glycan-targeting mAbs with enhanced functional affinity and direct cytotoxicity by establishing intermolecular cooperative binding may lead to better clinical applications. This approach may also be valuable for other mAbs targeting repetitive or high-density antigens. In addition, restoring the unusual pro-inflammatory cell killing pattern observed with many glycan-targeting mIgG3 mAbs to an IgG1 framework has the potential to be used in combination with other immunotherapies.

[0068] The modified IgG1 antibodies of the present invention can be used for the diagnosis and treatment of tumors in human or animal subjects. When used for diagnosis, the modified IgG1 antibodies of the present invention and their antigen-binding fragments can be labeled with detectable labels (e.g., radioactive labels, such as I or Tc), which can be attached to the modified IgG1 antibodies of the present invention using conventional chemistry known in the field of antibody imaging. Labels also include enzyme labels, such as horseradish peroxidase. Labels also include chemical moieties such as biotin, which can be detected by binding to specific homologous detectable moieties (e.g., labeled avidin). Although the modified IgG1 antibodies of the present invention and their antigen-binding fragments themselves have been shown to be effective in killing cancer cells, they can also be labeled with functional labels. Functional labels include substances designed to target cancer sites to cause their destruction. These functional labels include toxins such as ricin and enzymes such as bacterial carboxypeptidases or nitroreductases, which can convert prodrugs into active drugs. In addition, the modified IgG1 antibodies and antigen-binding fragments thereof may be attached or otherwise associated with chemotherapeutic or cytotoxic agents, such as maytansine (DM1 and DM4), neferol, auristatins, calicheamicin, duocarmycin, doxorubicin 90 131, or radiolabels, such as Y or I. In addition, the modified IgG1 antibodies and antigen-binding fragments thereof of the present invention may be administered alone or in combination with other treatments simultaneously or sequentially, depending on the condition to be treated. Therefore, the present invention further provides such products: containing the modified IgG1 antibodies and antigen-binding fragments thereof of the present invention and active agents as combined preparations for simultaneous, separate or sequential use in the treatment of tumors. Active agents may include chemotherapeutic or cytotoxic agents, including 5-fluorouracil, cisplatin, mitomycin C, oxaliplatin and tamoxifen, which may act synergistically with the modified IgG1 antibodies and antigen-binding fragments thereof of the present invention. Other active agents may include appropriate doses of analgesics such as nonsteroidal anti-inflammatory drugs (eg, aspirin, paracetamol, ibuprofen, or ketoprofen) or opiates such as morphine, or antiemetics.

[0069] Although not wishing to be bound by theory, the ability of the modified IgG1 antibodies and antigen-binding fragments thereof of the present invention to synergistically enhance tumor killing with an active agent may not be due to an immune effector mechanism, but may be a direct result of the binding of the modified IgG1 antibodies and antigen-binding fragments thereof to cell surface receptors. The modified IgG1 antibodies and antigen-binding fragments thereof of the present invention will generally be administered in the form of a pharmaceutical composition, which may include at least one component other than the modified IgG1 antibodies and antigen-binding fragments thereof.

[0070] In addition to the active ingredient, the pharmaceutical composition may also contain a pharmaceutically acceptable excipient, diluent, carrier, buffer, stabilizer or other materials known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other materials depends on the route of administration, which may be oral or by injection, such as intravenous injection. Preferably, the route of administration is intravenous injection.

[0071] Although delivery through a catheter or other medical hose is also used, it is expected that injection will be the primary route of therapeutic administration of the composition. Some suitable routes of administration include intravenous, subcutaneous, intraperitoneal, and intramuscular administration. Liquid formulations can be used after reconstitution from powder formulations.

[0072] For intravenous injection or injection at the site of pain, the active ingredient will be in the form of an aqueous solution acceptable to the parenteral, which is pyrogen-free and has a suitable pH, isotonicity and stability. Those skilled in the art are well able to use isotonic solvents, such as sodium chloride injection, Ringer's injection, lactated Ringer's injection to prepare suitable solutions. Preservatives, stabilizers, buffers, antioxidants and / or other additives may be included as needed.

[0073] Pharmaceutical compositions for oral administration can be in tablet, capsule, powder or liquid form. Tablets can include solid carriers, such as gelatin or adjuvants. Liquid pharmaceutical compositions typically include liquid carriers, such as water, petroleum, animal or vegetable oils, mineral oils or synthetic oils. Physiological saline solutions, glucose or other sugar solutions, or glycols, such as ethylene glycol, propylene glycol or polyethylene glycol can be included. When the formulation is liquid, it can be, for example, a physiological saline solution containing a non-phosphate buffered saline solution having a pH of 6.8-7.6, or a lyophilized powder.

[0074] The composition can also be administered via microspheres, liposomes, other microparticulate delivery systems, or sustained-release formulations placed in certain tissues, including the blood. Suitable examples of sustained-release carriers include semipermeable polymer matrices in the form of shared articles, such as suppositories or microcapsules. Implantable or microencapsulated sustained-release matrices include polylactic acid (U.S. Pat. No. 3,773,919; EP-A-0058481), copolymers of L-glutamic acid and γ-ethyl-L-glutamate

[43] , poly (2-hydroxyethyl-methacrylate). Liposomes containing polypeptides are prepared by well-known methods: (Eppstein et al., 1985; Hwang, Luk and Beaumier, 1980); EP-A-0052522; EP-A-0036676; EP-A-0088046; EP-A-0143949; EP-A-0142541; JP-A-83-11808; U.S. Patent Nos. 4,485,045 and 4,544,545. Typically, the liposomes are small (about 200 angstroms to 800 angstroms) unilamellar types, wherein the lipid content is greater than about 30 mole % cholesterol, a proportion selected to adjust for the optimal rate of polypeptide leakage. The composition can be applied topically to the tumor site or other desired site, or can be delivered in a manner targeted to the tumor or other cells.

[0075] The composition is preferably administered to an individual in a "therapeutically effective amount", an amount sufficient to show benefit to the individual. The actual amount, rate and time of administration will depend on the nature and severity of the disease to be treated. Treatment prescription, such as dosage decisions, is the responsibility of general practitioners and other physicians, generally taking into account the disease to be treated, the condition of the individual patient, the delivery site, the method of administration and other factors known to the physician. The composition of the present invention is particularly relevant to the treatment of existing tumors (especially cancer) and the prevention of recurrence of such conditions after initial treatment or surgery. Examples of the above techniques and protocols can be found in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A (ed) 1980.

[0076] The optimal dosage can be determined by the physician based on a number of parameters including, for example, age, sex, weight, severity of the condition being treated, the active ingredient being administered, and the route of administration. In general, serum concentrations of polypeptides and antibodies that allow receptor saturation are desirable. Concentrations in excess of about 1 nM are usually sufficient. For example, a dosage of 100 mg / m 2 The antibody provides a serum concentration of about 20 nM for about eight days. As a rough guide, the antibody can be given at a dose of 10 mg / m 2 Up to 300 mg / m 2 Equivalent doses of antibody fragments should be used at more frequent intervals to maintain serum levels above concentrations that allow glycan saturation.

[0077] The dosage of the composition will depend on the properties of the modified IgG1 antibody and antigen-binding fragments thereof, such as its binding activity and in vivo plasma half-life, the concentration of the polypeptide in the formulation, the route of administration, the site of administration and the rate, the clinical tolerance of the patient involved, the pathological conditions afflicting the patient, etc., which is within the skill of the physician. For example, a dosage of 3000 μg of antibody per administration per patient is preferred, although the dosage may be in the range of about 10 μg to 6 mg per dose. Different doses are used during a series of consecutive vaccinations; the physician may perform an initial vaccination and then boost with a relatively smaller dose of the antibody.

[0078] The modified IgG1 antibodies and antigen-binding fragments thereof of the present invention may be produced in whole or in part by chemical synthesis. The modified IgG1 antibodies and antigen-binding fragments thereof may be readily prepared according to well-established standard liquid or preferably solid phase peptide synthesis methods, general descriptions of which are widely available (e.g., see JM Stewart and JD Young, (1984)

[46] , M. Bodanzsky and A. Bodanzsky, (1984)); or they may be prepared in solution by liquid phase methods or by any combination of solid phase, liquid phase and solution chemistry, e.g., first completing the respective peptide portion and then, if necessary and appropriate, introducing residue X by reaction of the respective carbonic acid or sulfonic acid or its reactive derivatives after removal of any protecting groups present.

[0079] Another convenient method of producing modified IgG1 antibodies and antigen-binding fragments thereof according to the present invention is by expressing nucleic acids encoding them using nucleic acids in an expression system. The present invention also provides isolated nucleic acids encoding modified IgG1 antibodies and antigen-binding fragments thereof of the present invention. Nucleic acids include DNA and RNA. The skilled person will be able to determine that substitutions, deletions and / or additions to such nucleic acids will still provide modified IgG1 antibodies and antigen-binding fragments thereof of the present invention.

[0080] The present invention also provides a construct in the form of a plasmid, vector, transcript or expression cassette comprising at least one nucleic acid as described above. The present invention also provides a recombinant host cell comprising one or more constructs as described above. As previously mentioned, nucleic acids encoding the modified IgG1 antibodies and antigen-binding fragments thereof of the present invention form one aspect of the present invention, and methods for producing modified IgG1 antibodies and antigen-binding fragments thereof also form one aspect of the present invention, the method comprising expressing by nucleic acids encoding modified IgG1 antibodies and antigen-binding fragments thereof. Expression can be conveniently achieved by culturing recombinant host cells containing the nucleic acid under appropriate conditions. After production by expression, the modified IgG1 antibodies and antigen-binding fragments thereof can be isolated and / or purified using any suitable technique and then used appropriately. Systems for cloning and expressing polypeptides in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expressing heterologous polypeptides include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells and many other cells. A common and preferred bacterial host is Escherichia coli. The expression of antibodies and antibody fragments in prokaryotic cells (such as E. coli) is well established in the art. For review, see, for example, (Pluckthun, 1991). Those skilled in the art can also express in cultured eukaryotic cells as an option for producing modified IgG1 antibodies and antigen-binding fragments thereof, and recent reviews are provided, for example, (Reff 1993; Trill, Shatzman and Ganguly, 1995).

[0081] Suitable vectors can be selected or constructed, comprising suitable regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences (as appropriate). The vector can be a plasmid, a virus, such as a "phage" or a phagemid (as appropriate). For further details, see, for example (Sambrook, Fritsch and Maniatis, 1989). Many known techniques and protocols for manipulating nucleic acids, such as preparing nucleic acid constructs, mutagenesis, sequencing, introducing DNA into cells and gene expression and protein analysis, are described in detail in Ausubel et al., 1992 (Ausubel et al., 1992).

[0082] Therefore, another aspect of the present invention provides a host cell containing a nucleic acid as disclosed herein. Another aspect provides a method comprising introducing such a nucleic acid into a host cell. The introduction may be carried out using any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transduction using retrovirus or other viruses (e.g., vaccinia virus, or, for insect cells, baculovirus). For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation, and transfection using bacteriophage. After introduction, nucleic acid expression may be caused or allowed, for example, by culturing the host cell under conditions of expressing the gene. In one embodiment, the nucleic acid of the present invention is integrated into the genome (e.g., chromosome) of the host cell. According to standard techniques, integration may be promoted by including sequences that promote recombination with the genome. The present invention also provides a method comprising using a construct as described above in an expression system to express the modified IgG1 antibody and its antigen-binding fragment thereof.

[0083] The fragment crystal region (Fc region) is the tail region of the antibody that interacts with cell surface receptors called Fc receptors and certain proteins of the complement system. This property allows antibodies to activate the immune system. In the IgG antibody isotype, the Fc region consists of two identical protein fragments, which come from the second and third constant domains of the two heavy chains of the antibody. The Fc region of IgG contains highly conserved N-glycosylation sites.

[0084] The Fc (fragment, crystallizable) of IgG consists of a set of paired antibody HC domains, each with a CH2 fused to a CH3, forming a structure of approximately 50 kDa. The name "fragment, crystallizable" (Fc) comes from the fact that after cleavage of serum-derived myeloma IgG fractions with papain, the only fragments that can be crystallized are paired CH2-CH3 fragments.

[0085] Within the Fc, the two CH3 domains are tightly bound to each other, while the two CH2 domains have no direct protein-protein contacts with each other. Oligosaccharides bind to asparagine-297 (N297) within the two CH2 domains, filling part of the space between the two CH2s. In some crystal structures, hydrogen bonds were observed between the two carbohydrate chains, either directly or through bridging water molecules. Although antibodies appear to be highly segmented molecules, it has been shown that the structure of the Fc can affect the binding of the Fab to the target antigen, and similarly, the content of variable chains in the Fab can affect the binding of the Fc to various receptors. Recent circular dichroism studies have confirmed significant structural coupling between the FAb arm and the Fc of IgG. Therefore, the IgG molecule is a highly complex molecule in which different domains interact significantly, even over long distances.

[0086] While bivalent / multivalent antibodies contain a binding domain that interacts with an epitope, synthetic bivalent / multivalent ligands contain a pharmacophore that interacts with a target site. The term "functional affinity" may also be used to denote the bivalent / multivalent associated increase in apparent affinity. This term is synonymous with "avidity".

[0087] Avidity refers to the cumulative strength of multiple affinities of a single non-covalent binding interaction, such as the affinity between a protein receptor and its ligand, and can also be called "functional affinity". Therefore, affinity is different from intrinsic affinity, which describes the strength of a single interaction. However, since a single binding event increases the probability of other interactions (i.e., increases the local concentration of each binding partner near the binding site), affinity should not be considered as simply the sum of the affinities of the constituent elements, but the combined effect of all affinities involved in the biomolecular interaction. The utility of distinguishing "intrinsic affinity" and "functional affinity" comes from the different emphases involved in each term. The former is most useful when the structural relationship between the antibody binding site and the ligand complementary region is under close scrutiny or the kinetic mechanism of a specific interaction is under study. On the other hand, the latter is particularly important when quantitative measurements of affinity enhancement are being examined, as in the present invention.

[0088] Both "functional affinity" and "intrinsic affinity" refer to the same reversible process in form, as follows:

[0089] Complex(Unit Price)

[0090] Complex (multivalent)

[0091] Among them, F1 is a monovalent antibody fragment, L1 is a monovalent ligand, Ab n For multivalent antibodies, L m is a multivalent ligand with m groups. In each case, two kinetic units reversibly bind to form one unit, and in principle, each process can be characterized by an association constant. Since the association constant is a measure of the thermodynamic affinity, both processes can be assigned a quantitative value for affinity.

[0092] Fragments of antibodies and antigen-binding molecules

[0093] The antigen binding molecules of the present invention can be antibody fragments, specifically antigen binding fragments of antibodies. Antigen binding fragments include one or more antigen binding regions. It has been shown that fragments of whole antibodies can perform the function of binding antigens. Examples of binding fragments are (i) a Fab fragment consisting of the VL, VH, CL and CH1 domains; (ii) a Fd fragment consisting of the VH and CH1 domains; (iii) a Fv fragment consisting of the VL and VH domains of a single antibody; (iv) a dAb fragment consisting of the VH domain (Ward, ES et al., Nature 341: 544-546 (1989)); (v) isolated CDR regions; (vi) a F(ab′)2 fragment, a bivalent fragment comprising two linked Fab fragments; (vii) a single-chain Fv molecule (scFv) in which the VH domain and the VL domain are linked by a peptide linker that allows the two domains to associate to form an antigen binding site (Bird et al., Science 242: 423-426 (1988); Huston et al., PNAS USA 85:5879-5883 (1988)); (viii) bispecific single chain Fv dimers (PCT / US92 / 09965) and (ix) "diabodies", multivalent or multispecific fragments constructed by gene fusion (WO94 / 13804; P. Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)). Typically, the fragment is a Fab, F(ab')2 or Fv fragment or a scFv molecule.

[0094] A diabody is a multimer of polypeptides, each polypeptide comprising a first domain (comprising a binding region for an immunoglobulin light chain) and a second domain (comprising a binding region for an immunoglobulin heavy chain), the two domains being connected (e.g., by a peptide linker) but unable to associate with each other to form an antigen binding site: the antigen binding site is formed by the association of the first domain of one polypeptide in the multimer with the second domain of another polypeptide in the multimer (WO94 / 13804).

[0095] Where bispecific antibodies are used, these antibodies may be conventional bispecific antibodies, which may be made in a variety of ways (Hollinger & Winter, Current Opinion Biotechnol, 4: 446-449 (1993)), for example, chemically prepared or prepared from a hybridoma, or may be any of the bispecific antibody fragments described below. It is preferred to use scFv dimers or diabodies rather than whole antibodies. Diabodies and scFvs may be constructed without an Fc region, using only the variable domains, which may reduce the impact of anti-idiotypic responses. Other forms of bispecific antibodies include the single chain "Janusins" described in Traunecker et al., EMBO Journal 10: 3655-3659 (1991).

[0096] A bispecific antibody is an antibody that can bind to two target molecules (such as two antigens or two epitopes) at the same time. Bispecific antibodies can also be called dual binding antibodies. Examples of bispecific antibody formats include, but are not limited to; (mAb)2, Fcab, F(mAb')2, quadruple hybridoma, scFv (single chain variable fragment), bsDb (bispecific diabody), scBsDb (single chain bispecific diabody), BiTE (bispecific T cell engager), tandem antibodies, tandem scFv-Fc, Fab-scFv-Fc, Fab-scFv, miniantibodies, zybodies, DNL-F(ab)3 (dock and lock trivalent Fab), bssdAb (bispecific single domain antibody) and knob-and-hole structure.

[0097] Bispecific diabodies may also be useful relative to bispecific full antibodies because they can be easily constructed and expressed in E. coli. Diabodies (and many other polypeptides, such as antibody fragments) with appropriate binding specificities can be easily selected from libraries using phage display (WO94 / 13804). If one arm of the diabody is kept constant, for example, with specificity for antigen X, a library can be prepared where the other arm is varied and antibodies with appropriate specificity are selected.

[0098] In some aspects of the invention, it may be desirable to generate multispecific (e.g., bispecific) anti-target antibodies with binding specificity to at least two different epitopes of the same or different molecules. Exemplary bispecific antibodies can bind to two different epitopes of a target molecule. Alternatively, a target-specific antibody arm can bind to an arm that binds a cell surface molecule, such as a T cell receptor molecule (e.g., CD3). Exemplary bispecific antibodies of the invention can bind to an antigen from a T cell receptor molecule (e.g., CD3) and any other target.

[0099] The modified IgG1 antibody and antigen-binding fragment thereof of the present invention may be an antibody or antibody fragment Fab, (Fab')2, scFv, Fv, dAb, Fd or diabody. The antibody may be a polyclonal antibody. The antibody may be a monoclonal antibody (mAb). The modified IgG1 antibody and antigen-binding fragment thereof of the present invention may be a humanized, chimeric or veneered antibody, or may be a non-human antibody of any species. In a preferred embodiment, the modified IgG1 antibody and antigen-binding fragment thereof of the present invention is human or humanized.

[0100] Mouse or chimeric antibodies carry an increased risk of adverse anti-mouse antibody (HAMA) reactions in patients (Schroff et al., 1985; Azinovic et al., 2006; Miotti et al., 1999; D'Arcy and Mannik, 2001). Therefore, most approved therapeutic mAbs are humanized or fully human IgG antibodies.

[0101] Variants

[0102] The present invention also extends to variants of any peptide sequence disclosed herein. As used herein, the term "variant" refers to proteins with similar amino acid sequences and / or retaining the same function. For example, the term "variant" includes proteins or polypeptides, which include one or more amino acid additions, deletions, substitutions, etc. Examples of variants of the present invention are proteins including peptides defined as follows and replacing one or more amino acids with one or more other amino acids. Amino acid substitutions can be made, for example, to reduce or eliminate instability in the amino acid sequence. Alternatively, if desired, amino acid substitutions can be made to improve antigen affinity or to humanize or deimmunize antibodies. Affinity maturation variants, humanized variants, and deimmunization variants of specified antibodies are provided herein, as well as variants comprising amino acid substitutions to reduce or eliminate any instability in the antibody sequence.

[0103] As described above, in some embodiments, any substitution can only occur in the framework region. In these embodiments, the original CDR sequence is retained, but variations may occur in one or more framework regions.

[0104] Variant antigen binding molecules with one or more amino acid substitutions can retain the functional activity (e.g., EC50, IC50, IC90, Kd, ​​functional affinity and / or direct cell killing) of the antigen binding molecules from which the variant antigen binding molecules were derived. The variant antigen binding molecules of the present invention can be used and formulated in the same manner as described for the antigen binding molecules from which the variant antigen binding molecules were derived.

[0105] replace

[0106] The skilled artisan is aware that various amino acids have similar properties. One or more such amino acids of a substance can generally be substituted with one or more other such amino acids without eliminating the desired activity of the substance.

[0107] Thus, the amino acids glycine, alanine, valine, leucine, and isoleucine are generally interchangeable (amino acids with aliphatic side chains). Of these possible substitutions, it is preferred to use glycine and alanine as interchangeable (because they have relatively short side chains), and valine, leucine, and isoleucine as interchangeable (because they have larger hydrophobic aliphatic side chains). Other amino acids that are generally interchangeable include: phenylalanine, tyrosine, and tryptophan (amino acids with aromatic side chains); lysine, arginine, and histidine (amino acids with basic side chains); aspartate and glutamate (amino acids with acidic side chains); asparagine and glutamine (amino acids with amide side chains); and cysteine ​​and methionine (amino acids with sulfur-containing side chains).

[0108] Substitutions of this nature are often referred to as "conservative" or "semi-conservative" amino acid substitutions.

[0109] Using three-letter and one-letter codes, naturally occurring amino acids may be referred to as: glycine (G or Gly), alanine (A or Ala), valine (V or Val), leucine (L or Leu), isoleucine (I or Ile), proline (P or Pro), phenylalanine (F or Phe), tyrosine (Y or Tyr), tryptophan (W or Trp), lysine (K or Lys), arginine (R or Arg), histidine (H or His), aspartic acid (D or Asp), glutamic acid (E or Glu), asparagine (N or Asn), glutamine (Q or Gln), cysteine ​​(C or Cys), methionine (M or Met), serine (S or Ser), and threonine (T or Thr). Where the residue may be aspartic acid or asparagine, the symbols Asx or B may be used. Where the residue may be glutamic acid or glutamine, the symbols Glx or Z may be used. Unless the context dictates otherwise, aspartic acid includes aspartate and glutamic acid includes glutamate.

[0110] Amino acid deletion or insertion can also be completed relative to the amino acid sequence of the fusion protein described below. Thus, for example, amino acids that do not have a substantial effect on the activity of the polypeptide or at least do not eliminate such activity can be deleted. Such deletion may be advantageous because the total length and molecular weight of the polypeptide can be reduced while still maintaining activity. This can reduce the amount of the polypeptide required for a specific purpose, for example, can reduce dosage levels.

[0111] In some embodiments, the following amino acids can be exchanged with each other to make conservative amino acid substitutions:

[0112]

[0113] Thus, reference to a "conservative" amino acid substitution refers to an amino acid substitution in which one or more amino acids in an antibody sequence (e.g., in a CDR or in a VH or VL sequence) are replaced by another amino acid of the same class as described above. Conservative amino acid substitutions may be preferred in the CDR regions to minimize adverse effects on antibody function. However, conservative amino acid substitutions may also occur in the framework regions.

[0114] Amino acid changes relative to the sequences given below may be accomplished using any suitable technique, for example by site-directed mutagenesis or solid-state synthesis.

[0115] It should be understood that amino acid substitutions or insertions within the scope of the present invention may be performed using naturally occurring or non-naturally occurring amino acids, although naturally occurring amino acids may be preferred. Whether natural or synthetic amino acids are used, preferably only L-amino acids are present.

[0116] Manufacturing liability

[0117] Therapeutic proteins such as antibodies are inherently heterogeneous and complex due to chemical modifications and post-translational modifications (PTMs). Modifications may be caused by many factors, such as the host cell system, the processes used in manufacturing, or the conditions during storage or manufacturing. Modifications may be related to the chemical stability or aggregation potential of the molecule itself and the impact this has on the inherent physical stability of the antibody. The spontaneous modification of amino acid motifs or residues in a given antibody sequence that may occur during manufacturing or storage is called instability. Therefore, mutations can be made to the antibody sequence to address instability and thereby reduce the susceptibility of the antibody to modification and degradation.

[0118] Such modifications due to instability in the antibody sequence may include glycosylation, deamidation, oxidation, and changes in the C- and N-termini. Such modifications may occur during the manufacturing process. Certain residues and structural or sequence motifs are more susceptible to certain modifications. Examples of such instabilities to modification include Asn N-linked glycosylation, Ser / Thr O-linked glycosylation, Asn deamidation, Asp isomerization / fragmentation, Lys glycosylation, Met / Trp oxidation, free thiol groups, pyroglutamate, C-terminal Lys.

[0119] The skilled person knows that computational tools can be used to predict and identify structural and sequence instabilities that may lead to modifications. To minimize the occurrence of modifications, changes can be made to the manufacturing process. Protein engineering is also viewed as risk reduction. For example, selective mutations of these instabilities can help identify and reduce the risk of modifications that compromise antibody stability.

[0120] Aspartic acid residues (Asp) may undergo spontaneous modification. Motifs containing Asp, such as Asp-Gly sequences, can spontaneously isomerize to form isoaspartic acid. The formation of isoaspartate may weaken or completely eliminate the binding of the antibody. This is even more important if the Asp residue is present in the CDRs of the antibody.

[0121] Therefore, aspartic acid residue (Asp) can be replaced by any naturally occurring amino acid, to reduce this instability to modification. Alternatively, aspartic acid residue (Asp) can be replaced by alanine (Ala), glutamine (Gln) or glutamic acid (Glu), to reduce this instability to modification. The optimization of production / preparation can also be studied to reduce isomerization. Alternatively, the Asp-Gly motif can be modified to suppress deamidation by replacing the glycine residue with another naturally occurring amino acid, rather than by replacing the Asp residue.

[0122] Methionine residues (Met) may undergo spontaneous modification. The presence of Met in CDRs, especially when exposed to solvents, may cause problems if the Met is oxidized and interferes with binding. Therefore, the Met residues may be substituted with any other natural amino acid to reduce the instability of this modification. The Met residues are preferably substituted with Ala or Leu. Optimization of production / formulation may also be studied to reduce oxidation.

[0123] The preferred features of each aspect of the invention apply mutatis mutandis to each of the other aspects.The prior art documents described herein are incorporated to the maximum extent permitted by law. BRIEF DESCRIPTION OF THE DRAWINGS

[0124] The following detailed description, which is given by way of example and is not intended to limit the invention to the particular embodiments described, can be best understood in conjunction with the accompanying drawings.

[0125] Figure 1. 88IgG1 maintains cancer cell binding but significantly reduces direct cytotoxicity compared to 88mIgG3 and parental hybridoma mAb. A. Comparable HCT15 and COLO205 cell binding analysis by 88IgG1, 88mIgG3 and FG88 (hybridoma mAb); B. 88IgG1 significantly reduces direct cytotoxicity (PI uptake) on HCT15 compared to parental 88mIgG3 and FG88; 88IgG1 significantly reduces proliferation inhibition on COLO205 (C) and HCT15 (D) compared to parental 88mIgG3 and FG88. Significance was derived by two-way ANOVA with Dunnett's correction for multiple comparisons.

[0126] Figure 2. mIgG3 CH3 and a small amount of CH2 contribute to direct cytotoxicity and improve functional affinity. Constant domain changes show that CH1 does not contribute significantly to direct cytotoxicity (PI uptake, HCT15, Figure 2A ; Inhibition of proliferation of COLO205, Figure 2B In contrast, CH3 (1m3 and 3h3) contributed significantly to direct cytotoxicity, with a minor contribution from mCH2 being evident only in loss-of-function approaches (3h2) (PI uptake, HCT15, Figure 2C ; Proliferation inhibition, COLO205, Figure 2D ). 1m3 significantly increased the functional affinity and decreased the dissociation rate; 3h2 significantly decreased the functional affinity and increased the dissociation rate, which was more obvious for 3h3, confirming the main CH3 contribution and the secondary CH2 contribution ( Figure 2E , 2F ). Significance to each parental construct was derived by two-way ANOVA (direct cytotoxicity) or one-way ANOVA (functional affinity, off-rate) with Dunnett's correction for multiple comparisons.

[0127] Figure 3. SD286397 containing a CH2:CH3 linkage underlies the direct cytotoxicity and improved functional affinity of mIgG3. Subdomain partitioning of CH2CH3 identified two regions that did not contribute: SD232-294 and SD390-447, and two regions that significantly contributed to direct cytotoxicity and functional affinity: SD286-345 (CH2) and SD339-397 (CH3). Both constructs and the combination (SD286-397) significantly increased HCT15 ( Figure 3A ) and COLO205( Figure 3B ) PI uptake. Both constructs and combinations significantly increased COLO205 ( Figure 3C ) and HCT15( Figure 3D ) proliferation inhibition. The functional affinity (SPR) was significantly increased, which was mainly due to the above two constructs (respectively Figure 3E and Figure 3F ) due to reduced dissociation rate. Compared with 88IgG1, SD339-397 significantly reduced CDC activity (HCT15) ( Figure 3G ); the above constructs maintain ADCC activity (COLO205) ( Figure 3H ). Significance with respect to each parental construct was derived by two-way ANOVA (direct cytotoxicity) or one-way ANOVA (functional affinity, off-rate, effector function) with Dunnett's correction for multiple comparisons.

[0128] Figure 4. Discontinuous regions consisting of 286-306 combined with 339-378 confer direct cytotoxicity and enhanced functional affinity while maintaining immune effector function. Compared with 88IgG1, SD339-378 significantly increased PI uptake in HCT15 ( Figure 4A ) and proliferation inhibition ( Figure 4B SD307-345 significantly reduced PI uptake compared with SD286-345, indicating the contribution of SD286-306 ( Figure 4C Compared with 88IgG1, the combination of SD286-306+339-378 significantly increased the expression of HCT15 ( Figure 4D ) and COLO205( Figure 4E ) and COLO205( Figure 4F ) PI uptake. Compared with 88IgG1, SD286-306+339-378 significantly increased functional affinity (SPR) ( Figure 4G Compared with 88IgG1, SD286-306+339-378 maintained the inhibitory effect on HCT15 ( Figure 4H ) and the CDC activity against COLO205( Fig. 4I ) ADCC activity of each parental construct was derived by two-way ANOVA (direct cytotoxicity) or one-way ANOVA (functional affinity and effector function) with Dunnett's correction for multiple comparisons.

[0129] Figure 5 MHCII binding clusters containing several high-scoring binding epitopes. Cluster 1 consists of residues 294-315 and cluster 2 consists of residues 365-393.

[0130] Figure 6. i88G1, which has direct cytotoxicity and enhanced functional affinity while maintaining immune effector function, shows pore-forming ability. Compared with 88IgG1, i88G1 significantly increased the affinity for HCT15 ( Fig. 6A ) and COLO205( Figure 6B ) proliferation inhibition. Compared with 88IgG1, i88G1 significantly increased the functional affinity (SPR) ( Figure 6C Compared with 88IgG1, i88G1 slightly but significantly reduced the effect of COLO205 ( Fig.6D ) and compared with 88IgG1, i88G1 significantly increased ADCC against HCT15( Fig. 6E ) CDC activity. Evidence for the ability of i88G1 to detach, aggregate and form pores in HCT15 cells ( Fig. 6F ).

[0131] Figure 7. i129G1, which has direct cytotoxicity and enhanced functional affinity while maintaining immune effector function, exhibits pore-forming ability and in vivo tumor control. Compared with 129IgG1, i129G1 significantly increased the ability to inhibit COL0205 ( Fig. 7A ) Fig. 7A ) and PI uptake ( Figure 7B Compared with 129IgG1, i129G1 significantly increased functional affinity (SPR) ( Figure 7C ). i129G1 maintained some ADCC activity against COLO205, but was significantly reduced compared to h129IgG1 ( Fig.7D Compared with 129IgG1, i129G1 significantly increased its CDC activity against COLO205 ( Fig. 7E ). Evidence for the cell detachment, aggregation and pore formation of i129G1 on COLO205 ( Figure 7F In the COLO205 xenograft model (Balb / c mice), i129G1 significantly reduced in vivo tumor control compared to vehicle control and compared to 129IgG1 ( Figure 7G In studies with mice, there was no significant effect on mean body weight ( Figure 7H ). Significance compared to each parental construct was derived by two-way ANOVA (direct cytotoxicity and in vivo tumor control) or one-way ANOVA (functional affinity and effector function) with Dunnett's correction for multiple comparisons.

[0132] Figure 8. i27G1 shows significantly improved direct cytotoxicity and functional affinity compared to 27IgG1, while maintaining immune effector function. Compared to 27IgG1, i27G1 significantly increased the direct cytotoxicity against MCF7 ( Fig. 8A ) and AGS( Figure 8B ) proliferation inhibition. Compared with 27IgG1, i27G1 significantly increased the functional affinity (SPR) ( Figure 8C i27G1 maintained the same ADCC against MCF7 ( Fig.8D ) and CDC activity ( Fig. 8E ). Significance with respect to each parental construct was derived by two-way ANOVA (direct cytotoxicity) or one-way ANOVA (functional affinity) with Dunnett's correction for multiple comparisons.

[0133] Figure 9. Single revertant analysis shows that a minimum of 15 residues are required to maintain direct cell killing. 23 single i88G1 revertants for COLO205 ( Fig.9A ) and HCT15( Fig. 9B). Normalized cell killing (relative to i88G1) compared to COLO205 (seven residues) indicated that a greater number of mG3 residues were required to maintain direct cytotoxicity against HCT15 (15 residues). The improved constructs with 15 mG3 residues (i88G1v2, Fig. 9C and 9E , and i129G1v2, Fig.9D ) maintained the same direct cytotoxicity against COLO205 and HCT15.

[0134] Fig.10 Binding of FG27.10 and FG27.18 to ST16, HUVEC, and PBMC. For ST16 and HUVEC, the negative control for this assay was NSO supernatant, or for PBMC, cells alone, and the positive control was anti-sialyl-di-Lewis antibody recognizing HLA. a mAb, 505 / 4 or W6 / 32.

[0135] Fig.11 Binding of FG27.10 and FG27.18 to glycolipid extracts from colorectal (C170), ovarian (OVCAR-3, OVCAR-4, OVCA433, OAW28, OAW42), and gastric cell lines (ST16, MKN45, AGS). Glycolipids extracted from a range of cancer cell lines were plated on ELISA plates. Cells and glycolipids were then incubated with FG2710 or FG2718 supernatants. Binding to glycolipids was detected using anti-IgG-HRP / TMB.

[0136] Fig. 12A PI uptake in ST16 cells mediated by the mouse IgG3 mAb FG27.10 induced robust PI uptake even at 4°C, but the IgG1 variant FG27.18 did not. Fig. 12B : PI uptake by a panel of tumor cell lines (AGS, Colo201, C170, C170HM2, MCF7, OVCA4, LoVo, MKN45, 791T and Skov3) mediated by FG27.10 and FG27.18. Fig. 12C : PI uptake by C170 cells mediated by FG27.10, FG27.18, CH27 IgG2, and CH27 IgG1.

[0137] Figure 13. Binding of FG27.10, FG27.18, ch27IgG1 and the humanized variant hLewAC to a panel of cell lines: Fig.13A , HCT15; Fig. 13B, AGS; Fig. 13C , OVCAR3; Fig.13D , H322 and Fig.13E , MCF7, and FG27.10 ( Fig.13F ) and FG27.18( Figure 13G ) by SPR analysis.

[0138] Fig.14 .Compared with 27IgG1, i27G1 showed significantly improved direct cytotoxicity and functional affinity while maintaining immune effector function. Compared with 27IgG1, i27G1 significantly increased proliferation inhibition of MCF7 (panel i) and AGS (panel ii). Compared with 27IgG1, i27G1 significantly increased functional affinity (SPR) (panel iii). i27G1 maintained the same ADCC (panel iv) and CDC activity (panel v) for MCF7. Dunnett correction for multiple comparisons was used, and significance with each parental construct was derived by two-way variance (direct cytotoxicity) or one-way variance (functional affinity).

[0139] Figure 15. DNA and protein antibody sequences of antibody 88, including "improved" versions 1-3.

[0140] Figure 16. DNA and protein antibody sequences of antibody 129, including "improved" versions 1-3.

[0141] Fig.17 .DNA and protein antibody sequences of antibody 27, including the "improved" version 1.

[0142] FIG. 18 . iTrastuzumab (iTv1 and iTv2) exhibit improved functional affinity compared to wild-type trastuzumab (WT) in a ligand density-dependent manner (Biacore T200 analysis, Fig.18A Overview of cellular binding of trastuzumab constructs to a range of high-binding (SKBR3, BT474) and low- to medium-binding cell lines (MDA-MB231 and SKOV3) ( Fig.18B ). Compared with wild-type trastuzumab, the direct cytotoxicity against MDA-MB231 (iTv2) was significantly improved ( Fig.18C ). iTv1 and iTv2 (highest concentration) enhanced PI uptake by BT474 cells (reflecting membrane damage) compared to wild-type trastuzumab ( Fig.18D Directly labeled iTv2 improves solid-phase Her2 binding compared to wild-type trastuzumab ( Fig.18E ). Significance with respect to each parental construct was derived by two-way ANOVA (direct cytotoxicity) with Dunnett's correction for multiple comparisons.

[0143] Fig.19 .Protein Antibodies Trastuzumab antibody sequences, including "improved" versions 1 and 2.

[0144] Fig. 20 .IEDB screening of MHC class II binding epitopes. Top, sequence alignment (ClustalW) of hIgG1 and mIgG3 sequences containing SD286-306+339-378. The combined subdomains 286-306 and 339-378 are in the box; clusters 1 and 2 determined by IEDB analysis are indicated by arrows. Below, the middle column is the IEDB analysis table, including MHCII binding epitopes, DR alleles and scores. The right column lists residue changes that can address immunogenicity issues (mIgG3->hIgG1, underlined). DI, deimmunized construct. DETAILED DESCRIPTION

[0145] In describing embodiments of the present invention, the terminology is not intended to be limited to the specific term so selected, and it is to be understood that each specific term includes all technical equivalents that operate in a similar manner to accomplish a similar purpose.

[0146] The term "immunoglobulin" or Ig refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, a pair of low molecular weight light (L) chains and a pair of heavy (H) chains, all four chains possibly interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. For example, see Chapter 7 of Basic Immunology (Paul, W., ed., 2nd ed., Raven Press, NY (1989)).

[0147] method

[0148] Materials, cells, and antibodies

[0149] All cancer cell lines: COLO205, HCT15 (C170, C170HM2), AGS, COLO201, ST16, MCF7, OVCAR3, H322, OVCA4, LoVo, MKN45, 791T, BT474, MDA-MB231, SKBR3, SKOV3 and 791T, and mouse myeloma NS0 cell line were purchased from ATCC (Virginia, USA). All cell lines were authenticated using short tandem repeat analysis. Human serum albumin (HSA)-APD-sialyl-Lewis a and HAS-APD-Lewis aFrom IsoSepAB (Sweden). Erb2 His was from Abcam and Stratech. Cell lines were maintained in RPMI medium 1640 (Sigma) or DMEM high glucose supplemented with 10% fetal bovine serum, L-glutamine (2 mM) and sodium bicarbonate buffer. Parental mouse FG88 and FG129 mAbs were generated as described previously (Chua et al., 2015).

[0150] Cloning of modified mAb constructs

[0151] To create chimeric IgG1 variants of mAbs (FG88.2 and FG129) produced by our hybridomas, the heavy and light chain variable regions encoding the corresponding mAbs were introduced into the pDCOrig vector (Metheringham et al., 2009) using restriction enzymes BamHI / BsiWI (light chain site) or HindIII / AfeI (heavy chain site). Synthetic heavy chain constant regions, including complete mIgG3 constant regions as well as interchanged mIgG3-IgG1 domains and single residue changes, were designed and ordered by Eurofins MWG (Düsseldorf, Germany). Typically, this involves a 1054 bp cassette extending from the AfeI restriction site at the JH / CH junction 3′ to the XbaI site to the CH stop codon. The synthetic genes are provided in a proprietary Eurofins vector. After maxiprep (plasmid maxi kit, Qiagen), 15 μg of plasmid DNA was digested with AfeI and XbaI (both from NEB) and the insert was gel purified (QIAquick gel extraction kit, Qiagen). The insert was introduced into the AfeI / XbaI digested vector pOrigHiB (Metheringham et al., 2009) by ligation (T4 DNA ligase, NEB, following the manufacturer's recommendations). After sequence confirmation and maxiprep, 15 μg of plasmid DNA was digested with AfeI and AvrII (both from NEB) and the insert was gel purified. The insert was then introduced into the AfeI / AvrII digested vector pDCOrig by ligation.

[0152] HEK293 transfection and mAb purification

[0153] When using ExpiFectamine TM 293 transfection kit (Gibco, Life Technologies) transient transfection Expi293F TM In brief, 100 μg of DNA was used to transfect the suspension (100 ml, 2 × 10 6 / ml) in HEK293 cells, and the adjusted culture medium was collected on the 7th day after transfection. The adjusted transfection supernatant was filtered through a 0.22 μm bottle top filter (Merck Millipore), and sodium azide was added to a final concentration of 0.2% (w / v). Antibodies were purified on a protein G column (HiTrap ProteinG HP, GE Healthcare) using AKTAFPLC (GE Healthcare). The column was washed with PBS / Tris buffer (PBS with 50mM Tris / HCl, pH 7.0), and the antibody was then eluted to 100mM glycine, pH 12 (supplemented with 0.05% v / v Tween 20) using a fast (2ml) gradient, and 2ml fractions were collected. The mAb-containing portions were combined and neutralized (using 1M HCl) and the concentration was determined. All transiently expressed mAb constructs were then analyzed for cell binding compared to the parent mAb, using flow cytometry as a readout of the correctly folded mAb construct (data not shown).

[0154] Indirect immunofluorescence and flow cytometry

[0155] Cancer cells (1×10 5 ) were incubated with primary mAb (33.3 nmol / L or titrated) for 1 h at 4°C as described previously (Chua et al., 2015), followed by incubation with anti-mouse / anti-human-FITC-labeled secondary antibodies for 1 h at 4°C and fixed in 0.4% formaldehyde. The stained samples were analyzed on a MACSQuant10 flow cytometer and analyzed using FlowJo v10.

[0156] Functional affinity assay

[0157] The binding of 88 and 129 mAbs to Lewis was determined by surface plasmon resonance (SPR, Biacore 3000, GE Healthcare). a or Sialyl-Lewis a -APD-HAS binding kinetic parameters. mAbs with increasing concentrations (0.3nmol / L to 200nmol / L) were injected through a CM5 chip, and the data were fitted to a heterogeneous ligand binding model using BIAevaluation 4.1. The chip contained four pools, two of which were HSA coated (online reference pools), and the other two were coated with low amounts (30 to 80 response units (RU)) and high amounts (360RU to 390RU) of the respective polysaccharide-APD-HAS.

[0158] The kinetic parameters of trastuzumab antibody and trastuzumab engineered antibody variants binding to their ligand HER2 were determined by SPR (Biacore T200, Cytiva, formerly GE Healthcare). Injection of increasing concentrations (90.0 nM to 0.37 nM) of antibodies was passed through a CM5 chip coated with anti-His antibody and His-tagged HER2 was captured.

[0159] In vitro cytotoxicity

[0160] PI uptake and proliferation inhibition were performed to analyze the direct cytotoxic effects of mAbs. COLO205, HCT-15, or BT474 cells (5×10 4 ) were incubated with mAb for 2 h at 37°C, followed by the addition of 1 μg PI for 30 min. Cells were resuspended in PBS, run on a Beckman Counter FC-500 or MACSQuant 10 flow cytometer, and analyzed using WinMDI2.9 or FlowJo v10 software, respectively.

[0161] The proliferation inhibition of the constructs was evaluated by measuring the activity of cellular hydrogenases (proportional to the number of viable cells) using the water-soluble tetrazolium salt WST-8 (CCK8 kit, Sigma-Aldrich). Briefly, after plating cancer cells overnight (1000-2000 cells / 90u1 / well), different concentrations of the constructs were added in a final volume of 10μl / well and the plates were incubated at 37°C, (5% CO2) for 72 to 96 hours. The WST-8 reagent (10μl / well) was then added, and after a further incubation for 3 hours, the plates were read at 450nm (Tecan Infinite F50) and the percentage of inhibition was calculated. EC 50 Values ​​were determined by GraphPad Prism v 8.0 (GraphPad Inc, La Jolla, CA) using nonlinear regression (curve fitting).

[0162] Scanning electron microscopy

[0163] HCT-15 or COLO205 cells (1×105) were grown on sterile coverslips for 24 hours, and mAbs (0.2 μmol / L) were then added at 37°C for 18 hours. Controls included culture medium alone and 0.5% (v / v) hydrogen peroxide (H2O2) (Sigma). Cells were washed with pre-heated 0.1M sodium cacodylate buffer pH 7.4 (SDB) and fixed with 12.5% ​​(v / v) glutaraldehyde for 24 hours. Fixed cells were washed twice with SDB and then fixed with 1% (v / v) osmium tetroxide (pH7.4) for 45 minutes. After a final wash with H2O, cells were dehydrated in increasing ethanol concentrations and exposed to critical point drying, then sputtered with gold, followed by SEM analysis (JSM-840SEM, JEOL).

[0164] In vivo models

[0165] This study was conducted by CrownBio UK under UK Home Office Licence in accordance with NCRI, LASA and FELAS guidelines. Age-matched female BALB / c nude mice (Charles River, UK) were inoculated with 5×10 6 The COLO 205 human colorectal adenocarcinoma model was established by injecting 10 live cells into the left flank of each mouse. Based on the average tumor volume (approximately 103 mm 3 ±13mm 3 ) Mice (n = 10) were randomly assigned to treatment groups and administered mAb (0.1 mg) or vehicle (PBS, 100 u1) by intravenous injection (iv) every two weeks until week 5. Body weight and tumor volume were assessed three times a week, and the reduction in tumor volume was statistically analyzed using a two-way ANOVA with a Bonferroni post-test (interaction factor; GraphPad Prismv 7.4 (GraphPad Inc, La Jolla, CA)).

[0166] Example

[0167] The invention will now be further described with reference to the following examples and figures.

[0168] Example 1. In the absence of complement and immune effector cells, m88G3 exhibits polysaccharide binding and direct cytotoxicity (both reduced after chimerization to 88IgG1).

[0169] It has been previously demonstrated that the hybridoma-produced mIgG3 mAb FG88.2 exerts direct cytotoxic effects against high-binding cancer cell lines, such as COLO205 and HCT15, in the absence of complement or effector cells (Chua et al., 2015). This direct cytotoxicity involves mAb-induced cell aggregation, proliferation inhibition, and irregular pore formation through an oncolytic mechanism. We subsequently created a chimeric HEK293-expressed IgG1 mAb, 88IgG1, for clinical development. Compared with hybridoma-produced FG88.2 and HEK293-expressed 88mIgG3, 88IgG1 maintained the same level of binding to HCT15 and COLO205 cancer cells ( Figure 1A The latter mAb was generated to exclude expression system-related effects, such as differential Fc glycosylation, due to the use of mouse hybridoma cells and HEK293 cells. Surprisingly, 88IgG1 showed significantly reduced direct cytotoxicity against COLO205 and HCT15 cells by two functional assays (PI uptake and proliferation inhibition) compared to 88mIgG3 ( Figure 1B -D). 88mIgG3 also showed a modest reduction in direct cytotoxicity compared to hybridoma-produced FG88.2, suggesting that differential glycosylation of the Fc region resulting from the two expression settings (mouse hybridoma vs. HEK293 cells) may contribute to this effect. This suggests that direct cell killing is associated with kinetic binding of different isotypes. Therefore, the kinetic binding of our isotype-switched mAbs was analyzed using SPR on Lewisa-APD-HAS-coated -chips (Table 1). FG88.2 displayed on a high-density flow cell avidLewis a -APD-HSA binding with a fast apparent on-rate (k on Approx. 10 4 1 / smol / L) and a very slow dissociation rate (k off Approx. 10 -6 Compared with FG88.2, 88mIgG3 produced by HEK293 showed an apparent faster binding rate (k on Approx. ×10 5 1 / smol / L) and a slightly faster dissociation rate (k off Approx. 10 -4 1 / s), which may explain the slightly reduced cytotoxicity compared with FG88.2. In contrast, 88IgG1 has an apparently fast binding rate (k on Approx. 10 5 1 / smol / L) binds to its target, but exhibits a much faster dissociation phase (apparent koff of approximately 10 -21 / smol / L), which may be the reason for the reduced cytotoxic activity when binding to cancer cells. The mAb binding behaviors of the three mAbs on the low-density flow cell were largely comparable, and the equilibrium dissociation constants (K d ) are approximately 10 -8 mol / L.

[0170] Example 2. Domain analysis of the mIgG3 constant region showed that the mIgG3 CH3 domain has a major contribution, with slight involvement of CH2.

[0171] Collectively, the results suggest that the high Lewisa-APD-HSA functional affinity exhibited by FG88 and 88mIgG3 is primarily driven by their slow dissociation, likely due to intermolecular cooperativity of this isotype, and that this high affinity contributes to their direct cytotoxic effects against high-binding cancer cell lines. We therefore set out to design an IgG1 onco-glycan-targeting mAb with direct cytotoxic activity by transferring selected mIgG3 constant region residues into 88IgG1. First, the mIgG3 contributing regions were identified by creating hybrid 88IgG1 constructs containing either the mIgG3 CH1, CH2, or CH3 domains. Preliminary analyses determined that the contribution of mIgG3 CH1 to the direct cytotoxic capacity of 88mIgG3 was minimal, as the introduction of mIgG3 CH1 into 88IgG1(1m1) did not result in a significant increase in cytotoxicity ( Figure 2A and 2B ). Instead, IgG1 CH 1 Introducing 88mIgG 3 (3h1), also did not lead to a significant decrease in killing activity ( Figure 2A and 2B ). Next, the mIgG3 CH2 and CH3 domains were introduced into 88IgG1 using the gain-of-function approach. 3 88IgG1(1m3) showed a significant increase in PI uptake in HCT15 cells and a significant increase in proliferation inhibition in COLO205 cells ( Figure 2C and 2D ). The mice were 2 Introduction into 88IgG1(1m2) resulted in a small but non-significant increase in killing activity in both assays ( Figure 2C and 2D As confirmation of the contribution of these two domains, the opposite strategy was followed, whereby the loss of cytotoxic activity was assessed by introducing either the IgG1 CH2 or CH3 domain into 88mIgG3. IgG1 CH 3 88mG3 (3h3) showed a significant reduction in cytotoxicity, confirming previous gain-of-function results. Importantly, this strategy also identified a small contribution from mouse CH2, as 88mIgG3(3h2), which contains human CH2, showed a significant reduction in cytotoxic activity ( Figure 2C and 2D Next, the kinetic binding behavior of the hybrid constructs was analyzed. In both cases, the hybrid construct 1m3 showed a modest but significantly increased functional affinity (K d ), while 3h3 containing human CH3 showed a significant decrease in functional affinity (K d Increase, Figure 2E ), reflecting direct cytotoxicity. The human CH2 in construct 3h2 also resulted in a modest but significant decrease in functional affinity. In all cases, the changes in functional affinity were driven primarily by changes in the mAb off-rates, with 1m3 having a significantly reduced off-rate compared to 88IgG1 and 3h3, and 3h2 showing a significantly increased off-rate compared to 88mIgG3 ( Figure 2F The mouse CH2 in construct 1m2 did not result in an increase in functional affinity or a decrease in off-rate compared to 88IgG1, which is why the cytotoxicity of this construct was not significant ( Figure 2C and D). Taken together, the results suggest that mouse CH3 makes a more significant contribution to cytotoxicity and kinetic binding, whereas the contribution of mouse CH2 is smaller and only observed in a loss-of-function background.

[0172] Example 3. Discontinuous sequences within the CH2-CH3 region of aa 286-397 are critical for killing activity and increasing functional affinity.

[0173] Since the cytotoxic effect conferred by mouse CH3 is incomplete, and to further narrow down other contributing residues, we designed 88 hybrid mAb constructs in which the CH2 and CH3 domains were further subdivided into two subdomains (SD) with a 10-residue overlap in the connecting region: CH2: SD232-294 and SD286-345 and CH3: SD339-397 and SD390-447. For COLO205, both SD339-397 and SD286-345 provided similarly significant increased cytotoxicity, most evident at lower concentrations, while SD232-294 and SD390-447 were dispensable for cytotoxicity alone or in combination (not shown). Figure 3A and 3C However, for HCT15, the significant contribution of residues within SD339-397 is greater than that of SD286-345 ( Figure 3B and 3D), suggesting that subtle differences in carbohydrate antigen density and composition can modulate mAb binding and subsequent cytotoxic activity. Strikingly, 88IgG1 containing the combined mIgG3 SD286-345 and SD339-397 (SD286-397) restored nearly all of the cytotoxicity of 88mIgG3 in both cell lines and in both assays ( Figures 3A-3D ), obviating the need for additional subdomains. Functional affinity analysis of the subdomain constructs compared to 88IgG1 showed significant improvements in functional affinity for SD286-397 and SD339-397, both of which now match 88mIgG3 functional affinity, with a more modest improvement for SD286-345 ( Figure 3E The improvement in functional affinity was mainly due to the significant decrease in the apparent dissociation rate of SD286-397 and SD339-397 (about 10 -6 1 / s), SD286-345 showed a more modest improvement in dissociation rate (approximately 10 -3 1 / s)( Figure 3F ). These results add further weight to the cytotoxicity observations and suggest that creating mAbs with lower target off-rates supports direct cytotoxicity.

[0174] Although SD339-397, which contains 27 mIgG3 residues, reproduces up to 90% of the desirable properties of 88mIgG3, especially slow dissociation and enhanced cytotoxicity, it exhibits significantly reduced CDC activity compared to 88IgG1 ( Figure 3G ), so further development with SD286-397 is required. In contrast, the ADCC activity of SD339-397 was not reduced compared with 88IgG1 ( Figure 3H ). Although SD286-397 fully reproduced the cytotoxicity of 88mIgG3, it still contains 41 mIgG3 residues. Therefore, additional subdivisions of SD286-345 and SD339-397 were analyzed for cytotoxic activity and functional affinity. This analysis highlighted two regions in SD339-397 and SD286-345, respectively, that contribute to direct cytotoxicity. First, SD339-378, which contains 20 mIgG3-specific residues, significantly restored cytotoxicity to within about 80% to 90% of mIgG3 cytotoxicity in both tests after introduction into 88IgG1 ( Figure 4A and 4B This region also imparted a significant increase in functional affinity compared to 88IgG1, but the improvement was less pronounced than in the case of SD339-397 ( Figure 4GIn the same set of constructs, we identified another region of interest by loss-of-function approaches. SD307-345 showed significantly reduced cytotoxicity compared to SD286-345, implying a further contribution of residues 286-306 ( Figure 4C ). Overall, the results suggest that constructs containing a combination of residues 286-306 and 339-378 (a total of 26 mIgG3-specific residues) could fully recapitulate the direct cytotoxicity and functional affinity of 88mIgG3. To test this hypothesis, the cytotoxic activity and functional affinity of SD286-306+339-378 were evaluated. SD286-306+339-378 exhibited significantly improved direct cytotoxicity against both cell lines compared to 88IgG1, currently matching 88mIgG3 cytotoxicity ( Figures 4D-4F ). SPR analysis of SD286-306+339-378 showed that the functional affinity was significantly improved compared with 88IgG1, K d is (0.3×10 -9 nmol / L, and similar to 88mIgG3 ( Figure 4G Importantly, the CDC and ADCC activities of the SD286-306+339-378 construct were not significantly different from those of 88IgG1 ( Figure 4H and 4I The combination of improved functional affinity with direct cytotoxicity and maintained immune effector function suggests that our SD286-306+339-378 hybrid IgG1 mimics the desirable properties of 88mIgG3.

[0175] Example 4. Reversion of an immunogenic cluster identified in silico generated a lead candidate, improved "i"88g1, which possessed potent cell killing, pore-forming abilities, and good immune effector functions.

[0176] To evaluate the potential immunogenicity of our hybrid SD286-306+339-378 construct (generated by the presence of 26 mIgG3 residues), we performed an in silico screening of the SD286-306+339-378 sequence for MHCII binding epitopes (Immune Epitope Database, IEDB). Class II-restricted T helper cells are more relevant to humoral immune responses, and predicted binding clusters have been shown to be robust indicators of T cell responses (Jawa et al., 2013). Two MHCII binding clusters (containing several high-scoring binding epitopes) were identified: Cluster 1 (residues 294-315) and Cluster 2 (residues 365-393) ( Figure 5 / Fig. 20). Within cluster 1, reversion of three mouse residues 294 (A to E), 300 (F to Y), and 305 (A to V) to human residues produced a human sequence fragment that would be tolerated by individuals. Similarly, within the 339-378 region, reversion of two residues 351 (I to L) and 371 (N to G) removed two MHCII binding cores. Therefore, we created two additional constructs based on SD286-306+339-378 based on the above in silico screening: cluster 1 (DI1) and cluster 2 (DI2), containing three and two human reversion residues, respectively, and assayed their cytotoxicity. DI1 and DI2 maintained significantly improved cytotoxicity compared to 88IgG1, but DI2 showed a small but consistent decrease in activity compared to 88mIgG3 ( Fig. 6A and 6B In addition, in the case of 88DI1, direct cytotoxicity was consistent with a favorable functional affinity curve, with an apparent off-rate of (approximately 10 -4 1 / s), K d 0.5×10 -9 nmol / L, no significant difference with 88mIgG3 ( Figure 6C , Table 2). In contrast, DI2 showed significantly reduced functional affinity compared to 88mIgG3 ( Figure 6C Based on these findings, we focused on 88DI1 containing 23 mIgG3 residues, now renamed as improved “i” (improved) 88G1, to further analyze its immune effector function. 88IgG1 showed potent ADCC activity against COLO205 with a subnanomolar EC 50 ( Fig.6D ), consistent with the potent immune effector function of FG88 (Chua et al., 2015). Similarly, i88G1 showed potent ADCC with a subnanomolar EC 50 (0.35nmol / L), despite the difference with 88IgG1 (EC 50 Compared with 88IgG1, the CDC activity of i88G1 was moderately improved ( Fig. 6E , Table 2).

[0177] Earlier studies of the hybridoma-produced parental FG88 mAb have demonstrated its pore-forming ability, which is presumed to underlie its cytotoxicity (Chua et al., 2015). Therefore, we set out to analyze the pore-forming ability of i88G1 on HCT15 using SEM. Incubation of HCT15 with i88G1 or 88mIgG3 (but not 88IgG1) resulted in monolayer disruption, cell rounding, and aggregation. At high magnification, irregular pore formation was evident ( Fig. 6F), reflecting the original data observed for hybridoma-produced FG88 mAb (Chua et al., 2015).

[0178] Overall, the results indicate that transfer of selected regions from the mIgG3 constant region into the 88IgG1 backbone generates hybrid mAbs with direct cell killing capacity, increased functional affinity, and potent immune effector functions.

[0179] Example 5. Transfer of the "iG1" sequence into an alternative non-killer glycan-binding mAb (129IgG1) generates a cancer-targeting mAb with improved in vitro and in vivo anti-tumor activity.

[0180] We recently described a novel class of proteins that recognize sialyl-di-Lewis (Sialyl-di-Lewis) with potential for cancer immunotherapy development. a ) mAb (129mAb). Compared with the above-mentioned 88mAb, 129mAb has a more favorable tumor and normal human tissue distribution, mainly due to its very limited normal tissue reactivity. Neither hybridoma-produced FG129 (a mouse IgG1mAb) nor chimeric 129IgG1 showed direct cytotoxicity. This led us to test the hypothesis that the introduction of the 23 selected mIgG3 constant region residues into the Fc region of CH129 would produce "i" 129G1 with direct cytotoxicity and improved functional affinity, thereby showing superior clinical utility.

[0181] We evaluated the direct cytotoxic capacity of i129G1 against COLO205, a cancer cell line previously shown to be a high binding 129 mAb. i129G1 showed significantly improved (compared to 129 IgG1) dose-dependent proliferation inhibition ( Fig. 7A ), E.C. 50 45.6 nmol / L, and significantly improved but more modest PI uptake ( Figure 7B ). Next, we used sialyl Lewis a -APD-HAS coated chip and SPR analysis of the functional affinity of i129G1. Compared with 129IgG1, i129G1 mAb showed significantly improved functional affinity ( Figure 7C , Table 2), which was mainly due to the improvement of the dissociation rate by nearly two logs (2.6×10 -4 s -1 and 5.5×10 -6 s -1). 129IgG1 previously exerted a strong ADCC effector function against COLO205, so the ADCC and CDC activities of i129G1 against COLO205 were evaluated. Compared with 129IgG1, the ADCC activity of i129G1 was significantly reduced, but cell lysis remained in the nanomolar range (EC values ​​of i129G and 129IgG1 were 50 2.4×nmol / L and 1.7×nmol / L respectively)( Fig.7D , Table 2). However, compared with the parent 129IgG1, the CDC activity of i129G1 was significantly increased, and the EC 50 8.2×nmol / L and 75×mol / L respectively. Fig. 7E , Table 2). The direct cytotoxicity and improved functional affinity of i129G1 led us to analyze its pore-forming ability for COLO205. Incubation of COLO205 with i129G1 resulted in the formation of large cell clusters with uneven surfaces and the appearance of irregular pore-like structures ( Figure 7F ). Incubation with 129IgG1 at the same concentration also resulted in some degree of cell aggregation, but smaller and fewer clumps were observed with no evidence of pore formation.

[0182] The direct cytotoxicity and improved functional affinity of i129G1 led us to analyze the in vivo antitumor activity of i129G1 in the COLO205 xenograft model compared to the parental 129IgG1. The i129G1 mAb resulted in a significant reduction in tumor volume compared to vehicle control (two-way ANOVA, p < 0.0001), which remained significant compared to 129IgG1, confirming the in vitro results ( Figure 7G No effect on mean body weight was observed ( Figure 7H ).

[0183] The generated mAbs with direct cytotoxicity, improved functional affinity, and superior in vivo antitumor activity by introducing a selected number of mIgG3 constant region residues pave the way for applying our strategy to other cancer-targeting mAbs with clinical utility.

[0184] Example 6. Transfer of the "iG1" sequence to an alternative Lewis Y Glycan-binding mAb (27IgG1) generates cancer-targeting mAbs with improved in vitro direct cell killing ability.

[0185] We also created a monospecific binding Lewis y The mAb, 27mAb, has a broad tumor tissue distribution and good normal human tissue binding.y AGS and MCF7 cell lines showed direct cell killing, while chimeric 27IgG1 did not ( Fig. 8A and 8B We introduced our selected 23 mG3 residues into 27IgG1 to generate i27G1 and analyzed its direct cell killing effect. Compared with 27IgG1, i27G1 showed significantly improved direct cell killing for both MCF7 and AGS, equivalent to 27mG3 mAb ( Fig. 8A and 8B In addition, i27G1 exhibited significantly improved functional Lewis y Affinity Figure 8C Importantly, on MCF7 cells, the effector functions, both ADCC and CDC, of ​​i27G1 were comparable to those of 27IgG1 ( Fig.8D and 8E ).

[0186] Collectively, the results demonstrate that transfer of selected regions from the mIgG3 constant region into the 27IgG1 backbone generates hybrid mAbs with direct cell killing capacity, increased functional affinity, and potent immune effector functions.

[0187] Example 7. Further fine-tuning of the 23 "iG1" sequences revealed that 15 or 7 residues are critical for maintaining direct cell killing in a cell type-dependent manner.

[0188] In order to evaluate the individual contribution of each of the 23 selected mG3 residues, a single revertant strategy based on i88G1 was performed (Example 4). Each of the 23 mG3 residues in i88G1 was individually reverted to hG1 residues, and the resulting constructs were analyzed for direct cell killing of COL0205 and HCT15 using a cell proliferation assay (CCK-8, Sigma 96992), and compared with parental i88G1. In the same experiment, the percentage of killing at a fixed concentration was normalized to the corresponding percentage of killing of i88G1 at the same concentration in order to associate cell killing in multiple experiments. Most i88G1 revertant structures retain good direct cell killing of COLO205 cells ( Fig.9A). Residues 342, 343, 345, 361, 362, 374 and 376 (seven in total) had an average normalized killing of less than 1 and were considered to be essential for direct cell killing of the high binding COLO205 cell line. In contrast, if these seven residues were individually restored, the minimal construct did not express / fold, and thus this construct was not obtained. Direct cell killing of moderately bound HCT15 was shown to be more sensitive to reversion to hG1 residues, with only eight reversion residues (339, 344, 354, 356, 357, 358, 370, 373) maintaining cell killing within one standard deviation of i88G1, thus requiring retention of 15 mG3 residues: N286T, K288W, K290Q, Q342R, P343A, E345T, L351I, T359S, N361K, Q362K, G371N, P374S, S375E, D376A, A378S ( Fig. 9B The latter constructs were created for both 88 and 129 mAbs, i88G1v2 and i129G1v2, respectively, and analyzed for cell killing of COLO205 and HCT15. Direct cell killing of COLO205 by i88G1v2 and i129G1v2 was comparable to that observed for i88G1 and i129G1, respectively ( Fig. 9C and 9D The direct cell killing of HCT15 cells by i88G1v2 also matched that observed with i88G1 ( Fig.9E ).

[0189] Overall, the results indicate that 15 mG3 residues: N286T, K288W, K290Q, Q342R, P343A, E345T, L351I, T359S, N361K, Q362K, G371N, P374S, S375E, D376A, A378S, are sufficient to induce direct cell killing against high-binding as well as moderate-binding cancer cell lines.

[0190] Table 1. Summary of kinetic binding parameters for the parental 88 mAbs

[0191]

[0192] a Represents at least 3 analyses

[0193] b Results for high density surface binding are given

[0194] Table 2. Summary of functional characteristics of improved constructs

[0195]

[0196] a Inferred from the proliferation inhibition of COLO205

[0197] N / A: Not Applicable, ND: Not Determined

[0198] Example 8. Generation and initial characterization of FG27 mAb: FG27 enhances gastric tumor cell glycolipid immunity

[0199] Analysis of antibody response to immunization: Antibody titers were initially monitored by lipid enzyme-linked immunosorbent assay (ELISA). Thin layer chromatography (TLC) analysis was then performed using ST16 total and plasma membrane lipid extracts, flow cytometry analysis (FACS) was performed using ST16 tumor cells, and Western blot was performed using ST16 whole cell extracts and plasma membrane lipid extracts. Mice that were considered to have the best response were injected intravenously (iv) with ST16 plasma membrane lipid extracts before fusion to enhance compared to pre-bleeding serum controls.

[0200] The binding of FG27 hybridoma supernatants to a panel of tumor cell lines was analyzed by direct immunofluorescence and FACS analysis. Both FG27.10 and FG27.18 bound to ST16, but not to human umbilical vein endothelial cells (HUVEC) or peripheral blood mononuclear cells (PBMCs) compared to the positive control anti-HLA mAb, W6 / 32 (eBioscience, CA, USA) and the negative control. Fig.10 ). Both FG27.10 and FG27.18 were cloned. FG27.10 was of IgG3κ and FG27.18 was of IgG1κ subclass. To ensure that both mAbs bound to the glycolipids, glycolipids were extracted from a range of cell lines, dried on ELISA plates, and then incubated with FG27.10 and FG27.18. C170, ST16, and AGS-derived glycolipids were found to bind to both mAbs, but cell lines that lacked binding to whole cells (MKN-45, OAW28, OVCAR-3, OVCAR-4, and OAW42; Fig.11 ).

[0201] Example 9. Direct cell killing by parental FG27 mAb

[0202] Many anti-glycan mAbs have been shown to induce direct cell death in antigen-positive cell lines without the need for effector cells or complement. This has the potential to improve their efficacy in vivo, as tumors can develop mechanisms to avoid immune-mediated cell death. This ability is primarily associated with glycan-binding mAbs of the mIgG3 isotype, and thus this property is lost after chimerization or humanization.

[0203] To determine whether FG27.10 and FG27.18 have the ability to cause direct cell death, ST16 cells were incubated with FG27.10, FG27.18, and the anti-sialyl Lewis mAb SC104 for 2 h at room temperature, and cell death was then measured by uptake of PI, a DNA intercalating agent that is only taken up by dying cells ( Fig. 12A Interestingly, despite sharing the same variable region, only FG27.10 was able to induce direct cell death, while no PI uptake was observed with FG27.18. Fig. 12B Direct cell killing was demonstrated for both mAbs against a range of antigen positive (AGS, Colo201, C170, C170HM, MCF-7, OVCAR4) cell lines, showing variable amounts of killing. Neither mAb demonstrated killing against antigen negative cell lines (LoVo, MKN45, 791T, SKOV3).

[0204] Fig. 12C PI uptake by the colorectal cell line C170 is shown for a range of mAbs. FG27.10 and the positive control antibody SC101 showed titratable strong killing. FG27.18 showed non-titratable weak killing. The modified IgG1 chimera showed titratable modest killing, while the IgG2 variant did not.

[0205] Example 10. FG27 binding studies

[0206] Titration of humanized (humanized 27) and chimeric mAbs (CH27) against cancer cell lines showed similar cell binding patterns ( Fig.13A -E). The equilibrium affinity constants were generally of the same order of magnitude, but slightly higher compared to the parental mouse antibody ( Figures 13F-13G ), while the maximum binding capacity of humanized 27 and CH27 was greater than that of mouse mAb. This result indicates that the humanization process was successful.

[0207] Example 11. Direct cell killing by humanized FG27 mAb

[0208] To enhance direct cell killing of a human IgG1 27 chimera (27IgG1), we transferred selected mIgG3 constant region residues to the IgG1 Fc domain, generating an improved “i27G1” mAb that binds LewisY glycans and has improved direct cell killing ability in vitro.

[0209] Fig.14 Improved direct cell killing, functional affinity, and effector function of i27G1 were demonstrated. mIgG327 antibody was effective against cells expressing Lewis y MCF7( Fig.14i) and AGS cell lines ( Fig.14 ii) has direct cell killing ability, while chimeric 27IgG1 does not. Compared with 27IgG1, our i27G1 (containing selected mIgG3 constant region residues) has a direct cell killing ability against MCF7 ( Fig.14 i) and AGS( Fig.14 ii) Both showed significantly improved direct cell killing, equivalent to 27mG3 mAb. In addition, i27G1 showed significantly improved functional Lewis y Affinity Fig.14 Importantly, the effector functions, ADCC and CDC of i27G1 against MCF7 were comparable to those of 27IgG1 ( Fig.14 Overall, the results indicate that transfer of selected regions from the mIgG3 constant region into the 27IgG1 backbone generates hybrid mAbs with direct cell killing, increased functional affinity, and potent immune effector functions.

[0210] Example 12. Transfer of the "iG1" sequence into a protein-binding mAb (trastuzumab) generates a cancer-targeted mAb with improved functional affinity.

[0211] To evaluate whether changing the amino acid residues of antibodies targeting cancer-related proteins results in increased functional affinity, the "iG1" residues (23, 'v1' and 15, 'v2') were changed, and the affinity of the resulting "improved" antibody variants was determined by SPR analysis. To determine the functional affinity, three trastuzumab antibody constructs were titrated on a HER2-coated chip at three different ligand densities. A multi-cycle kinetic analysis (concentration range of 90nM to 0.37M) was performed, which showed that compared with WT, iTv1 and iTv2 had increased functional affinity for both low-density (20RU) and high-density (200RU) surfaces ( Fig.18A This improvement is more pronounced on high-density surfaces, mainly due to a decrease in the dissociation rate (8.5×10 for iTV1). -8 1 / s, iTv2 is 7.4×10 -8 1 / s, while WT is 6.0×10 -7 1 / s). Wild-type trastuzumab (WT) and "improved" variants (iTv1 and iTv2) expressed in HEK293 highly bound to the HER2-expressing cell lines SKBR3 and BT474, and had low to moderate binding to MDA-MB231 and SKOV3 ( Fig.18BTo evaluate whether the stronger binding of iTv1 and iTv2 would result in different cytotoxicity, proliferation inhibition of MDA-MB231 and PI uptake of BT474 were explored. iTv2 showed significantly improved proliferation inhibition of MDA-MB231 compared to WT ( Fig.18C ), both iTv1 and iTv2 showed improved PI uptake by BT474 cells compared to WT, most significantly at the highest tested concentration ( Fig.18D ). Titration of HRPO-labeled mAbs directly onto HER2-coated ELISA plates revealed improved binding of iTv2 over WT. Overall, the results demonstrate improved functional affinity of the “iG1” engineered trastuzumab construct, suggesting that our approach could benefit protein-targeted mAbs in addition to glycan targets. However, the ultimate outcome of our engineering approach depends on the interplay between mAb complex avidity and target density.

[0212] Implementation

[0213] Certain embodiments of the invention are described below. Various features of the following embodiments may be combined with features of other embodiments, for example, structural features such as sequence motifs may be combined with functional features of the claimed antibodies or antigen-binding fragments thereof.

[0214] 1. A modified IgG1 antibody or antigen-binding fragment thereof, comprising one or more residues of an immunoglobulin Fc-region and a binding region, wherein the one or more residues of the Fc-region are modified to corresponding residues from a mouse IgG3 antibody and wherein the modified IgG1 antibody or antigen-binding fragment thereof has enhanced functional affinity compared to a corresponding IgG1 antibody or antigen-binding fragment thereof comprising wild-type Fc-region residues.

[0215] 2. The modified IgG1 antibody or antigen-binding fragment thereof according to embodiment 1, wherein the functional affinity of the modified IgG1 antibody or antigen-binding fragment thereof is enhanced by at least about 10% compared to the corresponding IgG1 antibody or antigen-binding fragment thereof comprising wild-type Fc-region residues.

[0216] 3. A modified IgG1 antibody or antigen-binding fragment thereof according to embodiment 1 or embodiment 2, wherein the functional affinity of the modified IgG1 antibody or antigen-binding fragment thereof is enhanced by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 100% compared to the corresponding IgG1 antibody or antigen-binding fragment thereof comprising wild-type Fc-region residues.

[0217] 4. The modified IgG1 antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 3, wherein the modified IgG1 antibody or antigen-binding fragment thereof enhances direct cell killing compared to the corresponding IgG1 antibody or antigen-binding fragment thereof comprising wild-type Fc-region residues.

[0218] 5. The modified IgG1 antibody or antigen-binding fragment thereof according to embodiment 4, wherein the direct cell killing of the modified IgG1 antibody or antigen-binding fragment thereof is enhanced by at least about 10% compared to the corresponding IgG1 antibody or antigen-binding fragment thereof comprising wild-type Fc-region residues.

[0219] 6. A modified IgG1 antibody or antigen-binding fragment thereof according to embodiment 1 or embodiment 2, wherein direct cell killing of the modified IgG1 antibody or antigen-binding fragment thereof is enhanced by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 100% compared to a corresponding IgG1 antibody or antigen-binding fragment thereof comprising wild-type Fc-region residues.

[0220] 7. The modified IgG1 antibody or antigen-binding fragment thereof according to any preceding embodiment, wherein the one or more residues in the Fc-region are selected from the group consisting of: Q342, P343, E345, N361, Q362, P374, D376.

[0221] 8. The modified IgG1 antibody or antigen-binding fragment thereof according to any preceding embodiment, wherein the one or more modified residues in the Fc-region are selected from the group consisting of: Q342R, P343A, E345T, N361K, Q362K, P374S, D376A.

[0222] 9. A modified IgG1 antibody or antigen-binding fragment thereof according to any of the preceding embodiments, wherein one or more residues in the Fc-region are selected from: N286, K288, K290, Q342, P343, E345, L351, T359, N361, Q362, G371, P374, S375, D376, A378.

[0223] 10. A modified IGG1 antibody or antigen-binding fragment thereof according to any of the preceding embodiments, wherein the one or more modified residues in the Fc-region are selected from: N286T, K288W, K290Q, Q342R, P343A, E345T, L351I, T359S, N361K, Q362K, G371N, P374S, S375E, D376A, A378S.

[0224] 11. A modified IgG1 antibody or antigen-binding fragment thereof according to any of the preceding embodiments, wherein one or more residues in the Fc-region are selected from: N286, K288, K290, A339, Q342, P343, R344, E345, L351, S354, D356, E357, L358, T359, N361, Q362, K370, G371, Y373, P374, S375, D376, A378.

[0225] 12. A modified IgG1 antibody or antigen-binding fragment thereof according to any of the preceding embodiments, wherein the one or more modified residues in the Fc-region are selected from: N286T, K288W, K290Q, A339P, Q342R, P343A, R344Q, E345T, L351I, S354P, D356E, E357Q, L358M, T359S, N361K, Q362K, K370T, G371N, Y373F, P374S, S375E, D376A, A378S.

[0226] 13. A modified IgG1 antibody or antigen-binding fragment thereof according to any of the preceding embodiments, wherein one or more residues in the Fc-region are selected from: N286, K288, K290, E294, Y300, V305, A339, Q342, P343, R344, E345, L351, S354, D356, E357, L358, T359, N361, Q362, K370, G371, Y373, P374, S375, D376, A378.

[0227] 14. A modified IgG1 antibody or antigen-binding fragment thereof according to any of the preceding embodiments, wherein the one or more modified residues in the Fc-region are selected from: N286T, K288W, K290Q, E294A, Y300F, V305A, A339P, Q342R, P343A, R344Q, E345T, L351I, S354P, D356E, E357Q, L358M, T359S, N361K, Q362K, K370T, G371N, Y373F, P374S, S375E, D376A, A378S.

[0228] 15. The modified IgG1 antibody or antigen-binding fragment thereof according to any preceding embodiment, wherein the antibody or antigen-binding fragment thereof is a human antibody, a humanized antibody or a chimeric antibody.

[0229] 16. The modified IgG1 antibody or antigen-binding fragment thereof according to any preceding embodiment, wherein the IgG1 antibody or antigen-binding fragment thereof is a monospecific antibody, a bispecific antibody or a multispecific antibody.

[0230] 17. The modified IgG1 antibody or antigen-binding fragment thereof according to embodiment 16, wherein the IgG1 antibody or antigen-binding fragment thereof is a bispecific antibody comprising an Fc-region having a first heavy chain and a first antigen-binding region, a second heavy chain and a second antigen-binding region.

[0231] 18. The modified IgG1 antibody or antigen-binding fragment thereof according to embodiment 16 or 17, wherein the IgG1 antibody or antigen-binding fragment thereof is a bispecific antibody that binds to the CD3 antigen.

[0232] 19. A modified IgG1 antibody or antigen-binding fragment thereof comprising the motif RAXTXXXXXXXXXXXXXXXKKXXXXXXXXXXXSXA, wherein X is any amino acid.

[0233] 20. A modified IgG1 antibody or antigen-binding fragment thereof comprising the motif TXWXQXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXRAXTXXXXXIXXXXXXXSXKKXXXXXXXXNXXSEAXS, wherein X is any amino acid.

[0234] 21. A modified IgG1 antibody or antigen-binding fragment thereof comprising the motif TXWXQXXXXXXXXXXXXXXAXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXPXXRAQTXXXXXIXXPXEQMSXKKXXXXXXXTXXFSEAXS, wherein X is any amino acid.

[0235] 22. A modified IgG1 antibody or antigen-binding fragment thereof comprising the motif TXWXQXXXAXXXXXFXXXXAXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXPXXRAQTXXXXXIXXPXEQMSXKKXXXXXXXTNXFSEAXS, wherein X is any amino acid.

[0236] 23. A modified IgG1 antibody or antigen-binding fragment thereof comprising any one or more motifs selected from the following list:

[0237] (i)TXWXQ

[0238] (ii)RAXTXXXXXI

[0239] (iii)SXKKXXXXXXXXNXXSEAXS

[0240] (iv)NXXSEAXS

[0241] wherein X is any amino acid.

[0242] 24. A method for increasing the direct cell killing ability of an IgG1 antibody or an antigen-binding fragment thereof, which IgG1 antibody or antigen-binding fragment thereof comprises one or more residues of an immunoglobulin Fc-region and a binding region, wherein the method comprises replacing one or more residues of the CH2 and / or CH3 domains of the Fc-region with one or more corresponding residues from a mouse IgG3 CH2 and / or CH3 domain.

[0243] 25. A method for increasing the direct cell killing ability of an IgG1 antibody or an antigen-binding fragment thereof according to embodiment 24, wherein the method comprises modifying one or more residues in the Fc-region selected from the group consisting of: Q342, P343, E345, N361, Q362, P374, D376.

[0244] 26. A method according to embodiment 25, wherein one or more modified residues are selected from: Q342R, P343A, E345T, N361K, Q362K, P374S, D376A.

[0245] 27. A method for increasing the direct cell killing ability of an IgG1 antibody or its antigen-binding fragment according to embodiment 24, wherein the method comprises modifying one or more residues in the Fc-region selected from the following: N286, K288, K290, Q342, P343, E345, L351, T359, N361, Q362, G371, P374, S375, D376, A378.

[0246] 28. A method according to embodiment 27, wherein one or more modified residues are selected from: N286T, K288W, K290Q, Q342R, P343A, E345T, L351I, T359S, N361K, Q362K, G371N, P374S, S375E, D376A, A378S.

[0247] 29. A method for increasing the direct cell killing ability of an IgG1 antibody or its antigen-binding fragment according to embodiment 24, wherein the method comprises modifying one or more residues in the Fc-region selected from the following: N286, K288, K290, A339, Q342, P343, R344, E345, L351, S354, D356, E357, L358, T359, N361, Q362, K370, G371, Y373, P374, S375, D376, A378.

[0248] 30. A method according to embodiment 29, wherein one or more modified residues are selected from: N286T, K288W, K290Q, A339P, Q342R, P343A, R344Q, E345T, L351I, S354P, D356E, E357Q, L358M, T359S, N361K, Q362K, K370T, G371N, Y373F, P374S, S375E, D376A, A378S.

[0249] 31. A method for increasing the direct cell killing ability of an IgG1 antibody or its antigen-binding fragment according to embodiment 24, wherein the method comprises modifying one or more residues in the Fc-region selected from the following: N286, K288, K290, E294, Y300, V305, A339, Q342, P343, R344, E345, L351, S354, D356, E357, L358, T359, N361, Q362, K370, G371, Y373, P374, S375, D376, A378.

[0250] 32. A method according to embodiment 31, wherein one or more modified residues are selected from: N286T, K288W, K290Q, E294A, Y300F, V305A, A339P, Q342R, P343A, R344Q, E345T, L351I, S354P, D356E, E357Q, L358M, T359S, N361K, Q362K, K370T, G371N, Y373F, P374S, S375E, D376A, A378S.

[0251] 33. A pharmaceutical composition comprising the modified IgG1 antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 23, and a pharmaceutically acceptable carrier.

[0252] 34. The pharmaceutical composition according to embodiment 33, further comprising at least one other therapeutic agent.

[0253] 35. A pharmaceutical composition according to embodiment 33 or 34, comprising a modified IgG1 antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 23 and an immunomodulator.

[0254] 36. The modified IgG1 antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 23 or the composition according to any one of embodiments 33 to 35 for use as a medicament.

[0255] 37. The modified IgG1 antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 23 or the composition according to any one of embodiments 33 to 35 for use in treating cancer, an autoimmune disease, an inflammatory disease or an infectious disease.

[0256] 38. A method of treating an individual suffering from a disease, the method comprising administering to the individual a pharmaceutically effective amount of a modified IgG1 antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 23 or a composition according to any one of embodiments 33 to 35.

[0257] 39. The method of treating an individual according to embodiment 38, wherein the disease is selected from the group consisting of cancer, an autoimmune disease, an inflammatory disease, and an infectious disease.

[0258] 40. The method of treating an individual according to any one of 38 to 39, wherein the method further comprises administering an additional therapeutic agent to the individual.

[0259] 41. The method of treating an individual according to embodiment 40, wherein the additional therapeutic agent is an immunomodulatory agent.

[0260] 42. A kit comprising a modified IgG1 antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 23 or a composition according to any one of embodiments 33 to 35, wherein the modified IgG1 antibody or antigen-binding fragment thereof, or the composition is in one or more containers, such as vials.

[0261] 43. The kit according to embodiment 42, wherein the modified IgG1 antibody or antigen-binding fragment thereof or the composition is for simultaneous, separate or sequential use in therapy.

[0262] 44. Use of the modified IgG1 antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 23 or the composition according to any one of embodiments 33 to 35 for the manufacture of a medicament for treating a disease.

[0263] 45. The use according to embodiment 44, wherein the disease is cancer, an autoimmune disease, an inflammatory disease or an infectious disease.

[0264] 46. ​​A modified IgGl antibody or antigen-binding fragment thereof comprising one or more residue modifications to the Fc-region at residue positions selected from the group consisting of: Q342, P343, E345, N361, Q362, P374, D376.

[0265] 47. A modified IgGl antibody or antigen-binding fragment thereof, comprising one or more modifications to the Fc-region selected from the group consisting of: Q342R, P343A, E345T, N361K, Q362K, P374S, D376A.

[0266] 48. A modified IgG1 antibody or antigen-binding fragment thereof, comprising one or more residue modifications to the Fc-region at residue positions selected from the group consisting of: N286, K288, K290, Q342, P343, E345, L351, T359, N361, Q362, G371, P374, S375, D376, A378.

[0267] 49. A modified IgG1 antibody or antigen-binding fragment thereof, comprising one or more modifications to the Fc-region selected from the group consisting of: N286T, K288W, K290Q, Q342R, P343A, E345T, L351I, T359S, N361K, Q362K, G371N, P374S, S375E, D376A, A378S.

[0268] 50. A modified IgG1 antibody or antigen-binding fragment thereof, comprising one or more residue modifications to the Fc-region at residue positions selected from the group consisting of: N286, K288, K290, A339, Q342, P343, R344, E345, L351, S354, D356, E357, L358, T359, N361, Q362, K370, G371, Y373, P374, S375, D376, A378.

[0269] 51. A modified IgG1 antibody or antigen-binding fragment thereof, comprising one or more modifications to the Fc-region selected from the group consisting of: N286T, K288W, K290Q, A339P, Q342R, P343A, R344Q, E345T, L351I, S354P, D356E, E357Q, L358M, T359S, N361K, Q362K, K370T, G371N, Y373F, P374S, S375E, D376A, A378S.

[0270] 52. A modified IgG1 antibody or an antigen-binding fragment thereof, comprising one or more residue modifications to the Fc-region at residue positions selected from the group consisting of: N286, K288, K290, E294, Y300, V305, A339, Q342, P343, R344, E345, L351, S354, D356, E357, L358, T359, N361, Q362, K370, G371, Y373, P374, S375, D376, A378.

[0271] 53. A modified IgG1 antibody or an antigen-binding fragment thereof, comprising one or more modifications to the Fc-region selected from the group consisting of: N286T, K288W, K290Q, E294A, Y300F, V305A, A339P, Q342R, P343A, R344Q, E345T, L351I, S354P, D356E, E357Q, L358M, T359S, N361K, Q362K, K370T, G371N, Y373F, P374S, S375E, D376A, A378S.

[0272] 54. A modified IgG1 antibody or antigen-binding fragment thereof, comprising Fig.19 One or more sequences disclosed in .

[0273] 55. A modified IgG1 antibody or antigen-binding fragment thereof comprising Fig.19 The heavy and light chain sequences disclosed in .

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'Excess reactiveoxygen species production mediates monoclonal antibody-induced humanembryonic stem cell death via oncosis', Cell Death Differ, 24: 546-58 Series List <110> skansel gmbh <120> Modified Fc-region to enhance the functional affinity of antibodies and antigen-binding fragments thereof <130> P125250WO <150> GB1910900.8 <151> 2019-07-31 <160> 44 <170> PatentIn version 3.5 <210> 1 <211> 35 <212> PRT <213> Artificial sequence <220> <223> Modified Fc motif <220> <221> misc_feature <222> (3) <223> Xaa can be any amino acid <220> <221> misc_feature <222> (5)..(19) <223> Xaa can be any amino acid <220> <221> misc_feature <222> (22)..(32) <223> Xaa can be any amino acid <220> <221> misc_feature <222> (34)..(34) <223> Can be any amino acid <400> 1 Arg Ala Happy Thr Happy Happy Happy Happy Happy Happy Happy Hour 1 5 10 15 Happy Happy Happy Lys Lys Happy Happy Happy Happy Hour Happy Happy Hour 20 25 30 Ser Horn 35 <210> 2 <211> 93 <212> PRT <213> Artificial Sequence <220> <223> Modified Fc motif <220> <221> misc_feature <222> (2)…(2) <223> Can be any amino acid <220> <221> misc_feature <222> (4)..(4) <223> Can be any amino acid <220> <221> misc_feature <222> (6)..(56) <223> Can be any amino acid <220> <221> misc_feature <222> (59)..(59) <223> Can be any amino acid <220> <221> misc_feature <222> (61)..(65) <223> Can be any amino acid <220> <221> misc_feature <222> (67)..(73) <223> Can be any amino acid <220> <221> misc_feature <222> (75)..(75) <223> Can be any amino acid <220> <221> misc_feature <222> (78)..(85) <223> Can be any amino acid <220> <221> misc_feature <222> (87)..(88) <223> Can be any amino acid <220> <221> misc_feature <222> (92)..(92) <223> Can be any amino acid <400> 2 Thr No Trp No Gln No No No No 1 5 10 15 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes 20 25 30 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes 35 40 45 Happy Happy Happy Happy Hour Happy Happy Hour Happy Happy Hour Thr Happy Happy Happy Hour 50 55 60 Island Village Village Village Village Ser Village Lys Lys Village 65 70 75 80 Yes Yes Yes Yes Asn Yes Ser Glu Ala Yes Ser 85 90 <210> 3 <211> 93 <212> PRT <213> Artificial Sequence <220> <223> Modified Fc motif <220> <221> misc_feature <222> (2)…(2) <223> Can be any amino acid <220> <221> misc_feature <222> (4)..(4) <223> Can be any amino acid <220> <221> misc_feature <222> (6)..(19) <223> Can be any amino acid <220> <221> misc_feature <222> (21)..(53) <223> Can be any amino acid <220> <221> misc_feature <222> (55)..(56) <223> Xaa can be any amino acid <220> <221> misc_feature <222> (61)..(65) <223> Xaa can be any amino acid <220> <221> misc_feature <222> (67)..(68) <223> Xaa can be any amino acid <220> <221> misc_feature <222> (70)..(70) <223> Xaa can be any amino acid <220> <221> misc_feature <222> (75)..(75) <223> Xaa can be any amino acid <220> <221> misc_feature <222> (78)..(84) <223> Xaa can be any amino acid <220> <221> misc_feature <222> (86)..(87) <223> Xaa can be any amino acid <220> <221> misc_feature <222> (92)..(92) <223> Xaa can be any amino acid <400> 3 Thr No Trp No Gln No No No No 1 5 10 15 Haha Haha Ala Haha Haha Haha Haha Haha Haha Haha Haha Haha Haha 20 25 30 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes 35 40 45 Thank You Thank You Pro Thank You Arg Ala Gln Thr Thank You 50 55 60 No Island No No Pro No Glu Gln Met Ser No Lys Lys No No 65 70 75 80 Yes Yes Yes Thr Yes Yes Phe Ser Glu Ala Yes Ser 85 90 <210> 4 <211> 93 <212> PRT <213> Artificial Sequence <220> <223> Modified Fc motif <220> <221> misc_feature <222> (2)…(2) <223> Can be any amino acid <220> <221> misc_feature <222> (4)..(4) <223> Can be any amino acid <220> <221> misc_feature <222> (6)..(8) <223> Xaa can be any amino acid <220> <221> misc_feature <222> (10)..(14) <223> Xaa can be any amino acid <220> <221> misc_feature <222> (16)..(19) <223> Xaa can be any amino acid <220> <221> misc_feature <222> (21)..(53) <223> Xaa can be any amino acid <220> <221> misc_feature <222> (55)..(56) <223> Xaa can be any amino acid <220> <221> misc_feature <222> (61)..(65) <223> Xaa can be any amino acid <220> <221> misc_feature <222> (67)..(68) <223> Xaa can be any amino acid <220> <221> misc_feature <222> (70)..(70) <223> Xaa can be any amino acid <220> <221> misc_feature <222> (75)..(75) <223> Can be any amino acid <220> <221> misc_feature <222> (78)..(84) <223> Can be any amino acid <220> <221> misc_feature <222> (87)..(87) <223> Can be any amino acid <220> <221> misc_feature <222> (92)..(92) <223> Can be any amino acid <400> 4 Thr Not Trp Not Gln Not Ala No Not Phe Not 1 5 10 15 Haha Haha Ala Haha Haha Haha Haha Haha Haha Ha Haha Haha Haha Haha Haha 20 25 30 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes 35 40 45 Thank You Thank You Pro Thank You Arg Ala Gln Thr Thank You 50 55 60 Free Island Pro Free Glu Gln Met Ser Free Lys Lys Free 65 70 75 80 Yes Yes Yes Thr Asn Yes Phe Ser Glu Ala Yes Ser 85 90 <210> 5 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Modified Fc motif <220> <221> misc_feature <222> (2)…(2) <223> Can be any amino acid <220> <221> misc_feature <222> (4)..(4) <223> Can be any amino acid <400> 5 Thr Half Trp Half Gln 1 5 <210> 6 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Modified Fc motif <220> <221> misc_feature <222> (3)…(3) <223> Can be any amino acid <220> <221> misc_feature <222> (5)..(9) <223> Can be any amino acid <400> 6 Arg Ala Rock Thr Rock Rock Island 1 5 10 <210> 7 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Modified Fc motif <220> <221> misc_feature <222> (2)…(2) <223> Can be any amino acid <220> <221> misc_feature <222> (5)…(12) <223> Can be any amino acid <220> <221> misc_feature <222> (14)..(15) <223> Can be any amino acid <220> <221> misc_feature <222> (19)..(19) <223> Can be any amino acid <400> 7 Ser Have Lys Lys Have Have Have Have Have Asn Have Ser 1 5 10 15 Cheap Glue 20 <210> 8 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Modified Fc motif <220> <221> misc_feature <222> (2)…(3) <223> Can be any amino acid <220> <221> misc_feature <222> (7)…(7) <223> Can be any amino acid <400> 8 Asn No More Ser Glu Ala No Ser 1 5 <210> 9 <211> 699 <212> DNA <213> Artificial Sequence <220> <223> 88 light chain DNS sequence (variable region + kappa constant) <400> 9 atgagtgtgc tcactcaggt cctggcgttg ctgctgctgt gcttacagg tgccagatgt 60 gataccaga tgactcagtc tccaacctcc ctatctgcat ctgtgggaga aactgtcacc 120 atcacatgtc gaacaagtga gatattcac aattttaa catggtatca gcagaacag 180 ggaaaatctc ctcaggtcct ggtctataat gcaaaaacct taccagatgg tgtgccatca 240 aggttcagtg gcagtggatc aggaacacaa tattctca agatcacag cctgcagcct 300 gagattttg ggacttatta ctgtcacat tttggagta gtccgtggac gttcggtgga 360<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ggcaccaagc tggaaatcaa acgtacggta gcgccatctg tcttcatctt cccgccatct 420<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gatgagcagt tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctatccc 480<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> agagaggcca aagtacagtg gaaggtggat aacgccctcc aatcgggtaa ctcccaggag 540<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> agtgtcacag agcaggacag caaggacagc acctacagcc tcagcagcac cctgacgctg 600<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> agcaaagcag actacgagaa acacaaagtc tacgcctgcg aagtcaccca tcaggggcctg 660<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> agctcgcccg tcacaaagag cttcaacagg ggagagtgt 699<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 10<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 233<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> PRT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> Artificial sequence<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <220><h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <223> 88 light chain protein sequence (variable region + kappa<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> constant)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 10<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Met Ser Val Leu Thr Gln Val Leu Ala Leu Leu Leu Leu Trp Leu Thr<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1 5 10 15<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Thr Ser Leu Ser<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 20 25 30<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Ala Ser Val Gly Glu Thr Val Thr Ile Thr Cys Arg Thr Ser Glu Asn<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 35 40 45 Ile His Asn Phe Leu Thr Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro 50 55 60 Gln Val Leu Val Tyr Asn Ala Lys Thr Leu Pro Asp Gly Val Pro Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn 85 90 95 Ser Leu Gln Pro Glu Asp Phe Gly Thr Tyr Tyr Cys Gln His Phe Trp 100 105 110 Ser Ser Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 115 120 125 Thr Val Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 130 135 140 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 145 150 155 160 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 165 170 175 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 180 185 190 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 195 200 205 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 210 215 220 Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 11 <211> 1425 <212> DNA <213> Artificial sequence <220> <223> Heavy chain DNA sequence of i88v1 (variable + 'improved' v1 constant region) <400> 11 atgtacttgg gactgaactg tgtattcata gttttctct taaatggtgt ccagagtgaa 60 gtgaaactcg aggagtctgg aggaggcttg gtgcaacctg gaggatccat gaaactctct 120 tgtgctgcct ctggattcac ttttagtgac gcctggatga actgggtccg ccagtctcca 180 gagaaggggc ttgagtgggt tgctgaaatt agaagcaaag ttattaatcc tgcaatatac 240 tatgctgagt ctgtgaaaga gaggttcacc atattaagag atgattccaa aagtagtgtc 300 tacctgcaaa tgaacagctt aagagctgaa gacactggaa tttattactg ttccaggtct 360 actatgatta cgacaaggga cccgtcccgg tacttcgatg tctggggcgc agggaccacg 420 gtcaccgtct ccagcgcttc caccaagggc ccatcggtct tccccctggc accctcctcc 480 aagagcacct ctgggggcac agcggccctg ggctgcctgg tcaaggacta cttccccgaa 540 ccggtgacgg tgtcgtggaa ctcaggcgcc ctgaccagcg gcgtgcacac cttcccggct 600 gtcctacagt cctcaggact ctactccctc agcagcgtgg tgaccgtgcc ctccagcagc 660 ttgggcaccc agacctacat ctgcaacgtg aatcacaagc ccagcaacac caaggtggac 720 aagaaagttg agcccaaatc ttgtgacaaa actcacacat gcccaccgtg cccagcacct 780 gaactcctgg ggggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg 840 atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag 900 gtcaagttca actggtacgt ggacggcgtg gaggtgcata cagcctggac acagccccgt 960 gaagagcagt acaacagtac ctaccgagtg gtcagtgtcc tcaccgtcct gcaccaggac 1020 tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc 1080 gagaaaacca tctccaaacc caaaggaaga gcccagacac ctcaagtata caccataccc 1140 ccacctcgtg aaaaatgtc caagaag gttagtctga cctgcctggt caccaacttc 1200 ttctctgaag ccatcagtgt ggagtgggag agcaatgggc agccggagaa cactacaag 1260 accacgcctc ccgtgctgga ctccgacggc tccttctcc tctacagcaa gctcaccgtg 1320 gataagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctg 1380 cacaccact acacgcagaa gagccctcc ctgtctccgg gtaaa 1425 <210> 12 <211> 475 <212> PRT <213> Artificial sequence <220> <223> Heavy chain protein sequence of i88v1 (variable + 'improved' v1 constant region) <400> 12 Met Tyr Leu Gly Leu Asn Cys Val Phe Ile Val Phe Leu Leu Asn Gly 1 5 10 15 Val Gln Ser Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Gly Gly Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Asp Ala Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu 50 55 60 Glu Trp Val Ala Glu Ile Arg Ser Lys Val Ile Asn Pro Ala Ile Tyr 65 70 75 80 Tyr Ala Glu Ser Val Lys Glu Arg Phe Thr Ile Leu Arg Asp Asp Ser 85 90 95 Lys Ser Ser Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr 100 105 110 Gly Ile Tyr Tyr Cys Ser Arg Ser Thr Met Ile Thr Thr Arg Asp Pro 115 120 125 Ser Arg Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser 130 135 140 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 145 150 155 160 Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 165 170 175 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 180 185 190 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 195 200 205 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 210 215 220 Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 225 230 235 240 Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 245 250 255 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 260 265 270 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 275 280 285 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 290 295 300 Trp Tyr Val Asp Gly Val Glu Val His Thr Ala Trp Thr Gln Pro Arg 305 310 315 320 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 325 330 335 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 340 345 350 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Pro Lys 355 360 365 Gly Arg Ala Gln Thr Pro Gln Val Tyr Thr Ile Pro Pro Pro Arg Glu 370 375 380 Gln Met Ser Lys Lys Lys Val Ser Leu Thr Cys Leu Val Thr Asn Phe 385 390 395 400 Phe Ser Glu Ala Ile Ser Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 405 410 415 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 420 425 430 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 435 440 445 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 450 455 460 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470 475 <210> 13 <211> 1425 <212> DNA <213> Artificial sequence <220> <223> Heavy chain DNA sequence of i88v2 (variable + 'improved' v2 constant region) <400> 13 atgtacttgg gactgaactg tgtattcata gttttctct taaatggtgt ccagagtgaa gtgaaactcg aggagtctgg aggaggcttg gtgcaacctg gaggatccat gaaactctct tgtgctgcct ctggattcac ttttagtgac gcctggatga actgggtccg ccagtctcca gagaaggggc ttgagtgggt tgctgaaatt agaagcaaag ttattaatcc tgcaatatac 300. 300. 300. 300. 300. 300. 300. 300. 300. 300. 300 360. tacctgcaaa tgaacagctt aagagctga gacactgga tttattactg ttccaggtct actatgatta cgacaaggga cccgtcccgg tacttcgatg tctggggcgc agggaccacg 420 gtcaccgtct ccagcgcttc caccaagggc ccatcggtct tccccctggc accctcctcc 480 aagagcacct ctgggggcac agcggccctg ggctgcctgg tcaaggacta cttccccgaa540 ccggtgacgg tgtcgtggaa ctcaggcgcc ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact ctactccctc agcagcgtgg tgaccgtgcc ctccagcagc 660 ttgggcaccc agacctacat ctgcaacgtg aatcacaagc ccagcaacac caaggtggac aagaaagttg agcccaaatc ttgtgacaaa actcacacat gcccaccgtg cccagcacct 780 gaactcctgg ggggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg 840 atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag 900 gtcaagttca actggtacgt ggacggcgtg gaggtgcata cagcctggac acagccccgt 960 gaagagcagt acaacagtac ctaccgagtg gtcagtgtcc tcaccgtcct gcaccaggac 1020 tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc 1080 gagaaaacca tctccaaagc caaaggaaga gcccgaacac ctcaagtata caccataccc 1140 ccatcccgtg atgagctgtc caagaagaag gttagtctga cctgcctggt caaaaacttc 1200 tattctgaag ccatcagtgt ggagtgggag agcaatgggc agccggagaa caactacaag 1260 accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcaa gctcaccgtg 1320 gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg 1380 cacaaccact acacgcagaa gagcctctcc ctgtctccgg gtaaa 1425 <210> 14 <211> 475 <212> PRT <213> Artificial sequence <220> <223> Heavy chain protein sequence of i88v2 (variable + 'improved' v2 constant region) <400> 14 Met Tyr Leu Gly Leu Asn Cys Val Phe Ile Val Phe Leu Leu Asn Gly 1 5 10 15 Val Gln Ser Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Gly Gly Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Asp Ala Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu 50 55 60 Glu Trp Val Ala Glu Ile Arg Ser Lys Val Ile Asn Pro Ala Ile Tyr 65 70 75 80 Tyr Ala Glu Ser Val Lys Glu Arg Phe Thr Ile Leu Arg Asp Asp Ser 85 90 95 Lys Ser Ser Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr 100 105 110 Gly Ile Tyr Tyr Cys Ser Arg Ser Thr Met Ile Thr Thr Arg Asp Pro 115 120 125 Ser Arg Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser 130 135 140 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 145 150 155 160 Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 165 170 175 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 180 185 190 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 195 200 205 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 210 215 220 Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 225 230 235 240 Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 245 250 255 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 260 265 270 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 275 280 285 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 290 295 300 Trp Tyr Val Asp Gly Val Glu Val His Thr Ala Trp Thr Gln Pro Arg 305 310 315 320 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 325 330 335 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 340 345 350 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 355 360 365 Gly Arg Ala Arg Thr Pro Gln Val Tyr Thr Ile Pro Pro Ser Arg Asp 370 375 380 Glu Leu Ser Lys Lys Lys Val Ser Leu Thr Cys Leu Val Lys Asn Phe 385 390 395 400 Tyr Ser Glu Ala Ile Ser Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 405 410 415 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 420 425 430 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 435 440 445 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 450 455 460 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470 475 <210> 15 <211> 1425 <212> DNA <213> Artificial sequence <220> <223> Heavy chain DNA sequence of i88v3 (variable + 'improved' v3 constant region) <400> 15 atgtacttgg gactgaactg tgtattcata gttttctct taaatggtgt ccagagtgaa 60 gtgaaactcg aggagtctgg aggaggcttg gtgcaacctg gaggatccat gaaactctct 120 tgtgctgcct ctggattcac ttttagtgac gcctggatga actgggtccg ccagtctcca 180 gagaaggggc ttgagtgggt tgctgaaatt agaagcaaag ttattaatcc tgcaatatac 240 tatgctgagt ctgtgaaaga gaggttcacc atattaagag atgattccaa aagtagtgtc 300 tacctgcaaa tgaacagctt aagagctgaa gacactggaa tttattactg ttccaggtct 360 actatgatta cgacaaggga cccgtcccgg tacttcgatg tctggggcgc agggaccacg 420 gtcaccgtct ccagcgcttc caccaagggc ccatcggtct tccccctggc accctcctcc 480 aagagcacct ctgggggcac agcggccctg ggctgcctgg tcaaggacta cttccccgaa 540 ccggtgacgg tgtcgtggaa ctcaggcgcc ctgaccagcg gcgtgcacac cttcccggct 600 gtcctacagt cctcaggact ctactccctc agcagcgtgg tgaccgtgcc ctccagcagc 660 ttgggcaccc agacctacat ctgcaacgtg aatcacaagc ccagcaacac caaggtggac 720 aagaaagttg agcccaaatc ttgtgacaaa actcacacat gcccaccgtg cccagcacct 780 gaactcctgg ggggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg 840 atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag 900 gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccccgt 960 gaagagcagt acaacagtac ctaccgagtg gtcagtgtcc tcaccgtcct gcaccaggac 1020 tggctgaatg gcaggagta caagtgcaag gtctccaca aagccctccc agcccccatc 1080 gagaaaacca tctccaagc siaggaag gcccgaacc ctcaagtata caccctgccc 1140 ccatcccgtg atgagctgac caagaag gttagtctga cctgcctggt caaggcttc 1200 tattcttagcg ccatcgccgt ggagtgggag agcaatgggc agccgagaa caactacaag 1260 accacgcctc ccgtgctgga ctccgacggc tccttctcc tctacagcaa gctcaccgtg 1320 gataagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctg 1380 cacaccact acacgcagaa gagccctcc ctgtctccgg gtaaa 1425 <210> 16 <211> 475 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain protein sequence of i88v3 (variable + 'improved' v3 constant region) <400> 16 Met Tyr Leu Gly Leu Asn Cys Val Phe Ile Val Phe Leu Leu Asn Gly 1 5 10 15 Val Gln Ser Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Gly Gly Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Asp Ala Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu 50 55 60 Glu Trp Val Ala Glu Ile Arg Ser Lys Val Ile Asn Pro Ala Ile Tyr 65 70 75 80 Tyr Ala Glu Ser Val Lys Glu Arg Phe Thr Ile Leu Arg Asp Asp Ser 85 90 95 Lys Ser Ser Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr 100 105 110 Gly Ile Tyr Tyr Cys Ser Arg Ser Thr Met Ile Thr Thr Arg Asp Pro 115 120 125 Ser Arg Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser 130 135 140 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 145 150 155 160 Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 165 170 175 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 180 185 190 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 195 200 205 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 210 215 220 Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 225 230 235 240 Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 245 250 255 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 260 265 270 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 275 280 285 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 290 295 300 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 305 310 315 320 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 325 330 335 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 340 345 350 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 355 360 365 Gly Arg Ala Arg Thr Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 370 375 380 Glu Leu Thr Lys Lys Lys Val Ser Leu Thr Cys Leu Val Lys Gly Phe 385 390 395 400 Tyr Ser Ser Ala Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 405 410 415 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 420 425 430 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 435 440 445 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 450 455 460 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470 475 <210> 17 <211> 717 <212> DNA <213> Artificial sequence <220> <223> 129 Light chain DNA sequence (variable region + Kappa constant) <400> 17 atggaatcac agactcaggt cctcatgtcc ctgctgttct gggtatctac ctgtggggac 60 attgtgatga cacagtctcc atcctccctg actgtgacag caggagagaa ggtcactatg 120 agctgcaagt ccagtcagag tctgttaaac agtggaaatc aaaagaacta cttgacctgg 180 taccagcaga aaccagggca gcctcctaaa gtgttgatct actgggcatc cactagggaa 240 tctggggtcc ctgatcgctt cacaggcagt ggatctggaa cagatttcac tctcaccatc 300 agcagtgtgc aggctgaaga cctggcagtt tattactgtc agaatgatta tagttctcca 360 ttcacgttcg gctcggggac aaagttggaa ataaaacgta cggtagcggc cccatctgtc 420 ttcatcttcc cgccatctga tgagcagttg aaatctggaa ctgcctctgt tgtgtgcctg 480 ctgaataact tctatcccag agaggccaaa gtacagtgga aggtggataa cgccctccaa 540 tcgggtaact cccaggagag tgtcacagag caggacagca aggacagcac ctacagcctc 600 agcagcaccc tgacgctgag caaagcagac tacgagaaac acaaagtcta cgcctgcgaa 660 gtcacccatc agggcctgag ctcgcccgtc acaaagagct tcaacagggg agagtgt 717 <210> 18 <211> 239 <212> PRT <213> Artificial sequence <220> <223> 129 Light chain protein sequence (variable region + Kappa constant) <400> 18 Met Glu Ser Gln Thr Gln Val Leu Met Ser Leu Leu Phe Trp Val Ser 1 5 10 15 Thr Cys Gly Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val 20 25 30 Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu 35 40 45 Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys 50 55 60 Pro Gly Gln Pro Pro Lys Val Leu Ile Tyr Trp Ala Ser Thr Arg Glu 65 70 75 80 Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr 100 105 110 Cys Gln Asn Asp Tyr Ser Ser Pro Phe Thr Phe Gly Ser Gly Thr Lys 115 120 125 Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 165 170 175 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 180 185 190 Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 195 200 205 Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln 210 215 220 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 19 <211> 1401 <212> DNA <213> Artificial sequence <220> <223> Heavy chain DNA sequence of i129v1 (variable + 'improved' v1 constant region) <400> 19 atgctgttgg ggctgaagtg ggttttcttt gttgtttttt atcaaggtgt gcattgtgag 60 gtgcagcttg ttgagtctgg tggaggattg gtgcagccta aagggtcatt gaaactctca 120 tgtgcagcct ctggattcac cttcaatacc tacgccatga actgggtccg ccaggctcca 180 ggaaagggtt tggaatggt tgctcgcata agaagtaaa gtaataatta tgcaacatat 240 tatgccgatt cagtgaaaga caggttcacc atatccagag atgattcaca aagcatgctc 300 tatctgcaaa tgaacaactt gaaaaaggag gacacagcca tgtattactg tgtagggtac 360 ggtagtgggg gaaactactg gggtcaagga acctcagtca ccgtctccag cgcttccacc 420 aagggcccat cggtcttcc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 480 gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 540 ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 600 tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 660 aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt 720 ɣaaaactc acacatgcc accgtgccca gcacctgaac tcctgggggg accgtcagtc 780 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 840 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 900 ggcgtggagg tgcatacagc ctggacacag ccccgtgaag agcagtacaa cagtacctac 960 cgagtggtca gtgtcctcac cgtcctgcac caggactggc tgaatggca ggagtacaag 1020 tgcaaggtct ccaaaagc cctcccagcc cccatcgaga aaaccatctc caaccaaa 1080 ggaagagccc agacacctca agtatacacc ataccccac ctcgtgaaca aatgtccaag 1140 aagaaggtta gtctgacctg cctgtcacc aacttctct ctgaagccat cagtgtggag 1200 tgggagagca atgggcagcc ggagacaac tacaagacca cgcctcccgt gctggactcc 1260 gacggctcct tctcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 1320 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactac gcagaagagc 1380 ctctccctgt ctccgggtaa a 1401 <210> 20 <211> 467 <212> PRT <213> Artificial sequence <220> <223> Heavy chain Protein sequence of i129v1 (variable + 'improved' v1 constant region) <400> 20 Met Leu Leu Gly Leu Lys Trp Val Phe Phe Val Val Phe Tyr Gln Gly 1 5 10 15 Val His Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Lys Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Asn Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ala Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr Tyr 65 70 75 80 Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser 85 90 95 Gln Ser Met Leu Tyr Leu Gln Met Asn Asn Leu Lys Lys Glu Asp Thr 100 105 110 Ala Met Tyr Tyr Cys Val Gly Tyr Gly Ser Gly Gly Asn Tyr Trp Gly 115 120 125 Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 130 135 140 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 145 150 155 160 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 165 170 175 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 180 185 190 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 195 200 205 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 210 215 220 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 225 230 235 240 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 245 250 255 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300 His Thr Ala Trp Thr Gln Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 305 310 315 320 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys Pro Lys Gly Arg Ala Gln Thr Pro Gln Val 355 360 365 Tyr Thr Ile Pro Pro Pro Arg Glu Gln Met Ser Lys Lys Lys Val Ser 370 375 380 Leu Thr Cys Leu Val Thr Asn Phe Phe Ser Glu Ala Ile Ser Val Glu 385 390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460 Pro Gly Lys 465 <210> 21 <211> 1401 <212> DNA <213> Artificial sequence <220> <223> Heavy chain DNA sequence of i129v2 (variable + 'improved' v2 constant region) <400> 21 atgctgttgg ggctgaagtg ggttttcttt gttgtttttt atcaaggtgt gcattgtgag 60 gtgcagcttg ttgagtctgg tggaggattg gtgcagccta aagggtcatt gaaactctca 120 tgtgcagcct ctggattcac cttcaatacc tacgccatga actgggtccg ccaggctcca 180 ggaaagggtt tggaatgggt tgctcgcata agaagtaaaa gtaataatta tgcaacatat 240 tatgccgatt cagtgaaaga caggttcacc atatccagag atgattcaca aagcatgctc 300 tatctgcaaa tgaacaactt gaaaaaggag gacacagcca tgtattactg tgtagggtac 360 ggtagtgggg gaaactactg gggtcaagga acctcagtca ccgtctccag cgcttccacc 420 aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 480 gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 540 ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 600 tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 660 aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt 720 gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 780 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 840 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 900 ggcgtggagg tgcatacagc ctggacacag ccccgtgaag agcagtacaa cagtacctac 960 cgagtggtca gtgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 1020 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1080 ggaagagccc gaacacctca agtatacacc atacccccat cccgtgatga gctgtccaag 1140 aagaaggtta gtctgacctg cctggtcaa aacttctatt ctgaagccat cagtgtggag 1200 tgggagagca atgggcagcc ggagacaac tacaagacca cgcctcccgt gctggactcc 1260 gacggctcct tctcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 1320 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactac gcagaagagc 1380 ctctccctgt ctccgggtaa a 1401 <210> 22 <211> 467 <212> PRT <213> Artificial sequence <220> <223> Heavy chain Protein sequence of i129v2 (variable + 'improved' v2 constant region) <400> 22 Met Leu Leu Gly Leu Lys Trp Val Phe Val Phe Tyr Gln Gly 1 5 10 15 Will His Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Lys Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Asn Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ala Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr Tyr 65 70 75 80 Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser 85 90 95 Gln Ser Met Leu Tyr Leu Gln Met Asn Asn Leu Lys Lys Glu Asp Thr 100 105 110 Ala Met Tyr Tyr Cys Val Gly Tyr Gly Ser Gly Gly Asn Tyr Trp Gly 115 120 125 Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 130 135 140 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 145 150 155 160 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 165 170 175 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 180 185 190 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 195 200 205 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 210 215 220 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 225 230 235 240 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 245 250 255 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300 His Thr Ala Trp Thr Gln Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 305 310 315 320 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys Ala Lys Gly Arg Ala Arg Thr Pro Gln Val 355 360 365 Tyr Thr Ile Pro Pro Ser Arg Asp Glu Leu Ser Lys Lys Lys Val Ser 370 375 380 Leu Thr Cys Leu Val Lys Asn Phe Tyr Ser Glu Ala Ile Ser Val Glu 385 390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460 Pro Gly Lys 465 <210> 23 <211> 1401 <212> DNA <213> Artificial sequence <220> <223> Heavy chain DNA sequence of i129v3 (variable + 'improved' v3 constant region) <400> 23 atgctgttgg ggctgaagtg ggttttcttt gttgtttttt atcaaggtgt gcattgtgag 60 gtgcagcttg ttgagtctgg tggaggattg gtgcagccta aagggtcatt gaaactctca 120 tgtgcagcct ctggattcac cttcaatacc tacgccatga actgggtccg ccaggctcca 180 ggaaagggtt tggaatgggt tgctcgcata agaagtaaaa gtaataatta tgcaacatat 240 tatgccgatt cagtgaaaga caggttcacc atatccagag atgattcaca aagcatgctc 300 tatctgcaaa tgaacaactt gaaaaaggag gacacagcca tgtattactg tgtagggtac 360 ggtagtgggg gaaactactg gggtcaagga acctcagtca ccgtctccag cgcttccacc 420 aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 480 gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 540 ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 600 tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 660 aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt 720 gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 780 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 840 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 900 ggcgtggagg tgcataatgc caagacaaag ccccgtgaag agcagtacaa cagtacctac 960 cgagtggtca gtgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 1020 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1080 ggaagagccc gaacacctca agtatacacc ctgcccccat cccgtgatga gctgaccaag 1140 aagaaggtta gtctgacctg cctggtcaaa ggcttctatt ctagcgccat cgccgtggag 1200 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1260 gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 1320 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1380 ctctccctgt ctccgggtaa a 1401 <210> 24 <211> 467 <212> PRT <213> Artificial sequence <220> <223> Heavy chain Protein sequence of i129v3 (variable + 'improved' v3 constant region) <400> 24 Met Leu Leu Gly Leu Lys Trp Val Phe Phe Val Val Phe Tyr Gln Gly 1 5 10 15 Val His Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Lys Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Asn Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ala Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr Tyr 65 70 75 80 Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser 85 90 95 Gln Ser Met Leu Tyr Leu Gln Met Asn Asn Leu Lys Lys Glu Asp Thr 100 105 110 Ala Met Tyr Tyr Cys Val Gly Tyr Gly Ser Gly Gly Asn Tyr Trp Gly 115 120 125 Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 130 135 140 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 145 150 155 160 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 165 170 175 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 180 185 190 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 195 200 205 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 210 215 220 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 225 230 235 240 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 245 250 255 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 305 310 315 320 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys Ala Lys Gly Arg Ala Arg Thr Pro Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Lys Lys Val Ser 370 375 380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Ser Ser Ala Ile Ala Val Glu 385 390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460 Pro Gly Lys 465 <210> 25 <211> 714 <212> DNA <213> Artificial sequence <220> <223> 27 Light chain DNA sequence (variable region + Kappa constant) <400> 25 atgaagttgc ctgttaggct gttggtgctg atgttctgga ttcctgcttc catcagtgat 60 gttttgatga cccaaactcc actctccctg cctgtcagtc ttggagatca agcctccatc 120 tcttgcagat ctagtcagat cattgtacat actaatggaa acacctattt agaatggtac 180 ctgcagaaac caggccagtc tccaaacctc ctgatctaca aagtttccaa ccgattttct 240 ggggtcccag acaggttcag tggcagtgga tcagggacag atttcacact caagatcagc 300 agagtggagg ctgaggatct gggagtttat tactgctttc aaggttcaca tgttccattc 360 acgttcggct cggggacaaa gttggaaata aaacgtacgg tagcggcccc atctgtcttc 420 atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg 480 aataacttct atcccagaga ggccaaagta cagtggaagg tggataacgc cctccaatcg 540 ggtaactccc aggagagtgt cacagagcag gacagcaagg acagcaccta cagcctcagc 600 agcaccctga cgctgagcaa agcagactac gagaaacaca aagtctacgc ctgcgaagtc 660 acccatcagg gcctgagctc gcccgtcaca aagagcttca acaggggaga gtgt 714 <210> 26 <211> 238 <212> PRT <213> Artificial sequence <220> <223> 27 Light chain protein sequence (variable region + Kappa constant) <400> 26 Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Ala 1 5 10 15 Ser Ile Ser Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val 20 25 30 Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ile Ile 35 40 45 Val His Thr Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro 50 55 60 Gly Gln Ser Pro Asn Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser 65 70 75 80 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 85 90 95 Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys 100 105 110 Phe Gln Gly Ser His Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu 115 120 125 Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro 130 135 140 Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu 145 150 155 160 Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn 165 170 175 Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser 180 185 190 Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala 195 200 205 Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly 210 215 220 Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 27 <211> 1404 <212> DNA <213> Artificial sequence <220> <223> Heavy chain DNA sequence of i27v1 (variable + 'improved' v1 constant region) <400> 27 atgaacttct ggctcagctt gattttcctt gtccttgttt taaaaggtgt ccagtgtgaa 60 gtgaagctgg tggagtctgg gggaggctta gtgcagcctg gagggtccct gaaactctcc 120 tgtgcaacct ctggattcac tttcagtcac tattacatgt attgggttcg ccagactcca 180 gagaagaggc tggagtgggt cgcatacatt agtaatgatg gtgataacac ctattatcca 240 gacactataa ggggccgatt caccatctcc agagacaatg ccaggaacac cctgtacctg 300 caaatgagcc gtctgaagtc tgaggacaca gccatgtatt actgtgcaag agggaagtac 360 gacggggcct ggtttgctta ctggggccaa gggactctgg tcactgtctc tagcgcttcc 420 accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc tgggggcaca 480 gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 540 tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 600 tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc 660 tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agaaagttga gcccaaatct 720 tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca 780 gtcttcctct tccccccaaa acccaaggac acctcatga tctcccggac ccctgaggtc 840 acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 900 gacggcgtgg aggtgcatac agcctggaca cagccccgtg aagagcagta caacagtacc 960 taccgagtgg tcagtgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 1020 aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaaccc 1080 aaaggaagag cccagacacc tcaagtatac accatacccc cacctcgtga acaaatgtcc 1140 aagaagaagg ttagtctgac ctgcctggtc accaacttct tctctgaagc catcagtgtg 1200 gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1260 tccgacggct ccttctctcct ctacagcaag ctcaccgtgg acagagcag gtggcagcag 1320 gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaccacta cacgcagaag 1380 agccctctccc tgtctccgggg taaa 1404 <210> 28 <211> 468 <212> PRT <213> Artificial sequence <220> <223> Heavy chain Protein sequence of i27v1 (variable + 'improved' v1 constant region) <400> 28 Met Asn Phe Trp Leu Ser Leu Ile Phe Leu Val Leu Val Leu Lys Gly 1 5 10 15 Val Gln Cys Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe 35 40 45 His Tyr Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg Leu 50 55 60 Glu Trp Val Ala Tyr Ile Ser Asn Asp Gly Asp Asn Thr Tyr Tyr Pro 65 70 75 80 Asp Thr Ile Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn 85 90 95 Thr Leu Tyr Leu Gln Met Ser Arg Leu Lys Ser Glu Asp Thr Ala Met 100 105 110 Tyr Tyr Cys Ala Arg Gly Lys Tyr Asp Gly Ala Trp Phe Ala Tyr Trp 115 120 125 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 130 135 140 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 145 150 155 160 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 165 170 175 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 180 185 190 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 195 200 205 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 210 215 220 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 225 230 235 240 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 245 250 255 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 260 265 270 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 275 280 285 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 290 295 300 Val His Thr Ala Trp Thr Gln Pro Arg Glu Glu Gln Tyr Asn Ser Thr 305 310 315 320 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 325 330 335 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 340 345 350 Ile Glu Lys Thr Ile Ser Lys Pro Lys Gly Arg Ala Gln Thr Pro Gln 355 360 365 Val Tyr Thr Ile Pro Pro Pro Arg Glu Gln Met Ser Lys Lys Lys Val 370 375 380 Ser Leu Thr Cys Leu Val Thr Asn Phe Phe Ser Glu Ala Ile Ser Val 385 390 395 400 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 405 410 415 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 420 425 430 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 435 440 445 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 450 455 460 Ser Pro Gly Lys 465 <210> 29 <211> 214 <212> PRT <213> Artificial sequence <220> <223> Light chain protein sequence of iTrastuzumabv1 and iTrastuzumabv2 <400> 29 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 30 <211> 450 <212> PRT <213> Artificial sequence <220> <223> Heavy chain protein sequence of iTrastuzumabv1 <400> 30 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Thr Ala Trp Thr Gln Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Pro Lys Gly Arg Ala Gln Thr Pro Gln Val Tyr 340 345 350 Thr Ile Pro Pro Pro Arg Glu Gln Met Ser Lys Lys Lys Val Ser Leu 355 360 365 Thr Cys Leu Val Thr Asn Phe Phe Ser Glu Ala Ile Ser Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 31 <211> 450 <212> PRT <213> Artificial sequence <220> <223> Heavy chain protein sequence of iTrastuzumabv2 <400> 31 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Thr Ala Trp Thr Gln Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Arg Ala Arg Thr Pro Gln Val Tyr 340 345 350 Thr Ile Pro Pro Ser Arg Asp Glu Leu Ser Lys Lys Lys Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Asn Phe Tyr Ser Glu Ala Ile Ser Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 32 <211> 93 <212> PRT <213> Homo sapiens <220> <221> misc_feature <223> Human IgG1 sequence 286-278 <400> 32 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 1 5 10 15 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 20 25 30 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 35 40 45 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 50 55 60 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 65 70 75 80 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 85 90 <210> 33 <211> 93 <212> PRT <213> Mus musculus <220> <221> misc_feature <223> Mouse IgG3 sequence 286-378 <400> 33 Thr Ala Trp Thr Gln Pro Arg Glu Ala Gln Tyr Asn Ser Thr Phe Arg 1 5 10 15 Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Arg Gly Lys 20 25 30 Glu Phe Lys Cys Lys Val Asn Asn Lys Ala Leu Pro Ala Pro Ile Glu 35 40 45 Arg Thr Ile Ser Lys Pro Lys Gly Arg Ala Gln Thr Pro Gln Val Tyr 50 55 60 Thr Ile Pro Pro Pro Arg Glu Gln Met Ser Lys Lys Lys Val Ser Leu 65 70 75 80 Thr Cys Leu Val Thr Asn Phe Phe Ser Glu Ala Ile Ser 85 90 <210> 34 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Subdomain 286-306 <400> 34 Phe Arg Val Val Ser Ala Leu Thr Val 1 5 <210> 35 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Subdomain 286-306 <400> 35 Thr Phe Arg Val Val Ser Ala Leu Thr 1 5 <210> 36 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Subdomain 286-306 <400> 36 Val Val Ser Ala Leu Thr Val Leu His 1 5 <210> 37 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Subdomain 286-306 <400> 37 Leu Val Leu His Gln Asp Trp Leu Asn 1 5 <210> 38 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Subdomain 286-306 <400> 38 Tyr Arg Val Val Ser Val Leu Thr Val 1 5 <210> 39 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Subdomain 286-306 <400> 39 Ala Gln Tyr Asn Ser Thr Phe Arg Val 1 5 <210> 40 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Subdomain 286-306 <400> 40 Glu Gln Tyr Asn Ser Thr Tyr Arg Val 1 5 <210> 41 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Subdomain 339-378 <400> 41 Leu Thr Cys Leu Val Thr Asn Phe Phe 1 5 <210> 42 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Subdomain 339-378 <400> 42 Leu Thr Cys Leu Val Thr Gly Phe Phe 1 5 <210> 43 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Subdomain 339-378 <400> 43 Val Tyr Thr Ile Pro Pro Pro Arg Glu 1 5 <210> 44 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Subdomain 339-378 <400> 44 Val Tyr Thr Leu Pro Pro Pro Arg Glu 1 5

Claims

1. A modified IgG1 antibody or antigen-binding fragment thereof comprising residues of an immunoglobulin Fc-region and a binding region, wherein residues comprising N286, K288, K290, Q342, P343, E345, L351, T359, N361, Q362, G371, P374, S375, D376 and A378 of the Fc-region are modified to the corresponding residues from a mouse IgG3 antibody and wherein the modified IgG1 antibody or antigen-binding fragment thereof has an enhanced functional affinity compared to a corresponding IgG1 antibody or antigen-binding fragment thereof comprising the wild-type Fc-region residues.

2. The modified IgG1 antibody or antigen-binding fragment thereof according to claim 1, characterized in that The functional affinity of the modified IgG1 antibody or antigen-binding fragment thereof is enhanced by at least about 10% compared to a corresponding IgG1 antibody or antigen-binding fragment thereof comprising wild-type Fc-region residues.

3. The modified IgG1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, characterized in that The modified IgG1 antibody or antigen-binding fragment thereof enhances direct cell killing compared to a corresponding IgG1 antibody or antigen-binding fragment thereof comprising wild-type Fc-region residues.

4. The modified IgG1 antibody or antigen-binding fragment thereof according to claim 3, characterized in that: The modified IgG1 antibody or antigen-binding fragment thereof has at least about 10% enhanced direct cell killing compared to a corresponding IgG1 antibody or antigen-binding fragment thereof comprising wild-type Fc-region residues.

5. The modified IgG1 antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The modified residues in the Fc-region are selected from the group consisting of N286T, K288W, K290Q, Q342R, P343A, E345T, L351I, T359S, N361K, Q362K, G371N, P374S, S375E, D376A, A378S.

6. The modified IgG1 antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The residues comprising N286, K288, K290, A339, Q342, P343, R344, E345, L351, S354, D356, E357, L358, T359, N361, Q362, K370, G371, Y373, P374, S375, D376, and A378 in the Fc-region were modified to the corresponding residues of mouse IgG3 antibody.

7. The modified IgG1 antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The modified residues include: N286T, K288W, K290Q, A339P, Q342R, P343A, R344Q, E345T, L351I, S354P, D356E, E357Q, L358M, T359S, N361K, Q362K, K370T, G371N, Y373F, P374S, S375E, D376A, A378S.

8. The modified IgG1 antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The residues comprising N286, K288, K290, E294, Y300, V305, A339, Q342, P343, R344, E345, L351, S354, D356, E357, L358, T359, N361, Q362, K370, G371, Y373, P374, S375, D376, and A378 of the Fc-region were modified to the corresponding residues of mouse IgG3 antibody.

9. The modified IgG1 antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that The modified residues include: N286T, K288W, K290Q, E294A, Y300F, V305A, A339P, Q342R, P343A, R344Q, E345T, L351I, S354P, D356E, E357Q, L358M, T359S, N361K, Q362K, K370T, G371N, Y373F, P374S, S375E, D376A, A378S.

10. A method for increasing the direct cell killing ability of an IgG1 antibody or an antigen-binding fragment thereof, wherein the IgG1 antibody or the antigen-binding fragment thereof comprises residues of an immunoglobulin Fc-region and a binding region, characterized in that: The method comprises replacing residues of the CH2 and / or CH3 domain of the Fc-region with corresponding residues from mouse IgG3 CH2 and / or CH3 domain, and wherein the substituted residues include N286, K288, K290, Q342, P343, E345, L351, T359, N361, Q362, G371, P374, S375, D376, A378.

11. The method of claim 10, wherein the modified residues comprise: N286T, K288W, K290Q, Q342R, P343A, E345T, L351I, T359S, N361K, Q362K, G371N, P374S, S375E, D376A, A378S.

12. The method of claim 10 or 11, wherein the substituted residues include N286, K288, K290, A339, Q342, P343, R344, E345, L351, S354, D356, E357, L358, T359, N361, Q362, K370, G371, Y373, P374, S375, D376, A378.

13. The method of claim 12, wherein the modified residues comprise: N286T, K288W, K290Q, A339P, Q342R, P343A, R344Q, E345T, L351I, S354P, D356E, E35 7Q, L358M, T359S, N361K, Q362K, K370T, G371N, Y373F, P374S, S375E, D376A, A378S.

14. The method of claim 10 or 11, wherein the substituted residues include N286, K288, K290, E294, Y300, V305, A339, Q342, P343, R344, E345, L351, S354, D356, E357, L358, T359, N361, Q362, K370, G371, Y373, P374, S375, D376, A378.

15. The method of claim 14, wherein the modified residues comprise: N286T, K288W, K290Q, E294A, Y300F, V305A, A339P, Q342R, P343A, R344Q, E345T, L351I, S354P, D356E, E357Q, L358M, T359S, N361K, Q362K, K370T, G371N, Y373F, P374S, S375E, D376A, A378S. 16 . A pharmaceutical composition comprising the modified IgG1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier.

17. The pharmaceutical composition of claim 16, further comprising at least one other therapeutic agent, wherein the at least one other therapeutic agent is an immunomodulator.

18. A kit comprising the modified IgG1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 or the composition according to claim 16 or 17, characterized in that: The modified IgG1 antibody or antigen-binding fragment thereof or composition is located in one or more containers.

19. Use of the modified IgG1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 or the composition according to claim 16 or 17 for the manufacture of a medicament for treating a disease.

20. The use according to claim 19, characterized in that The disease is cancer, an autoimmune disease, an inflammatory disease or an infectious disease.

21. The use according to claim 19, characterized in that The medicament also includes an additional therapeutic agent, which is an immunomodulator.

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