Improved bispecific antitumor antigen / anti-HSG antibodies for pretargeting hyperproliferative disorders

Abstract

The present invention relates to bispecific anti-tumor antigen / anti-HSG antibodies with selected light and heavy chain variable domains that enable improved tumor pretargeting for specific delivery of therapeutic or diagnostic agents. The antibodies have significant affinity for tumor antigens and sufficient residence time at the desired location. Antibodies that do not bind to the antigen are rapidly cleared from the body, minimizing exposure of normal tissues. Humanized anti-HSG antibodies bind with high affinity to both histamine-succinyl-glycyl (HSG)-containing moieties and tumor antigens, respectively. Bispecific antibodies can also be Fc-silenced for additional properties, such as reduced binding to FcγR and FcRn, modulating effector function and half-life. The antibodies can be used for the diagnosis and treatment of subjects with malignant tumors.

Description

[Technical Field]

[0001] The present invention relates to bispecific anti-tumor antigen / anti-HSG antibodies with selected light and heavy chain variable domains that enable improved pretargeting of malignant tumors, particularly tumors, more particularly solid tumors. The humanized anti-HSG (histamine-succinyl-glycyl) binding site binds with high affinity to molecules containing the histamine-succinyl-glycyl (HSG) moiety. Methods for producing bispecific anti-tumor antigen / anti-HSG antibodies containing humanized anti-HSG variable domains are also disclosed. Bispecific antibodies can further possess properties such as reduced aggregation potential and reduced hydrophobicity through selected amino acid substitutions within the variable domains. Anti-tumor antigen / anti-HSG antibodies can be used for the diagnosis and treatment of subjects suffering from cancer. [Background technology]

[0002] Tumor targeting with monoclonal antibodies is an attractive approach for selective cancer treatment. Radiolabeled antibodies have proven effective in patients with hematological malignancies, but applying radioimmunotherapy (RIT) to solid tumors has been challenging. The slow blood clearance and delayed tumor uptake of directly radiolabeled antibodies result in continuous radiation exposure of normal organs and bone marrow, resulting in high background signals when used as a diagnostic tool. Pretargeting methods have been developed to overcome these limitations. In pretargeting, a non-radiolabeled humanized bispecific monoclonal antibody (bsMAb) is first administered intravenously. After the bsMAb localizes within the tumor and clears from the circulation, a radiolabeled hapten peptide is administered. The peptide is rapidly captured within the tumor by the bsMAb, while the remainder is rapidly cleared from the blood and excreted via the kidneys (Stawski RS et al., 2015). Pretargeting reduces radiation exposure of radiosensitive normal tissues, such as bone marrow, and other tissues (Stawski RS et al., 2015).

[0003] The tri-Fab bispecific monoclonal antibody TF2, bivalent against carcinoembryonic antigen 5 (CEACAM-5, or CEA) and monovalent against the histamine-succinyl-glycine (HSG) hapten peptide, was described in Schoffelen R. et al., 2013, and Rossi EA et al., 2006. TF2 binds to the tumor-associated antigen CEACAM-5, which is expressed on the cell surface of colon tumors. The radionuclide-bearing HSG hapten peptide IMP288 is then administered, which binds to the anti-HSG arm of the bispecific mAb (Schoffelen R. et al., 2013). The radionuclide ( 111 In, or 177 The phase I study NCT00860860, in which CEACAM-5-expressing tumor cells were radioimmunized and / or treated with radioimmunotherapy depending on their characteristics, demonstrated for the first time that pretargeting with TF2 and radiolabeled IMP288 in patients with CEACAM-5-expressing colorectal cancer is feasible and safe. This pretargeting method specifically and rapidly targets tumors (Schoffelen R. et al., 2013).

[0004] The anti-HSG mAb sequence of TF2 is derived from humanized anti-HSG mAb clone 679, originally identified from a mouse hybridoma (Morel A et al., 1990). Mouse anti-HSG mAb 679 and humanized mAb 679 are described in US20090246131 and US20090240037A1, respectively. In US2009 / 0240037A1, humanized mAb 679 was produced and tested in a single-chain variable fragment (scFv) format as an anti-CEACAM-5 x anti-HSG diabody with a C-terminal 6xHis tag. However, humanized HSG mAbs in the art still have some disadvantages. Although the mAb still closely resembles the mouse amino acid sequence, this results in undesirable immunogenicity of the antibody. Furthermore, the production of Tri-Fab (anti-tumor antigen x anti-HSG) antibodies requires the use of a specialized technique known as the dock-and-lock (DNL) method. DNL requires the use of a pair of distinct protein domains involved in the natural association between cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) and A-kinase anchoring proteins (AKAPs). The dimerization and docking domains found in the regulatory subunit of PKA and the anchoring domain (AD) of the interacting AKAP are each conjugated to a biological entity, and the resulting derivatives readily form stably tethered complexes of defined composition when combined. The attachment of an additional non-antibody subunit to the Fab arm further increases the risk of immunogenicity of the antibody. Furthermore, large-scale production of DNL Tri-Fabs is considered extremely challenging.

[0005] The cytokine macrophage migration inhibitory factor (MIF) was described as early as 1966 (David, JR, 1966; Bloom BR and Bennett, B., 1966). The biochemical properties and physiological roles of MIF were elucidated after its cloning and recombinant expression (Bernhagen et al., 1993; Bernhagen et al., 1994). It is now widely accepted that MIF is a central regulator of innate immunity, playing a central role in inflammatory responses and cancer.

[0006] MIF has been shown to be upregulated in a wide variety of human tumors, including those of pancreatic, breast, prostate, colon, brain, skin, and lung origin (Bando et al., 2002; Chen et al., 2010; Kamimura et al., 2000; Meyer-Siegler and Hudson, 1996; Shimizu et al., 1999; Takahashi et al., 1998; Winner et al., 2007). Several studies have reported that MIF expression is closely correlated with tumor aggressiveness and metastatic potential, suggesting that MIF may play an important role in the extent of disease and cell survival (Rendon et al., 2009). Recent data suggest that extracellular MIF may contribute to a more aggressive tumor phenotype compared with intracellular MIF (Verjans et al., 2009). MIF contributes to a microenvironment that supports tumor growth, angiogenesis, invasiveness, and metastasis. In addition to its pro-inflammatory function, MIF exerts anti-apoptotic and pro-inflammatory effects, including inhibition of p53 (Hudson et al., 1999; Mitchell and Bucala, 2000), activation of the central kinases ERK1 / 2 (Mitchell et al., 1999), and AKT (Lue et al., 2007). MIF has been further described as a pro-angiogenic factor that promotes angiogenesis (Coleman et al., 2008), as well as tumor angiogenesis through stabilization of HIF-1α (Winner et al., 2007), and upregulation of pro-angiogenic factors such as VEGF and IL-8 (Ren et al., 2004). MIF also acts as a chemokine and is expected to contribute to inflammatory cell recruitment within the tumor environment via the chemokine receptors CXCR2 and CXCR4 (Bernhagen et al., 2007; Rendon et al., 2007).

[0007] However, MIF is significantly different from other cytokines and chemokines because it is constitutively expressed and present in the circulation of healthy subjects: preformed MIF is stored in cytoplasmic pools in macrophages, T cells, and many other cells in the body, including the hypothalamic-pituitary-adrenal axis, allowing for rapid release upon stimulation without de novo synthesis (Bernhagen et al., 1993; Bacher et al., 1997; Fingerle-Rowson et al., 2003).

[0008] Due to the ubiquitous nature of this protein, MIF may be considered an inappropriate target for therapeutic intervention. However, MIF occurs in two immunologically distinct structural isoforms, termed reduced MIF (redMIF) and oxidized MIF (oxMIF) (Thiele M. et al., 2015). redMIF was found to be an abundantly expressed isoform of MIF that can be found in the cytoplasm and circulation of all subjects. redMIF appears to be a latent, inactive storage form (Schinagl A. et al., 2018).

[0009] In contrast, oxMIF appears to be a physiologically relevant disease-associated isoform, as it can be detected in tumor tissues, specifically from patients with colorectal, pancreatic, ovarian, and lung cancer (Schinagl et al., 2016), but also in the circulation and inflamed tissues of patients with inflammatory diseases (Thiele et al., 2015), demonstrating the high tumor specificity of oxMIF.

[0010] Antibodies targeting oxMIF have shown efficacy in in vitro and in vivo models of inflammation and cancer (Hussain F. et al., 2013; Schinagl. A. et al., 2016; Thiele et al., 2015).

[0011] WO2019 / 234241A1 discloses an anti-oxMIF / anti-CD3 bispecific antibody. WO2009 / 086920A1 describes the anti-oxMIF antibody Bax69 (imalumab).

[0012] WO2022069712A1 describes anti-oxMIF antibodies with reduced aggregation ability and reduced hydrophobicity. Protein aggregation, specifically antibody aggregation, is frequently observed during several stages of bioprocessing, including protein expression, purification, and storage. Antibody aggregation can affect the overall yield of therapeutic protein manufacturing processes and can contribute to the stability and immunogenicity of therapeutic antibodies.

[0013] Protein aggregation of antibodies therefore remains a significant issue in their exploitability and remains a major focus area for antibody production. Antibody aggregation can be triggered by partial unfolding of their domains, leading to monomer-monomer association and subsequent nucleation and growth of aggregates. The aggregation tendency of antibodies and antibody-based proteins can be influenced by external experimental conditions, but is strongly dependent on intrinsic antibody properties determined by their sequence and structure.

[0014] For example, resistance to aggregation can be achieved by stabilizing the native state (i.e., resisting unfolding) or by reducing the aggregation tendency of the unfolded or partially folded state of the protein. The disadvantage of stabilizing the native state is that the protein is more likely to be exposed to an environment that unfolds it. Generally, when a protein denatures or unfolds, amino acid residues that normally mediate intramolecular contacts within the protein are exposed. Such exposure often makes the protein more susceptible to forming intermolecular contacts and aggregation. In contrast to proteins that resist unfolding, proteins with reduced aggregation tendency, when unfolded, only refold to a biologically active, non-aggregated state after exposure to such an environment.

[0015] The aggregation resistance or aggregation tendency of proteins, including antibodies and their antigen-binding domains, is usually limited by the most aggregation-prone domain contained therein and by the strength of its interactions with surrounding domains, if any.

[0016] The reason for this is that when a domain unfolds, if it cannot refold, it may interact with other domains in the same protein or other proteins and form aggregates. The constant domains of antibodies generally do not aggregate and do not change significantly. Therefore, the weakest domains of antibodies in terms of aggregation potential and stability are generally the variable domains (e.g., heavy chain variable domains (V H ) and / or a light chain variable domain (V L ), Ewert S. et al., 2003). In this regard, aggregation-prone V H or V L Incorporating domains into otherwise stable recombinant antibody products often imparts these generally undesirable traits to the new recombinant design. Therefore, modifying a variable domain to be aggregation-resistant will most likely render the entire protein, including that variable domain, aggregation-resistant. Various strategies have been proposed to reduce variable domain aggregation (e.g., rational design of aggregation-resistant proteins, complementarity-determining region (CDR) grafting, or introduction of disulfide bonds into variable domains). Rational design of aggregation-resistant proteins generally requires the use of in silico analysis to predict the impact of point mutations on the aggregation propensity of a protein. However, purely in silico prediction does not necessarily result in actual improvement in this regard.

[0017] A reduction in aggregation tendency has been shown to be accompanied by an increase in expressed titer. This indicates that reducing protein aggregation can be beneficial throughout the development process and may lead to a more efficient route to clinical trials. For therapeutic proteins, aggregates are a significant risk factor for adverse immune responses in patients and can form through a variety of mechanisms. Controlling aggregation can improve protein stability, manufacturability, failure rates, safety, formulation, potency, immunogenicity, and solubility (Wei Li et al., 2016; Van der Kant R. et al., 2017).

[0018] There is also growing interest in modulating antibody effector functions to serve specific therapeutic purposes. Specifically, because potent immune effector functions through FcγR and complement interactions can sometimes be detrimental to antibody mechanisms, Fc-null or Fc-silencing antibodies could be a strategy to suppress Fc effector functions.

[0019] WO2023 / 031397A1 describes Fc-silencing anti-oxMIF antibodies containing amino acid substitutions in the light and heavy chain variable domains. Schinagl et al. (published April 1, 2023) report pretargeting immunotherapy with anti-oxMIF / HSG bispecific antibodies.

[0020] Fc gamma receptors (FcγRs) are a well-described family of proteins that include membrane-bound surface receptors, heterotypic intracellular receptors, and cytoplasmic glycoproteins. FcγRs regulate humoral and innate immunity and are essential for the appropriate response to infection and the prevention of chronic inflammatory or autoimmune diseases. Membrane-bound receptors include FcγR11a, FcγR11b, FcγR111a, and FcγRIa receptors. Antibodies can regulate immune responses through interactions with FcγRs.

[0021] In innate immune effector cells, activating and inhibitory FcγRs establish a threshold for cell activation by immune complexes. Important examples of effector responses regulated by FcγRs are phagocytosis, ADCC, and the release of inflammatory mediators. In dendritic cells (DCs), paired FcγR expression regulates cell maturation and antigen presentation, thereby indirectly controlling cellular immune responses. In B cells, inhibitory FcγRIIB is essential for maintaining humoral tolerance. Inhibitory FcγRIIB acts as a late checkpoint at the level of class-switched memory B cells, plasmablasts, or plasma cells. Furthermore, FcγRIIB plays an important role in regulating plasma cell homeostasis and survival. Antibody-FcγR interactions are influenced by several factors that affect the expression levels of activating and inhibitory FcγRs (e.g., cytokines) or alter the affinity of antibody-FcγR interactions (e.g., differences in antibody glycosylation). Depending on the particular glycosylation pattern, IgG molecules can have enhanced pro- or anti-inflammatory activity. Importantly, antibody glycosylation is regulated during the immune response.

[0022] Atypical FcγRs are the neonatal Fc receptor (FcRn) and cytoplasmic glycoproteins, such as complement factor C1q. FcRn is expressed by endothelial cells and internalizes serum components, including soluble IgG, from the bloodstream via pinocytosis. IgG binding to FcRn is pH-dependent; the acidic pH (pH 6.0) inside the endosomal compartment allows IgG to bind to FcRn. After recycling to the cell surface, IgG dissociates from FcRn at physiological pH (approximately pH 7.2) and is released back into the circulation, thereby protecting it from lysosomal degradation and extending its half-life. Thus, FcRn functions as a recycling transcytosis receptor responsible for maintaining IgG and albumin in the circulation. Modifications of the Fc region that result in the reduction or silencing of FcRn binding are known in the art and are described, for example, in Kenanova V. et al., 2005 and Pyzik M. et al., 2019.

[0023] Similarly, modulating neonatal Fc receptor (FcRn) binding of IgG antibodies to modulate pharmacokinetics is gaining increasing popularity. In particular, the above has made the comprehensive adaptation of antibodies to each application field a challenge and remains a challenge.Therefore, there remains a need in the art for improved bispecific antibodies, particularly for use in more effective targeted delivery methods of therapeutic and diagnostic agents, particularly in pretargeting. Summary of the Invention [Problem to be solved by the invention]

[0024] It is an object of the present invention to provide improved bispecific antibodies for tumor pretargeting and subsequent specific delivery of therapeutic or diagnostic agents. [Means for solving the problem]

[0025] Said object is solved by the subject matter of the present invention. The antibodies of the present invention selectively bind to tumor antigens and HSG hapten moieties. They have significant affinity for tumor antigens and sufficient residence time at the desired tumor location. Antibodies that do not bind to tumor antigens are cleared from the circulation, minimizing exposure of normal tissues to subsequently administered therapeutic or diagnostic agents.

[0026] Provided herein is a bispecific anti-tumor antigen / anti-HSG antibody comprising a binding site that specifically recognizes a tumor antigen and a binding site that specifically recognizes HSG, - a light chain variable (VL) domain comprising a sequence selected from the group consisting of SEQ ID NOs: 17, 19, 89, 90, and 91, or - a light chain variable (VL) domain comprising a sequence selected from the group consisting of SEQ ID NOs: 17, 19, 89, 90, and 91, which has one or two additional amino acid substitutions and has a glutamine at position 100 (Q100), a glutamic acid at position 105 (E105), and an isoleucine at position 106 (I106) according to the Kabat numbering, and - a heavy chain variable (VH) domain comprising a sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, and 10, or - a heavy chain variable (VH) domain comprising a sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, and 10, with one or two additional amino acid substitutions and with an arginine at position 19 (R19) according to the Kabat numbering; and a bispecific anti-tumor antigen / anti-HSG antibody comprising:

[0027] The bispecific anti-tumor antigen / anti-HSG antibodies of the present invention have extremely low immunogenicity, particularly due to modifications in the anti-HSG VL and VH domains. The antibodies of the present invention also have increased expression titers, high monomer purity, and reduced shedding of the anti-HSG heavy chain, making them highly suitable for manufacturing.

[0028] The antibodies of the present invention have highly selective binding specificity for tumor antigens and HSG haptens and exhibit high storage stability. Targeted antibody modification can reduce FcRn binding, thereby optimizing the half-life of the antibodies of the present invention, particularly when used in pretargeting.

[0029] According to a particular embodiment, the antibodies of the invention are in the Fab-scFv-Fc format. According to certain embodiments of the invention, the antibodies described herein comprise a single-chain variable fragment (scFv) that specifically recognizes HSG and has a formula selected from the group consisting of VH-linker-VL-linker, VL-linker-VH-linker, wherein the linker comprises SEQ ID NO: 39 or SEQ ID NO: 42.

[0030] According to an alternative specific embodiment, the antibody has a CrossMab (CH1-CL crossover) format, ie a Fab with a CH1-CL domain crossover.

[0031] Specifically, the antibody of the invention comprises VL and VH domains that specifically recognize HSG, wherein the VL domain is linked at its C-terminus to a CH1-crosslink comprising SEQ ID NO: 30, and the VH domain is linked at its C-terminus to a CL-crosslink comprising SEQ ID NO: 38.

[0032] In a further embodiment, the antibodies described herein comprise an Fc region comprising SEQ ID NO: 31, or an Fc region comprising a sequence having at least 95%, particularly 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 31, comprising asymmetric mutations in each CH3 domain that allow heterodimerization of two CHs from different antibodies, wherein one CH comprises "knob" mutations T366W and S354C according to the EU numbering index, and one CH comprises "hole" mutations T366S, L368A, Y407V, and Y349C.

[0033] In particular, the antibodies of the invention comprise variant Fc regions with reduced or eliminated effector function and / or FcRn binding. Specifically, the antibodies of the invention have a variant Fc region with an amino acid substitution at any one or more of positions E233, L234, L235, G236, G237, P238, I253, D265, S267, H268, N297, S298, T299, H310, E318, L328, P329, A330, P331, H435 of SEQ ID NO: 31 according to the EU numbering index, and optionally an aglycosylated Fc region.

[0034] Specifically, the antibodies of the present invention contain two constant heavy chain domains having hinge and Fc regions comprising SEQ ID NO:35 and SEQ ID NO:32. In an alternative embodiment, an antibody of the invention contains two constant heavy chain regions having hinge and Fc domains comprising SEQ ID NO:36 and one of SEQ ID NO:33 or SEQ ID NO:34.

[0035] In further embodiments of the invention, the anti-tumor antigen binding of the antibodies described herein is directed to oxMIF, mesothelin (MSLN), and folate receptor alpha (FRα), and specifically, the antibodies bind to oxMIF.

[0036] In a particular embodiment of the present invention, the binding site that specifically recognizes oxMIF is - a light chain variable domain comprising SEQ ID NO: 27 or a light chain variable domain comprising SEQ ID NO: 27 further comprising the amino acid substitutions M30L and / or P80S, and - a heavy chain variable domain comprising SEQ ID NO: 46, specifically with the amino acid substitutions L5Q and / or W97Y where amino acid positions are numbered according to Kabat.

[0037] According to certain embodiments, the antibodies described herein are selected from the group consisting of Fab-scFv-Fc, CrossMab, (scFv)2-Fc, scFv / scFv-Fc, Fab / (scFv)2-Fc, Fab / Fab-scFv-Fc (IgG-central scFv), Fab / Fab-crossFab-Fc, Fab / crossFab-Fc, IgG-scFv, IgG-(scFv)2.

[0038] In a further embodiment, the antibody is for use in treating or detecting malignant tumors, and said antibody is administered to a subject in a first step and an HSG moiety is administered in a second step, said HSG moiety binding to the antibody.

[0039] More specifically, the HSG moiety is conjugated to or labeled with one or more diagnostic and / or therapeutic agents, and even more specifically, the HSG moiety comprises one or more HSG haptens, one or more diagnostic and / or therapeutic agents, and a chelator.

[0040] In further embodiments, the antibodies described herein bind to an HSG moiety conjugated to one or more diagnostic and / or therapeutic agents or labeled with one or more diagnostic and / or therapeutic agents, specifically the HSG moiety comprises one or more HSG haptens, one or more diagnostic and / or therapeutic agents, and a chelator.

[0041] Specifically, the chelator conjugated to the HSG moiety binds to a radionuclide, specifically DOTA, DTPA, deferoxamine B (DFO), and DFO. * is selected from the group consisting of:

[0042] Specifically, the radionuclides are: 67 Ga, 89 Zr, 111 In, 124 I, 131 I, 177 Lu, and 225 Ac. In a further embodiment of the invention, the therapeutic agent is a radionuclide (radioisotope) or a cytotoxic agent and the diagnostic agent is a radionuclide.

[0043] Further provided herein is an antibody for use in the preparation of a medicament. Also provided herein is a pharmaceutical composition comprising the antibody together with a pharmaceutical excipient. Specifically, the pharmaceutical compositions provided herein are formulated for intravenous administration.

[0044] In a further embodiment of the invention, the pharmaceutical composition is for use in the treatment of patients suffering from cancer, in particular in the treatment of tumors, solid tumors, more particularly in the treatment of colorectal, ovarian, breast, prostate, pancreatic, and lung cancer.

[0045] Also provided herein are isolated nucleic acids encoding the antibodies described herein. Also provided herein are expression vectors containing the nucleic acids.

[0046] In a further embodiment of the invention, provided herein is a method for diagnosing cancer in a subject in vivo, wherein the antibodies described herein are used to detect tumor cells.

[0047] In a further embodiment of the present invention, there is also provided herein a method for diagnosing cancer in vitro, wherein the antibodies described herein are used to detect tumor cells in a sample. Also provided herein are methods of treating cancer using the antibodies described herein or the pharmaceutical compositions described herein. [Brief explanation of the drawings]

[0048] [Figure 1] Figure 1 is a schematic diagram of anti-target X × anti-HSG bispecific mAb with Fab-scFv-Fc, CrossMab (CH1-CL crossover), and IgG-central scFv formats. Left: Fab-scFv-Fc, center: CrossMab (CH1-CL crossover), right: IgG-central scFv. [Figure 2]Figure 2 shows the evaluation of the purity of mAbs C0132 and C0176-C0180 and the extent of cleavage of the anti-HSG scFv-Fc heavy chain by SDS-PAGE and Coomassie staining. A total of 3 μg of Protein A-purified mAb was separated by NuPAGE™ 4-12% SDS-PAGE under reducing conditions ("red"). Spectra Multicolor Broad Range Protein Ladder was used as a standard. Arrows indicate the anti-oxMIF LC and cleaved anti-HSG heavy chain. [Figure 3] Figure 3 shows the deconvoluted mass spectrum of C0132 mAb. The two major peaks correspond to the intact antibody (126882.3 Da) and the anti-HSG scFv-Fc heavy chain truncated antibody (100701.9 Da). [Figure 4A] Figure 4 shows the stability of the newly humanized anti-oxMIF x anti-HSG bispecific Fab-scFv-Fc mAb compared to the previously humanized C0132 mAb containing the anti-HSG sequence. Stability was assessed by SEC before (day 0) and after 83 days of storage at -80°C (A) or 4°C (B). % monomer values ​​for each antibody were normalized to day 0 (=100%) to allow direct comparison of samples. [Figure 4B] Same as above. [Figure 5]Figure 5 shows the binding of bispecific anti-oxMIF x anti-HSG mAbs to immobilized HSG. OD values ​​at 450 nm (mean ± SEM, n = 3) were plotted against mAb concentration, and curve fitting was performed by a four-parameter logistic fit using GraphPad Prism. (A) Binding curves of Fab-scFv-Fc BsMAbs C0132, C0176, C0181, C0182, C0186, and C0192 to HSG; (B) EC50 values ​​of A (mean ± SEM, n = 3); (C) EC50 values ​​for binding of CrossMAbs (C0255, C0238, C0239, C0240, C0241, C0245, and C0250) to HSG (mean ± SEM, n = 3). The dotted line represents the EC50 value of the reference bispecific anti-oxMIF x anti-HSG antibody. [Figure 6] Figure 6 shows the maintenance of binding of anti-oxMIF × anti-HSG bispecific mAbs to immobilized MIF (oxMIF). Anti-oxMIF × anti-HSG Fab-scFv-Fc mAb (A, B) and anti-CrossMab (C) bound to immobilized oxMIF and were detected with goat anti-human IgG (Fc-specific)-HRP conjugate. Binding curves (A) and EC50 values ​​(mean ± SEM, n = 2–3; B–C) of the Fab-scFv-Fc bsAb are shown. C0008 (imalumab) was used as a reference anti-oxMIF mAb. [Figure 7] Figure 7 shows the evaluation of off-target binding of anti-oxMIF × anti-HSG bsMAbs to A2780 MIF knockout cells. A2780 MIF- / - cells were stained with serial dilutions of anti-oxMIF × anti-HSG bispecific mAbs C0176, C0181, C0182, C0186, and C0192, and the control therapeutic mAb rituximab. Binding was detected in live cells using an AF488-conjugated goat anti-human IgG (H+L) secondary antibody. Geometric mean fluorescence intensity (MFI) values ​​in the AF488 channel were plotted against mAb concentration in GraphPad Prism. The dotted line represents staining (MFI) with the secondary antibody alone. [Figure 8]Figure 8 shows the thermal stability of newly humanized anti-oxMIF x anti-HSG crossMAbs. The thermal stability of newly humanized anti-oxMIF x anti-HSG crossMAbs C0238, C0239, C0240, C0241, C0245, and C0250, as well as crossMAb C0255, which has the sequence of previously humanized anti-HSG Ab 679, was assessed by nanoscale differential scanning fluorimetry (nanoDSF). The monitored emission intensity ratio (350 nm / 330 nm) was plotted as a function of temperature, and its first derivative was calculated to determine the inflection point temperature of the first unfolding transition (TIP, °C, mean ± SD, n = 4). DETAILED DESCRIPTION OF THE INVENTION

[0049] Unless otherwise indicated or defined, all terms used herein have their ordinary meaning in the art and will be clear to those skilled in the art.For example, refer to standard handbooks such as Sambrook et al., "Molecular Cloning: A Laboratory Manual" (4th edition), vol. 1-3, Cold Spring Harbor Laboratory Press (2012); Krebs et al., "Lewin's Genes XI", Jones and Bartlett Learning (2017), and Murphy and Weaver, "Janeway's Immunobiology" (9th edition, or more recent edition), Taylor and Francis Inc., 2017.

[0050] The subject matter of the claims specifically refers to artificial products or methods of using or producing such artificial products, which may be variants of naturally occurring (wild-type) products. While there may be some sequence identity with naturally occurring structures, it is well understood that the materials, methods, and uses of the invention, which specifically refer to, for example, isolated nucleic acid sequences, amino acid sequences, fusion constructs, expression constructs, transformed host cells, and modified proteins, are "artificial" or synthetic and therefore not considered the result of the "laws of nature."

[0051] The terms "comprise," "contain," "have," and "include," as used herein, can be used interchangeably and are to be understood as open definitions that allow for additional members, parts, or elements. "Consisting of" is considered the closest definition, with no additional elements characteristic of the definition that it consists of. Thus, "comprising" is broader and includes the "consisting of" definition.

[0052] The term "about," as used herein, refers to a value that is the same as a given value or that differs by + / - 5% from the given value. As used in this specification and claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0053] As used herein, amino acid refers to the 20 naturally occurring amino acids encoded by 61 triplet codons. These 20 amino acids can be classified as neutrally, positively, and negatively charged: The "neutral" amino acids are shown below with their respective three-letter and one-letter codes and polarities: alanine (Ala, A; non-polar, neutral), asparagine (Asn, N; polar, neutral), cysteine ​​(Cys, C; non-polar, neutral), glutamine (Gln, Q; polar, neutral), glycine (Gly, G; non-polar, neutral), isoleucine (Ile, I; non-polar, neutral), leucine (Leu, L; non-polar, neutral), methyl (methyl-, ... Thionine (Met, M; non-polar, neutral), phenylalanine (Phe, F; non-polar, neutral), proline (Pro, P; non-polar, neutral), serine (Ser, S; polar, neutral), threonine (Thr, T; polar, neutral), tryptophan (Trp, W; non-polar, neutral), tyrosine (Tyr, Y; polar, neutral), valine (Val, V; non-polar, neutral), and histidine (His, H; polar, positive (10%) and neutral (90%).

[0054] The "positively" charged amino acids are: arginine (Arg, R; polar, positive), and lysine (Lys, K; polar, positive). The "negatively" charged amino acids are: aspartic acid (Asp, D; polar, negative), and glutamic acid (Glu, E; polar, negative).

[0055] The bispecific antibody of the present invention comprises at least one binding site that specifically recognizes oxMIF and one binding site that specifically recognizes the HSG (histamine-succinyl-glycyl) hapten, and has particularly high affinity for HSG and oxMIF.

[0056] The VL and VH domains of the HSG binding sites of the bispecific anti-oxMIF / anti-HSG antibodies described herein are extensively modified compared to known anti-HSG variable domains. While the murine anti-HSG mAb 679 (mo679) and humanized mAb 679 (hz679) are described in US20090240037A1 and US20090246131, respectively, further sequence modifications were required to provide the improved antibodies described herein. As shown below, not only were the sequence modifications performed according to known techniques, but careful and complex selection of the acceptor domains was also required to accommodate specific VL and VH sequences that were advantageous in terms of manufacturing, stability, and immunogenicity, but still exhibited high affinity for the HSG hapten-containing moiety.

[0057] The anti-HSG binding site of the antibody of the invention comprises a light chain variable (VL) domain comprising one of SEQ ID NOs: 17, 19, 89, 90, or 91, and a heavy chain variable (VH) domain comprising a sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, and 10.

[0058] In alternative embodiments, one or both of the VL and VH comprise a sequence having at least 95%, particularly 96%, 97%, 98%, 99% sequence identity. Alternatively, one or both of the VL and VH comprise one or two additional amino acid substitutions. In this regard, the following is particularly true: The following amino acid positions of SEQ ID NO: 17 remain conserved: E105, I106, and optionally Q100.

[0059] The following amino acid positions of SEQ ID NO: 19 remain conserved: E105, I106, and optionally Q100. The following amino acid positions of SEQ ID NO:89 remain conserved: E105, I106, and optionally Q100.

[0060] The following amino acid positions of SEQ ID NO:90 remain conserved: E105, I106, and optionally Q100. The following amino acid positions of SEQ ID NO: 91 remain conserved: E105, I106, and optionally Q100. The following amino acid position of SEQ ID NO: 4 remains conserved: R19.

[0061] The following amino acid position of SEQ ID NO:6 remains conserved: R19. The following amino acid position of SEQ ID NO:8 remains conserved: R19. The following amino acid position of SEQ ID NO: 10 remains conserved: R19. The numbering is by Kabat.

[0062] SEQ ID NOs: 17, 19, 89, 90, 91, 4, 6, 8, and 10 encompass all of the CDRs and framework regions of the bispecific anti-tumor antigen / anti-HSG antibodies described herein.

[0063] In certain embodiments, the carboxyl terminus of the VH is linked to the amino terminus of the VL by a peptide linker Gly-Gly-Ser (G2S), thereby forming a single-chain variable fragment (scFv). Specifically, the linker is (G2S) n (where n is 1, 2, 3, 4, or 5, specifically n is 3, 4, or 5).

[0064] In a particular embodiment, the linker is SEQ ID NO:39. In particular, said linkers are used for antibodies of the invention in the formats scFv-Fab-Fc and IgG-central scFv.

[0065] ScFv is (G4S) n (where n is 1, 2, 3, 4, or 5, and specifically n is 1 to 3).

[0066] In a particular embodiment, the linker is SEQ ID NO:42. In particular, said flexible linkers are used for antibodies of the invention in the format scFv-Fab-Fc and in the format IgG-central scFv.

[0067] Specifically, when used with antibodies of the invention in the formats scFv-Fab-Fc and IgG-central scFv, the flexible linker is then linked to the Fc region of the antibody heavy chain (e.g., of SEQ ID NO: 32 or SEQ ID NO: 34) via the truncated hinge region of SEQ ID NO: 40.

[0068] VH and VL can also be linked to the respective antibody constant regions (e.g., CL kappa cross (SEQ ID NO: 38), and CH1 cross (e.g., SEQ ID NO: 30)), whereas, specifically when used in antibodies of the present invention in the format CrossMab (CH1-CL), CL kappa cross is then linked via a truncated hinge (of SEQ ID NO: 40) to the Fc region of SEQ ID NO: 32, or to the truncated hinge-Fc of SEQ ID NO: 33. According to particular embodiments of the present invention, antibodies comprising the following combinations of VH and VL: SEQ ID NO: 4 and SEQ ID NO: 17 (VH1+VL1) SEQ ID NO: 4 and SEQ ID NO: 19 (VH1+VL2) SEQ ID NO: 4 and SEQ ID NO: 89 (VH1+VL3.1) SEQ ID NO: 4 and SEQ ID NO: 90 (VH1+VL4.1) SEQ ID NO: 4 and SEQ ID NO: 91 (VH1+VL5.1) SEQ ID NO: 6 and SEQ ID NO: 17 (VH2+VL1) SEQ ID NO: 6 and SEQ ID NO: 19 (VH2+VL2) SEQ ID NO: 6 and SEQ ID NO: 89 (VH2+VL3.1) SEQ ID NO: 6 and SEQ ID NO: 90 (VH2+VL4.1) SEQ ID NO: 6 and SEQ ID NO: 91 (VH2+VL5.1) SEQ ID NO: 8 and SEQ ID NO: 17 (VH3+VL1) SEQ ID NO: 8 and SEQ ID NO: 19 (VH3+VL2) SEQ ID NO: 8 and SEQ ID NO: 89 (VH3+VL3.1) SEQ ID NO: 8 and SEQ ID NO: 90 (VH3+VL4.1) SEQ ID NO: 8 and SEQ ID NO: 91 (VH3+VL5.1) SEQ ID NO: 10 and SEQ ID NO: 17 (VH4+VL1) SEQ ID NO: 10 and SEQ ID NO: 19 (VH4+VL2) SEQ ID NO: 10 and SEQ ID NO: 89 (VH4+VL3.1) SEQ ID NO: 10 and SEQ ID NO: 90 (VH4+VL4.1) SEQ ID NO: 10 and SEQ ID NO: 91 (VH4+VL5.1) In certain embodiments, the bispecific antibodies described herein contain a variant Fc domain with reduced or eliminated effector function and / or FcRn binding.

[0069] "Effector function," as used herein, refers to a biochemical event that results from the interaction of an antibody Fc region with an Fc receptor or ligand. Effector functions include, but are not limited to, ADCC, ADCP, and CDC.

[0070] As used herein, "effector cell" refers to a cell of the immune system that expresses one or more Fc receptors and mediates one or more effector functions. Effector cells include, but are not limited to, monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, and T cells, and may be derived from any organism, including, but not limited to, humans, mice, rats, rabbits, and monkeys. According to the present invention, the bispecific antibodies described herein have silenced effector functions through amino acid substitutions at selected positions in the heavy chain constant region, specifically the Fc region. Reduction or complete silencing of the effector functions of these antibodies by reducing complement- and FcγR-mediated activity can include reduced or eliminated complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and / or antibody-dependent cellular phagocytosis (ADCP).

[0071] The term "Fc" or "Fc region" (or fragment crystallizable region), as used herein, refers to a polypeptide comprising the constant region of an antibody excluding the first constant region immunoglobulin domain (CH1 domain) (and in some cases, a portion of the hinge). The Fc region refers to the C-terminal region of an antibody. The Fc region consists of two (specifically identical) protein fragments derived from the second and third constant domains of the two heavy chains of an antibody: the A chain and the B chain. The second and third constant domains are known as the CH2 domain and CH3 domain, respectively. The CH2 domain contains the CH2 domain sequence of the A chain and the CH2 domain sequence of the B chain. The CH3 domain contains the CH3 domain sequence of the A chain and the CH3 domain sequence of the B chain. As used herein, the Fc region includes the hinge region or a portion thereof.

[0072] The CH2 domain of a human IgG Fc region sequence typically spans from approximately amino acid 231 to approximately amino acid 340 (EU numbering). The CH2 domain sequence is unique in that it is not closely paired with another domain sequence. Rather, two N-linked branched glycans are inserted between the two CH2 domain sequences in an intact native IgG molecule.

[0073] The CH3 domain comprises a stretch of residues at the C-terminus of the CH2 domain sequence of the Fc region sequence (ie, from about amino acid residue 341 to about amino acid residue 447 of IgG according to EU numbering).

[0074] A "functional Fc region" or "functional Fc domain" possesses the "effector functions" and FcRn binding of a native Fc region. Exemplary "effector functions" include C1q binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); and the like. Such effector functions generally require combining an Fc region with a binding domain (e.g., an antibody variable domain) and can be assessed using a variety of assays known in the art and disclosed herein.

[0075] A "native Fc region" or "native Fc domain" comprises an amino acid sequence identical to that of an Fc region found in nature. Native-sequence human Fc regions include native-sequence human IgG1 Fc regions (non-A and A allotypes); native-sequence human IgG2 Fc regions, native-sequence human IgG3 Fc regions, and native-sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0076] A "variant Fc region" or "variant Fc domain" comprises an amino acid sequence that differs from that of a native Fc region sequence by "one or more amino acid substitutions." The variant Fc region sequence comprises at least one amino acid substitution compared to the native Fc region sequence or the Fc region sequence of a parent polypeptide, e.g., from about 1 to about 20 amino acid substitutions, preferably from about 1 to about 17 amino acid substitutions, in the native Fc region sequence or the Fc region sequence of a parent polypeptide. In certain embodiments, the variant Fc region sequences herein are at least about 80% identical to the native Fc region sequence and / or the parent polypeptide Fc region sequence, most preferably at least about 90% identical, and more preferably at least about 95% identical.

[0077] In certain embodiments, the amino acid substitutions are at any one or more of positions E233, L234, L235, G236, 1253, G237, P238, D265, S267, H268, N297, S298, T299, H310, E318, L328, P329, A330, P331 and H435 according to EU numbering relative to IgG1 or SEQ ID NO: 31.

[0078] In a further specific embodiment, the amino acid substitutions are at any one or all of positions L234, L235, and H310 in the CH2 domain and H435 in the CH3 domain (all relative to IgG1 (SEQ ID NO: 31) according to EU numbering). More specifically, the substitutions are L234A, L235A, H310A, and / or H435Q. These residues are located in the Fc region and can result in near-complete inhibition of FcγR interactions, thus resulting in near-complete or complete Fc silencing.

[0079] Modifications of the Fc region that result in the reduction or silencing of Fc effector function are known in the art and are described in Saunders K., 2019 and Liu R. et al., 2020.

[0080] In alternative embodiments, the amino acid substitutions are at any one or all of positions L234F, H268Q, K274Q, Y296F, A327G, A330S, P331S in the CH2 domain, and any one or all of positions R355Q, K409R, Q419E, P445L in the CH3 domain.

[0081] Specifically, the Fc-silenced bispecific antibodies described herein comprise one or more of the following combinations of amino acid substitutions or deletions: i) L235, G237 and E318, specifically L235A, G237A and E318A; ii) L234, L235, specifically L234A, L235A; iii) S228, L235, specifically S228P, L235E; iv) G236, L328, specifically G236R, L328R; v) S298, T299, specifically S298G, T299A; vi) L234, L235, P331, specifically L234F, L235E, P331S; vii) H268, V309, A330, P331, specifically H268Q, V309L, A330S, P331S; viii) E233, L234, L235, G236, S267, specifically E233P, L234V, L235A, G236del, S267K; ix) L234, L235, P329, specifically L234A, L235A, P329G; x) V234, G237, P238, H268, A330, P331, specifically V234A, G237A, P238S, H268A, A330S, P331S; xi) L234, L235, D265, specifically L234F, L235E, D265A; xii) D265, specifically D265A; xiii) G237, specifically G237A; xiv) E318, specifically E318A; xv) E233, specifically E233P; xvi) G236, L328, specifically G236R, L328R; xvii) L235, specifically L235E; xviii) L234, L235, P331, specifically L234Q, L235F, P331S; xix) L234, L235, G237, P238, H268, A330, P331, specifically L234A, L235A, G237A, P238S, H268A, A330S, P331S; xx) N297, specifically N297A, N297Q or N297G (resulting in an aglycosylated antibody).

[0082] Glycosylation (O- and N-glycosylation) is a post-translational modification of Abs and can be regulated by various B cell stimuli, including, for example, environmental factors such as stress or disease, cytokine activity, and innate immune signaling receptors such as Toll-like receptors. The glycosylation pattern of a parent antibody can be modified by methods well known in the art. Specifically, O-linked glycosylation sites are located in the CH2 and hinge regions.

[0083] Specifically, the bispecific antibodies described herein contain one or more of the following combinations of amino acid substitutions: i)I253A; ii) H310A; iii) H435, specifically H435A, H435Q, or H435R; iv) I253A and H310A; v) I253A and H310A, and one of H435Q, H435A, and H435R; vi) H310A and one of H435Q, H435A, and H435R.

[0084] However, the silencing and further mutations described herein in the CH domain can also be introduced into the Fc of wild-type IgG2, IgG3 or IgG4 at the corresponding positions according to EU numbering.

[0085] The term "aglycosylated" indicates that the Fc region is not glycosylated. All human constant regions of the IgG isotype are known to be glycosylated at the Asp residue at position 297, which forms part of the N-glycosylation motif Asp 297-X 298-Ser 299 or Thr 299 (where X is any amino acid residue except proline). The glycan has a heptasaccharide core and variable extensions such as fucose, galactose, and / or sialic acid. The antibodies of the present invention can therefore be made aglycosylated by replacing Asp 297 of such constant regions with another amino acid that cannot be glycosylated or deglycosylated by enzymatic means. While any other amino acid residue could potentially be used, Ala is most preferred. Alternatively, glycosylation of Asp 297 can be prevented by altering one of the other residues in the motif, for example, by substituting residue 298 with Pro or residue 299 with any amino acid other than Ser or Thr. Techniques for performing this site-directed mutagenesis are well known to those skilled in the art and may be performed, for example, using commercially available site-directed mutagenesis kits.

[0086] The term "silenced Fc" refers to an antibody Fc region in which effector function and / or FcRn binding has been reduced or eliminated by amino acid substitutions or modified glycosylation patterns that result in glycan modifications that reduce or eliminate antibody binding to any of the FcγR receptors (such as FcγRIIaH, FcγR11aR, FcγR11b, FcγR11laF, FcγR11laV, and FcγRIa and / or FcRn receptors), as well as binding of the antibody to complement factor C1 q protein. Such reduction or elimination of this binding results in the reduction or elimination of effector function and / or FcRn binding typically mediated by wild-type IgG Fc regions.

[0087] The term "Fc null" may be used herein when FcγR binding to complement factor C1 q protein (such as any one of FcγRlla, FcγRll FcγRllla, and FcγRIa and / or FcRn receptors) is completely abolished.

[0088] Significant Fc silencing can be achieved by combining the mutations L234 and L235. These residues are located near the hinge region and, when substituted with alanine, reduce FcγR binding. For example, the combination of L234A and L235A with P329G can result in near-complete inhibition of FcγR interactions across all FcγR isoforms.

[0089] The Fc-silencing bispecific antibodies described herein that have significantly reduced, silenced, negligible, or eliminated FcγR and C1q binding affinities are antibodies that have reduced FcγR and C1q binding activity compared to the parent polypeptide or a polypeptide comprising a native Fc region sequence. In some embodiments, the Fc-silencing bispecific antibodies that have significantly reduced, silenced, negligible, or eliminated FcR and C1q binding affinities also have significantly reduced, silenced, negligible, or eliminated ADCC, ADCP, and CDC activity compared to the parent polypeptide or a polypeptide comprising a native Fc region sequence. Fc-silencing bispecific antibodies described herein that exhibit reduced or undetectable binding to FcγRs may bind to all FcγRs with lower affinity than the parent polypeptide. An exemplary parent polypeptide may be the antibody imalumab. Such variants that exhibit reduced binding to FcγRs may exhibit little or no detectable binding to FcγRs. In one specific embodiment, the variant exhibits 0-20% of the binding to FcγR compared to the native IgG Fc region, e.g., as measured by a change in equilibrium constant. In one embodiment, the variant exhibits 0-10% of the binding to FcγR compared to the native IgG Fc region. In one embodiment, the variant exhibits 0-5% of the binding to FcγR compared to the native IgG Fc region. In one embodiment, the variant exhibits 0-1% of the binding to FcγR compared to the native IgG Fc region.

[0090] The antibodies described herein that have silenced complement activity can be determined by cell-based CDC assays, and reduced or absent binding to C1q can be determined, i.e., by SPR or ELISA.

[0091] Reduced or silenced CDC activity is determined to be downregulated by at least 1.5-fold, particularly at least 2-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, and more particularly at least 10-fold compared to the reference, i.e., unmodified wild-type Fc.

[0092] Decreased ADCC or ADCP activity is determined to be at least 2-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or more particularly at least 10-fold reduced in potency compared to the reference antibody, i.e., the unmodified wild-type Fc.

[0093] In certain embodiments, the anti-tumor antigen / anti-HSG antibody exhibits reduced binding to FcRn due to amino acid substitutions at selected positions in the heavy chain constant region. The reduced binding of the Fc silencing bispecific antibody of the present invention to FcRn results in a reduced half-life in circulation and faster in vivo clearance. Determination of FcRn binding and in vivo clearance / half-life can also be performed using methods known in the art (see, for example, Petkova, SB et al., 2006).

[0094] The Fc domain of the bispecific antibodies described herein that exhibit reduced FcRn binding compared to an anti-tumor antigen / anti-HSG antibody comprising a wild-type IgG Fc region may preferably comprise an Fc domain comprising one, two or three amino acid substitutions at any one of positions I253, H310, and H435.

[0095] The bispecific antibodies described herein may also have L234A / L235A ("LALA") mutations at the beginning of the CH2 region of the heavy chain. Introduction of the L234A / L235A mutation has been demonstrated to result in almost complete elimination of mAb binding to FcγR and a strong reduction in complement binding (Lo M. et al., 2017; Wang X. et al., 2018; Zhou Q. et al., 2020).

[0096] Fc silencing also allows the binding of the antibodies described herein to Fcγ receptor-bearing immune cells and complement to minimize potential undesirable effects during radioimaging, particularly when performing pretargeting.

[0097] A decrease in FcRn binding (i.e., affinity) is determined as at least a 2-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or more particularly at least a 10-fold decrease in binding (i.e., affinity) compared to a reference antibody, i.e., an unmodified wild-type antibody.

[0098] According to certain embodiments, two different heavy chains may be combined in the antibodies described herein. Heterodimerization of two different heavy chains can be achieved using techniques such as engineering heterodimeric Fc mutants by replacing the interactions that favor homodimerization with those that favor heterodimerization at the CH3 domain interface by introducing asymmetric mutations into each CH3 domain that promote the assembly of HCs from different antibodies. Thus, structure-based rational design and directed evolution can be used to ensure that mutant pairs thermodynamically favor the formation of heterodimers over homodimers. Strategies known from the art are, for example, symmetric-to-asymmetric steric complementarity design (e.g., KiH, HA-TF, and ZW1); charge-to-charge swap (e.g., DD-KK); charge-to-steric complementarity swap plus additional long-range electrostatic interactions (e.g., EW-RVT); and isotype strand swap (e.g., Strand Exchange Engineered Domain (SEED)).

[0099] Knob-into-hole (KiH) technology: One heavy chain has a "knob" mutation T366W and a disulfide bond stabilizing S354C mutation in its CH3 region (Merchant AM et al., 1998), which specifically contains the anti-tumor antigen binding site, e.g., anti-oxMIF binding site. The other heavy chain has a "hole" mutation T366S / L368A / Y407V and a disulfide bond stabilizing Y349C mutation in its CH3 region (Merchant AM et al., 1998), which specifically contains the anti-HSG binding site.

[0100] According to certain embodiments of the invention, the antibodies described herein comprise SEQ ID NOs: 35 and 32. According to an embodiment of the invention, the antibodies described herein comprise SEQ ID NOs: 36 and 33 or SEQ ID NOs: 36 and 34.

[0101] The antibodies described herein can be used to detect and localize tumors that have tumor-associated or tumor-specific antigens on their cell surface. The anti-tumor antigen binding site can specifically target and bind to any tumor antigen or tumor-associated antigen.The antigen can include, but is not limited to, melanoma cell surface antigen, breast cancer cell surface antigen such as CA15-3, lung cancer cell surface antigen, colorectal cancer cell surface antigen, gastric cancer cell surface antigen, pancreatic cancer cell surface antigen, glioma cell surface antigen, common sarcoma cell surface antigen, gastrointestinal cancer cell surface antigen, brain tumor cell surface antigen, esophageal cancer cell surface antigen, common epithelial cancer cell surface antigen, osteosarcoma cell surface antigen, fibrosarcoma cell surface antigen, bladder cancer cell surface antigen, prostate cancer cell surface antigen, kidney cancer cell surface antigen, ovarian cancer cell surface antigen, testicular cancer cell surface antigen, endometrial cancer cell surface antigen, cervical cancer cell surface antigen, Hodgkin's disease cell surface antigen, lymphoma cell surface antigen, leukemia cell surface antigen, trophoblastic tumor cell surface antigen, tumor necrosis antigen, and oxMIF. Where the disease state is cancer, for example, many antigens expressed by or otherwise associated with tumor cells are known in the art, including carbonic anhydrase IX, alpha-fetoprotein, alpha-actinin-4, A3, antigen specific for the A33 antibody, ART-4, B7, Ba 733, BAGE, BrE3-antigen, CEA, cancer antigen CA-125, PSA, CAP-1, CASP-8 / m, CCL19, CCL21, CEACEACAM5, CEACAM6, CEACAM8, c-met, CDK-4 / m, CDKN2A, CXCR4, CXCR7, CXCL12, HIF-1α, colon-specific antigen-p (CSAp), Flt-1, Flt-3, folate receptor, G250 antigen, GAGE, gp100, GRO-β, HLA-DR, HM1.24, human chorionic gonadotropin (HCG) and its subunits, HER2 / neu, HMGB-1, hypoxia-inducible factor (HIF-1), HS P70-2M, HST-2, Ia, IGF-1R, IFN-γ, IFN-α, IFN-β, IL-2, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12, I L-15, IL-17, IL-18, IL-25, insulin-like growth factor-1 (IGF-1), KC4-antigen, KS-1-antigen, KS1-4, Le-Y, LDR / FUT, HER2, DAM, EGFR, EGFRvIII, EGP -1, EGP-2, ELF2-M, EpCAM, mesothelin (MSLN), oxidized macrophage migration inhibitory factor (oxMIF), MAGE, MAGE-3, MART-1, MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B, folate receptor alpha (FRα), EGFR, Trop-2, MUC1, MUC2, MUC3, MUC4, MUC5, MUM-1 / 2, MUM-3, NCA66, NCA95, NCA 90, pancreatic cancer mucin, placental growth factor, p53, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, PIGF, ILGF, ILGF-1R, RS5, RANTES, T101, SAGE, S100, survivin, survivin-2B, TAC, TAG-72, tenascin, Thomson-Friedenreich antigen, VEGFR, ED-B fibronectin, WT-1, 17-1A-antigen, complement factors C3, C3a, C3b, C5a, C5, anAngiogenesis markers, bcl-2, bcl-6, Kras, cMET, oncogene markers and oncogene products, tumor necrosis antigen, TNF-α, TRAIL receptor (R1 and R2), NCA-90, NCA-95, transmembrane activator and interactor of CAML (TACI), B-cell maturation antigen (BCMA), APRIL, TALL-I (also known as BLyS or BAFF), CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e , CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154, CXCR4, CD3, ADAM17, CD2, CD6, CD11a, CD11b, CD16, CD16b, CD28, CD30, CD32a, CD44, CD56, CD57, CD64, CD69, CD74, CD89, CD9 0, CD137, CD177, CDC27, HLA-DR alpha chain, KIR, LSECtin or SLC44A2, B7, Ia, Ii, HM1.24, HLA-DR, tenascin, VEGF, PlGF, ED-B fibronectin, oncogenes, oncogene products (e.g., c-met or PLAGL2), IL-2, T101, TAG, and the like.

[0102] According to certain embodiments, the anti-tumor antigen binding site of the antibodies described herein specifically recognizes oxMIF. oxMIF antibodies are described in PCT / EP2021 / 077106 and PCT / EP2022 / 052463.

[0103] The oxMIF binding site is specific for the oxidized form of MIF, specifically human oxMIF, and does not show substantial cross-reactivity with reduced MIF. oxMIF is a disease-associated structural isoform of MIF that can be specifically and primarily detected in the circulation of subjects with inflammatory diseases and in tumor tissues of cancer patients.

[0104] oxMIF binding specificity can be determined by any assay suitable for determining selective binding to oxMIF, for example, any competitive assay against a control antibody such as imalumab for binding to oxMIF, or various assays known in the art and disclosed herein.

[0105] The bispecific antibodies of the invention comprise at least one binding site that specifically recognizes oxMIF and, according to certain embodiments, exhibit a reduced tendency to aggregate and a reduced hydrophobicity compared to the unmodified antibody lacking said amino acid substitutions due to targeted amino acid substitutions in the variable heavy and light domains.

[0106] The reduced aggregation ability is due to amino acid substitutions at selected positions within the variable domains of the antibodies described herein. The level of antibody aggregation can be measured using a variety of known techniques, including mass spectrometry, size exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC), dynamic light scattering (DLS), light obscuration (LO), dynamic imaging particle analysis (DIPA) methods such as microflow imaging (MFI), and Coulter Counter (CC), differential scanning fluorimetry (DSF).

[0107] As used herein, reduced hydrophobicity and reduced aggregation ability refer to reduced surface hydrophobicity and reduced aggregation ability of an antibody compared to a reference antibody, such as the antibody imalumab, which is published in Proposed INN List 111 (WHO Drug Information, Vol. 28, No. 2, 2014) but lacks a C-terminal lysine. Measurements can be performed using various known techniques, including, but not limited to, hydrophobic interaction chromatography (HIC) or affinity capture self-interaction nanoparticle spectroscopy (AC-SINS, Estep P. et al., 2015).

[0108] In one embodiment, the oxMIF binding site of the antibody of the invention specifically comprises a light chain variable domain having SEQ ID NO: 45 with one or more amino acid substitutions, specifically 1, 2, 3, 4 or 5 amino acid substitutions at positions M30, F49, A51, P80, W93 according to the Kabat numbering, specifically M30L, F49Y, A51G, P80S, W93F, more specifically F49Y, A51G, W93F, or at position 36. and further comprising at least one, particularly 1, 2, 3, 4, 5 amino acid substitutions at positions M30, F49, A51, P80, W93, particularly M30L, F49Y, A51G, P80S, and W93F, more particularly F49Y, A51G, and W93F. Either light chain variable domain is combined with a heavy chain variable domain comprising SEQ ID NO: 46, or a heavy chain variable domain comprising SEQ ID NO: 46 with an amino acid substitution at position L5 and / or W97 according to Kabat numbering, specifically L5Q and / or W97Y, or a heavy chain variable domain with 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 46 with an amino acid substitution at position L5 and / or W97, specifically L5Q and / or W97Y.

[0109] According to a particular embodiment, the amino acid W93 is replaced by F, Y, or H. In a further embodiment, the anti-oxMIF antibody of the present invention with reduced aggregation ability and reduced hydrophobicity specifically comprises a heavy chain variable domain comprising SEQ ID NO: 46 and an amino acid substitution at position W97, specifically W97Y, or an amino acid substitution at position L5, specifically L5Q, or amino acid substitutions L5Q and W97Y, or a heavy chain variable domain comprising 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 46 and further comprising the amino acid substitution W97Y, optionally in combination with L5Q.

[0110] According to a particular embodiment, the amino acid W97 is substituted with F, Y, or H. In a preferred embodiment, the anti-oxMIF antibodies of the invention with reduced aggregation ability and reduced hydrophobicity specifically comprise amino acid substitutions at positions W93 and W97.

[0111] The tyrosine at position 36 of the light chain is specifically left unmodified to preserve the binding properties of the antibodies described herein, as any modification at this amino acid position may result in an undesirable decrease in binding properties.

[0112] In certain embodiments, the antibodies described herein: - a light chain variable domain comprising SEQ ID NO: 27, or a light chain variable domain comprising SEQ ID NO: 27 and further comprising the amino acid substitutions M30L and / or P80S, and - a heavy chain variable domain comprising SEQ ID NO: 46, specifically with the amino acid substitutions L5Q and / or W97Y where amino acid positions are numbered according to Kabat.

[0113] The term "antibody" as used herein is used in the broadest sense and includes polypeptides or proteins consisting of or including antibody domains, understood to be the constant and / or variable domains of immunoglobulin heavy and / or light chains, with or without linker sequences. The term also includes fusion proteins, such as fusions with immunotoxins, or antibody conjugates, such as antibody-drug conjugates that bind to HSG and oxMIF.

[0114] An antibody domain may be in its native structure or may be modified, for example, by mutagenesis or derivatization to modify antigen binding properties or any other property, such as stability or functional properties, e.g., binding to an Fc receptor, such as FcRn and / or Fc gamma receptor. A polypeptide sequence is considered an antibody domain if it comprises a beta-barrel structure consisting of at least two beta strands of the antibody domain structure connected by a loop sequence.

[0115] The term "antibody" is understood to include antigen-binding derivatives, variants, and fragments thereof. A derivative or variant is any combination of one or more antibody domains or antibodies of the invention and / or fusion proteins. In fusion proteins, any domain of an antibody of the invention can be fused at any position to one or more other proteins, such as other antibodies or antibody formats (e.g., binding structures including CDR loops, receptor polypeptides, but also ligands, scaffold proteins, enzymes, labels, toxins, etc.).

[0116] The term "antibody" is intended to refer in particular to a polypeptide or protein that exhibits binding properties for target tumor antigens, in particular oxMIF, and HSG. The terms "antibody fragment, antigen-binding fragment, antigen-binding variant, or antibody variant" can be used interchangeably and refer to molecules other than intact antibodies, including antigen-binding portions of intact antibodies that bind to the antigen to which the intact antibody binds, and multispecific antibodies formed from antibody fragments or variants, and further comprising a variant Fc region as described herein. Examples of antigen-binding portions include, but are not limited to, Fv, Fab, Fab', Fab'-SH, single-chain antibody molecules (e.g., scFv), diabodies, crossFab fragments; and linear antibodies. According to the present invention, the antibody fragment or variant is fused to a silenced Fc portion or a silenced Fc domain via a hinge region and / or a linker, in particular a hinge region preceded by a linker (e.g., (scFv)-Fc, (scFv)2-Fc, scFv / scFv-Fc, Fab / scFv-Fc, Fab / (scFv)2-Fc, Fab / Fab-scFv-Fc (IgG-central scFv), Fab / Fab-crossFab-Fc, IgG-scFv and IgG-(scFv)2, Furthermore, antibody fragments include single-chain polypeptides that have characteristics of a VH domain (i.e., capable of assembling with a VL domain into a functional antigen-binding site), or characteristics of a VL domain (i.e., capable of assembling with a VH domain into a functional antigen-binding site), thereby providing the antigen-binding properties of a full-length antibody. Antibody fragments as referred to herein also encompass full-length antibody formats having a silenced Fc domain containing one or more structural loop regions containing the antigen-binding region, such as an Fcab™, or an IgG structure in which the silenced Fc region is replaced by an Fcab™ containing a second, different antigen-binding site.

[0117] As used herein, "Fab fragment or Fab" refers to an antibody fragment comprising a light chain fragment containing the VL domain and constant domain (CL) of the light chain, and a VH domain and first constant domain (CH1) of the heavy chain. The antibody of the present invention may comprise at least one Fab fragment in which either the variable or constant regions of the heavy and light chains are exchanged. With either the variable or constant regions exchanged, the Fab fragment is also referred to as a "crossFab fragment" or "crossover Fab fragment." Two different chain compositions of crossover Fab molecules are possible and are included in the antibodies of the present invention. The variable regions of the Fab heavy and light chains may be exchanged, i.e., the crossover Fab molecule comprises peptide chains. According to the present invention, the Fab is fused to a silenced Fc portion or a silenced Fc domain via the hinge region.

[0118] As used herein, "Fab arm" refers to a silenced Fc portion or a Fab fragment fused by the hinge region to a silenced Fc domain.

[0119] The term "functional variant" or "functionally active variant" includes naturally occurring allelic variants, as well as mutants or any other non-naturally occurring variants. As known in the art, allelic variants (also called homologs) are alternative forms of nucleic acids or peptides characterized by one or more nucleotide or amino acid substitutions, deletions, or additions that do not essentially change the biological function of the nucleic acid or polypeptide. Specifically, functional variants may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residue substitutions, deletions, and / or additions, or combinations thereof, where the substitutions, deletions, and / or additions are conservative modifications that do not alter the antigen binding properties. Specifically, the functional variants described herein may contain up to, or up to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions, deletions, and / or additions, which are conservative modifications and do not alter antibody function. Specifically, the functionally active variants described herein contain up to 15, and preferably up to 10 or 5, amino acid substitutions, deletions, and / or additions, which are conservative modifications and do not alter antibody function.

[0120] Functional variants can be obtained by altering the polypeptide or nucleotide sequence, for example, by one or more point mutations, such that the sequence alterations retain or improve the function of the unaltered polypeptide or nucleotide sequence when used in the combinations of the present invention. Such sequence alterations can include, but are not limited to, (conservative) substitutions, additions, deletions, mutations, and insertions. Conservative substitutions occur within a family of amino acids that are related in side chains and chemical properties. Examples of such families are amino acids with basic side chains, acidic side chains, nonpolar aliphatic side chains, nonpolar aromatic side chains, uncharged polar side chains, small side chains, large side chains, etc.

[0121] According to certain embodiments, the antibodies described herein may comprise one or more tags for purification and / or detection, such as, but not limited to, affinity tags, solubility-enhancing tags, and monitoring tags.

[0122] Specifically, the affinity tag is selected from the group consisting of a polyhistidine tag, a polyarginine tag, a peptide substrate for an antibody, a chitin-binding domain, an RNAse S peptide, protein A, β-galactosidase, a FLAG tag, a Strep II tag, a streptavidin-binding peptide (SBP) tag, a calmodulin-binding peptide (CBP), a glutathione S-transferase (GST), a maltose-binding protein (MBP), an S tag, an HA tag, and a c-Myc tag; specifically, the tag is a His tag containing one or more Hs, such as a hexahistidine tag.

[0123] By "fused" or "linked" or "conjugated" is meant that the components (e.g., a Fab molecule and an Fc domain subunit) are linked by a peptide bond (either directly or via one or more peptide linkers).

[0124] As used herein with respect to the linkage between an HSG hapten and a chelator and / or therapeutic / diagnostic agent, the term "linker" refers to a peptide linker, preferably a peptide comprising an amino acid sequence having a length of 2, 3, 4, 5, 6, 7 or more amino acids, preferably 3-15, more preferably 3-5 amino acids. Specific linkers are described herein, such as SEQ ID NOS: 39 and 42, and may be of variable length.

[0125] The term "immunoglobulin" refers to a protein with the structure of a naturally occurring antibody. For example, IgG class immunoglobulins are heterotetrameric glycoproteins of approximately 150,000 daltons consisting of two disulfide-bonded light chains and two heavy chains. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also known as the variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also known as the heavy chain constant region. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also known as the variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain, also known as the light chain constant region. IgG class immunoglobulins essentially consist of two Fab molecules and an Fc domain connected via an immunoglobulin hinge region. The heavy chains of immunoglobulins can be assigned to one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which are further subtyped, e.g., γ1 (IgG1), γ2 (IgG2), γ 3( IgA1) and α2 (IgA2). The light chains of immunoglobulins can be assigned to one of two types, called kappa (κ) and lambda (λ).

[0126] The term "chimeric antibody" refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species, usually prepared by recombinant DNA techniques. Chimeric antibodies may contain rabbit or mouse variable regions and human constant regions. Chimeric antibodies are the product of expression of immunoglobulin genes containing DNA segments encoding immunoglobulin variable regions and DNA segments encoding immunoglobulin constant regions. Methods for producing chimeric antibodies require conventional recombinant DNA and gene transfection techniques well known in the art (Morrison, SL et al., 1984).

[0127] A "human antibody" has an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or an antibody derived from a non-human source that utilizes a human antibody repertoire or other human antibody-encoding sequence. This definition of human antibody specifically excludes humanized antibodies, which comprise non-human antigen-binding residues. As used herein, the term "human antibody," as also referred to in reference to chimeric and humanized antibodies, also includes such antibodies that have been modified in the constant region, for example, by "class switching," i.e., alteration or mutation of the Fc portion (e.g., IgG1 to IgG4 and / or IgG1 / IgG4 mutations).

[0128] The term "recombinant human antibody," as used herein, is intended to include all human antibodies prepared, expressed, generated, or isolated by recombinant means, such as antibodies isolated from host cells such as HEK, NS0, or CHO cells, or from animals (e.g., mice) that are transgenic for human immunoglobulin genes, or antibodies expressed using recombinant expression vectors transfected into host cells. The amino acid sequences of the VH and VL regions of the recombinant antibody are derived from and related to human germline sequences, but are sequences that may not naturally exist within the human antibody repertoire in vivo.

[0129] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is made from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup described in Kabat et al., 1991.

[0130] A "humanized" antibody refers to a humanized chimeric antibody that contains amino acid residues from non-human hypervariable regions (HVRs) and human framework regions (FRs). In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, with all or substantially all of the HVRs (e.g., CDRs) corresponding to HVRs of a non-human antibody and all or substantially all of the FRs corresponding to FRs of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. Specifically, forms of humanized antibodies in which the constant region has been further modified or altered from the constant region of the original antibody to generate new properties (i.e., with respect to reduced or abolished C1q binding and / or FcR binding) are encompassed by the present invention.

[0131] Methods and details for humanizing anti-HSG are described herein, specifically in the Examples. The term "bispecific" as used herein refers to a binding reaction with at least an anti-tumor antigen, such as an oxMIF antigen, and an additional antigen, an HSG hapten antigen. A bispecific antibody can specifically contain at least two sites with specific binding properties, and two different target antigens, at least one tumor antigen and HSG, are recognized by the antibody. A bispecific antibody format contains two binding sites, each capable of specifically binding to a tumor antigen and HSG. Alternatively, a further exemplary bispecific format may contain more than two binding sites, for example, three, four, five, or more binding sites, where one or more binding sites bind to one or more of the same or different tumor antigens, and one or more binding sites can specifically bind to HSG.

[0132] A "bispecific antibody" according to the present invention is an antibody with two different binding specificities. Bispecific antibodies can be prepared as full-length antibodies as described herein, or as antibody fragments that still contain a silenced Fc region. Immunoglobulin Fc heterodimers can be engineered by modifying the CH3 domain interface with different mutations in each domain, such that engineered Fc fragments with CH3 variant pairs preferentially form heterodimers rather than homodimers (Ha JH. et al., 2016). Examples of bispecific antibody formats can be, but are not limited to, bispecific IgG (BsIgG) as described in Spiess C. et al., 2015, and Brinkmann U. and Kontermann RE, 2017, IgG with additional antigen-binding moieties appended, BsAb fragments, bispecific fusion proteins, BsAb conjugates, hybrid bsIgG, modified Fc fusion proteins, appended IgG-HC fusions, appended IgG-LC fusions, appended IgG-HC&LC fusions, Fc fusions, CH3 fusions, F(ab')2 fusions, CH1 / CL, modified IgG, Fc-modified IgG, diabodies, etc.

[0133] In alternative embodiments encompassing the bispecific antibodies described herein, the term "IgG-scFv" refers to a type of bispecific antibody in which the bispecificity has been engineered by fusing one scFv to a monospecific immunoglobulin G (IgG). According to embodiments of the invention, the bispecific antibody is Fc-silenced. That is, the bispecific antibody comprises a mutant Fc region of wild-type human IgG with one or more amino acid substitutions or glycosylation modifications described herein. The specificity of the IgG can be for a tumor antigen, and the specificity of the scFv can be for an HSG hapten, or vice versa. Furthermore, scFvs can be added to either the amino or C-terminus of one of the light or heavy chains, resulting in the production of various types of IgG-scFv bispecific antibodies (BsAbs): (i) IgG(H)-scFv (scFv linked to the C-terminus of one of the full-length IgG HCs); (ii) scFv-(H)IgG (same as IgG(H)-scFv except that the scFv is linked to the N-terminus of the HC); (iii) IgG(L)-scFv or (iv) scFv-(L)IgG (scFv linked to the C-terminus or N-terminus of the IgG light chain, forming IgG(L)-scFv or scFv-(L)IgG, respectively). Specifically, IgG-scFvs range from 165 kDa to 185 kDa, specifically approximately 175 kDa.

[0134] In certain embodiments, the term Fab / bs(scFv)2-Fc refers to a bispecific antibody in which one Fab arm is replaced with bs(scFv)2, while the second IgG arm is preserved.

[0135] In certain embodiments, the term Fab / scFv-Fc refers to a bispecific antibody in which one Fab arm is replaced with an scFv, while the second IgG arm is preserved. Antibody C0181, described herein and shown schematically in Figure 1, serves as a non-limiting example of a Fab / scFv-Fc.

[0136] In certain embodiments, the term Fab / Fab-scFv-Fc (IgG-central scFv) refers to a bispecific antibody in which one Fab arm is replaced by a Fab-scFv, while the second IgG arm is preserved.

[0137] The term "CrossMab" (where Mab refers to monoclonal antibody) refers to a bispecific Ab format derived from independent parent antibodies. Heavy chain mispairing is avoided by applying the knobs-into-holes (KIH) method. Bispecific antibodies are produced by antibody domain exchange, where either the variable domain or the constant domain (CL and CH1) of one Fab arm is exchanged between the light and heavy chains, thus avoiding light chain mispairing. This "crossover" preserves antigen-binding affinity and also preserves the two distinct arms to avoid light chain mispairing. Examples of CrossMab include, but are not limited to, Fabs with different regions exchanged, VH-VL, and CH1-CL. In CrossMab Fab, the entire VH-CH1 and VL-CL regions are exchanged. In CrossMab VH-VL format, only the VH and VL regions are exchanged. In CrossMab CH1-CL1 format, the CH1 and CL regions of the bispecific antibody are exchanged. Specifically, CrossMab is approximately 150 kDa.

[0138] The term "antigen," which is used interchangeably herein with the terms "target" or "target antigen," is intended to refer to the entire target molecule or a fragment of such a molecule that is recognized by an antibody binding site. Specifically, substructures of antigens, such as polypeptide or carbohydrate structures, commonly referred to as "epitopes" (e.g., immunologically relevant B-cell or T-cell epitopes), can be recognized by such binding sites.

[0139] The term "epitope," as used herein, specifically refers to a molecular structure that may completely constitute or be part of a specific binding partner for the binding site of an antibody format of the present invention. Epitopes may be composed of carbohydrates, peptide structures, fatty acids, organic biochemicals, or inorganic substances, or derivatives thereof, and any combination thereof. When an epitope is contained in a peptide structure such as a peptide, polypeptide, or protein, the epitope typically contains at least three amino acids, specifically 5 to 40 amino acids, specifically less than 10 amino acids, specifically 4 to 10 amino acids. Epitopes can be either linear or conformational. Linear epitopes consist of a single segment of the primary sequence of a polypeptide or carbohydrate chain. Linear epitopes can be contiguous or overlapping. Conformational epitopes consist of amino acids or carbohydrates linked by folding of a polypeptide to form a tertiary structure, where the amino acids are not necessarily adjacent to each other in the linear sequence. An exemplary oxMIF epitope may be the sequence EPCALCS (SEQ ID NO: 53), which is located within the central region of oxMIF.

[0140] The term "antigen-binding domain" or "binding domain" or "binding site" refers to a portion of an antigen-binding moiety that comprises a region that specifically binds to and is complementary to a part or all of an antigen. When an antigen is large, an antigen-binding molecule may bind only to a specific portion of the antigen, which is called an epitope. An antigen-binding domain may be provided, for example, by one or more antibody variable domains (also called antibody variable regions). Preferably, the antigen-binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0141] The term "binding site," as used herein with respect to an antibody of the present invention, refers to a molecular structure capable of binding interaction with an antigen. Typically, a binding site is located within the complementarity-determining region (CDR) of an antibody, also referred to herein as a "CDR-binding site," which is a specific region with different structures that confer binding function to different antigens. The different structures may be derived from a natural repertoire of antibodies, e.g., a murine or human repertoire, or may be produced recombinantly or synthetically, e.g., by mutagenesis, particularly by randomization methods. These include mutagenized CDR regions, loop regions of variable antibody domains, particularly CDR loops of antibodies, e.g., the CDR1, CDR2, and CDR3 loops of either the VL and / or VH antibody domains. The antibody formats used according to the present invention typically contain one or more CDR-binding sites, each specific for an antigen.

[0142] The oxMIF binding sites of the antibodies described herein are specific for oxidized MIF, i.e., animal oxMIF, particularly mammalian oxMIF, such as, but not limited to, mouse, rat, monkey, and human oxMIF, particularly human oxMIF, but do not exhibit substantial cross-reactivity with reduced MIF. In one embodiment, the humanized or human anti-oxMIF binding site comprises the light chain complementarity-determining regions of one or more (e.g., all three) of the humanized or human anti-oxMIF binding domains described herein.

[0143] The term "specific," as used herein, refers to a binding reaction that determines the cognate ligand of interest in a heterogeneous population of molecules. Herein, the binding reaction is a binding reaction with at least the oxMIF antigen. Thus, under specified conditions, e.g., immunoassay conditions, an antibody that specifically binds to its particular target does not significantly bind to other molecules present in the sample, and in particular does not exhibit substantial cross-reactivity with reduced MIF.

[0144] A specific binding site is typically not cross-reactive with other targets, yet the binding site may specifically bind to one or more epitopes, isoforms, or variants of a target, or may be cross-reactive with other related target antigens, e.g., homologs or analogs.

[0145] Specific binding means that the binding is selective in terms of selected target identity, high, moderate, or low binding affinity or avidity. Selective binding is usually achieved when the binding constant or kinetics for a target tumor antigen, such as oxMIF or HSG hapten, differs by at least 10-fold compared to the binding constant or kinetics for an antigen that is not the target antigen, preferably by at least 100-fold, more preferably by at least 1000-fold.

[0146] The term "valent" as used within this application refers to the presence of a specific number of binding sites in an antibody molecule. Thus, the terms "bivalent," "tetravalent," and "hexavalent" refer to the presence of two, four, and six binding sites in an antibody molecule, respectively.

[0147] The term "monovalent," as used herein with respect to antibody binding sites, is intended to refer to a molecule that contains only one binding site for a target antigen. The term "valency" is therefore understood to be the number of binding sites for the same target antigen that specifically bind either to the same epitope of the antigen or to different epitopes.

[0148] The antibodies of the present invention are understood to include monovalent, bivalent, tetravalent or multivalent binding sites that specifically bind to oxMIF and HSG. The term "hypervariable region" or "HVR" as used herein refers to each region of an antibody variable domain that is hypervariable in sequence and / or forms a structurally defined loop ("hypervariable loop"). Generally, a natural four-chain antibody contains six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). HVRs generally contain amino acid residues from the hypervariable loops and / or "complementarity-determining regions" (CDRs), the latter of which have the highest sequence variability and / or are involved in antigen recognition (Kabat et al., 1991). Hypervariable regions (HVRs) are also referred to as complementarity-determining regions (CDRs), and these terms are used interchangeably herein to refer to the portions of the variable region that form the antigen-binding region. The exact number of residues encompassing a particular CDR varies depending on the sequence and size of the CDR. Those skilled in the art can routinely determine the residues that comprise a particular CDR given the amino acid sequence of the variable region of an antibody.

[0149] Kabat defined a numbering system for variable region sequences that is applicable to any antibody. One skilled in the art can unambiguously assign this system of "Kabat numbering" to any variable region sequence without relying on any experimental data beyond the sequence itself. The Kabat numbering of residues can be determined for a given antibody by aligning the antibody sequence with a "standard" Kabat numbering sequence at the homologous region. As used herein, "Kabat numbering" refers to the numbering system described in Kabat et al., 1983, US Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest." Unless otherwise specified, references to the numbering of specific amino acid residue positions in antibody variable regions are according to the Kabat numbering system. The numbering of constant regions is according to the EU numbering index.

[0150] CDRs also contain "specificity-determining residues" or "SDRs," which are residues that contact the antigen. SDRs are contained within regions of CDRs called abbreviated-CDRs, or a-CDRs. Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra. CDR determinations can also be made according to IMGT (Lefranc MP. 1997). IMGT has its own definitions of framework regions (designated FR-IMGT) and CDRs (designated CDR-IMGT). The IMGT numbering method counts residues consecutively from 1 to 128 based on a germline V sequence alignment.

[0151] CDRs (or SDRs) may be further determined according to MacCallum RM et al., 1996. Herein, antigen-contacting residues are analyzed and combined with site geometries of antibody Fv and Fab crystal structures available from the Protein Data Bank. Antigen contact propensity is shown for each antibody residue, allowing CDR definitions to be proposed based on observed antigen contacts. Contacts are more prevalent with CDR residues located centrally within the binding site; some less central CDR residues are contacted only by large antigens. Non-contacting residues within CDRs correspond to residues identified by Chothia and colleagues as important in defining the "canonical" conformation (Chothia C et al., 1987).

[0152] A "point mutation" is specifically understood as a modification of a polynucleotide that results in the expression of an amino acid sequence that differs from the unmodified amino acid sequence by substitution or exchange, deletion, or insertion of one or more single (non-consecutive) or double amino acids of different amino acids. Preferred point mutations refer to the exchange of amino acids of the same polarity and / or charge.

[0153] "Percent (%) sequence identity" with respect to the polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the particular polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity; any conservative substitutions are not considered as part of the sequence identity. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0154] According to the present invention, the sequence identity of the variable or constant region sequences with the respective sequences described herein is at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%.

[0155] As noted above, the bispecific antibodies described herein contain a first binding site for a tumor antigen or tumor-associated antigen and a second binding site for the HSG (histamine-succinyl-glycyl) hapten, specifically located in the HSG moiety. In alternative embodiments, the antibody may contain two, three, or more HSG hapten binding sites and one or more binding sites for a target antigen associated with a disease or condition.

[0156] Such HSG moieties can be of various structures, preferably comprising peptides having only two amino acid residues, more preferably two to ten residues, which may be conjugated to other moieties, such as chelators. The HSG moiety is preferably a low molecular weight molecule, preferably having a molecular weight of less than 50,000 daltons, advantageously less than about 20,000, 10,000, or 5,000 daltons, and comprises a chelate and a chelated metal, and optionally a diagnostic and / or therapeutic agent. In a preferred embodiment, the HSG moiety has four or more residues, such as the peptide DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH2 (SEQ ID NO: 63), where DOTA is 1,4,7,10-tetraazacyclododecanetetraacetic acid. The HSG moiety can also include unnatural amino acids, such as D-amino acids, in the peptide backbone structure to enhance the stability of the peptide in vivo.

[0157] Some specific embodiments of the HSG portion may include, but are not limited to: -DOTA-D-Asp-D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH2 (IMP 271; SEQ ID NO: 64) -DOTA-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2 (IMP 277; SEQ ID NO: 65); -DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2 (IMP 288; SEQ ID NO: 66); -DOTA-D-Ala-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2 (IMP 281; SEQ ID NO: 67); -DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH2(IMP 284; SEQ ID NO: 68) -DOTA-D-Lys(HSG)-D-Glu-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH2(IMP 301; SEQ ID NO: 69) -[DOTA-D-Lys(HSG)-D-Ala-D-Lys(HSG)-D-Glu-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH2 (IMP 302; SEQ ID NO: 70) -DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-D-Cys-NH2 (IMP 305; SEQ ID NO: 71) -Ac-D-Lys(In-DTPA)-D-Tyr-D-Lys(In-DTPA)-D-Lys(Tscg-Cys)-NH2 (IMP 297; SEQ ID NO: 72) -HCO-CO-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2 (IMP 289; SEQ ID NO: 73); -Ac-D-Phe-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-NH2 (SEQ ID NO: 74); -Ac-D-Phe-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2 (SEQ ID NO: 75); -Ac-D-Phe-D-Lys(Bz-DTPA)-D-Tyr-D-Lys(Bz-DTPA)-NH2 (SEQ ID NO: 76); -Ac-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH2 (SEQ ID NO: 77); -DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH2 (SEQ ID NO: 78); -(Tscg-Cys)-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(DOTA)-NH2 (SEQ ID NO: 79); -Tscg-D-Cys-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2 (SEQ ID NO: 80); -(Tscg-Cys)-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2 (SEQ ID NO: 81); -Ac-D-Cys-D-Lys(DOTA)-D-Tyr-D-Ala-D-Lys(DOTA)-D-Cys-NH(SEQ ID NO: 82); -Ac-D-Cys-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2 (SEQ ID NO: 83); -Ac-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-D-Lys(Tscg-Cys)-NH2 (SEQ ID NO: 84); and -Ac-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-D-Lys(Tscg-Cys)-NH2 (SEQ ID NO: 85).

[0158] Peptides used as targetable constructs are conveniently synthesized on an automated peptide synthesizer using a solid support and standard techniques of iterative orthogonal deprotection and coupling. Free amino groups in peptides subsequently used for chelate conjugation are advantageously blocked with standard protecting groups, such as Boc groups, while the N-terminal residue may be acetylated (Ac-) to enhance serum stability. Such protecting groups will be known to those skilled in the art. See Greene and Wuts, Protective Groups in Organic Synthesis, 1999 (John Wiley and Sons, NY). In preferred embodiments, the HSG moiety may contain one or more hydrophilic chelating moieties, which can bind to metal ions and may also help ensure rapid in vivo clearance. Chelators may be selected for their specific metal-binding properties and can be easily exchanged.

[0159] Particularly useful metal chelating combinations include 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs. Macrocyclic chelators such as NOTA (1,4,7-triaza-cyclononane-N,N',N''-triacetic acid), DOTA, TETA (p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid), and NETA are also useful with a variety of metals. DTPA- and DOTA-type chelators, whose ligands contain hard-base chelating functionalities, such as carboxylate or amine groups, are most effective at chelating hard acid cations, particularly group IIa and group IIIa metal cations. Such metal chelate complexes can be made extremely stable by tailoring the ring size to the metal of interest. Other cyclic-type chelators, such as macrocyclic polyethers, are of interest for stably binding nuclides. Porphyrin chelators can be used with numerous metal complexes. More than one type of chelator can be conjugated to a carrier to bind multiple metal ions. Chelators such as thiosemicarbazonylglyoxylcysteine ​​(Tscg-Cys) and thiosemicarbazinyl-acetylcysteine ​​(Tsca-Cys) are advantageously used to bind soft acid cations of Tc, Re, Bi, and other transition metals, lanthanides, and actinides that are tightly bound to soft base ligands. It may be useful to attach more than one type of chelator to a peptide. In certain embodiments, two different hard or soft acid chelators may be incorporated into a targetable construct, e.g., with different chelator ring sizes, to preferentially bind two different hard or soft acid cations due to differences in cation size, chelator ring geometry, and preferred cation complex structures. This allows for the incorporation of two different metals into the HSG moiety and their eventual capture by the bispecific antibodies of the invention.

[0160] The HSG moiety may be labeled with or conjugated to one or more diagnostic and / or therapeutic agents. Specifically, the HSG moiety can include one or more HSG haptens, one or more therapeutic / diagnostic agents, and one or more chelators.

[0161] Some useful non-limiting examples are cyclic or bifunctional chelators such as diethylenetriaminepentaacetic acid (DTPA), and 1,4,7,10-tetra-azacyclododecane-N,N',N'',N''-tetraacetic acid, ca-DTPA, ibca-DTPA, 1B4M-DTPA, lys-DTPA, vinylDTPA, glu-DTPA, p-SCN-bn-DOTA, DOTA-NHS-ester, deferoxamine B or derivatives thereof; or linear or bifunctional chelators such as p-SCN-Bn-DTPA, HOPO and CHX-A''-DTPA, ethylenediaminetetraacetic acid (EDTA), DTPA, EDTMP, NOTA, TETA, DOTMP, NS, NS, HEDP. Further examples of suitable chelating agents include DOTA derivatives such as p-isothiocyanatobenzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (p-SCN-Bz-DOTA) and DTPA derivatives such as p-isothiocyanatobenzyl-diethylenetriaminepentaacetic acid (p-SCN-Bz-DTPA), the former being a cyclic chelating agent and the latter being a linear chelating agent.

[0162] Further examples of deferoxamine (desferrioxamine, DFO) and its derivatives include p-NCS-Bz-DFO, DFOSq, DFO * , oxoDFO * , DFO * Sq, DFO * -NCS, DFO * These include, but are not limited to, pPhe-NCS.

[0163] Specifically, the DFO may have the following structure:

[0164] [ka]

[0165] Specifically, DFO * can have the following structure:

[0166] [ka]

[0167] The peptide backbone and DOTA may be directly linked or may be linked via a cleavable or non-cleavable linker. In certain embodiments, the HSG moieties disclosed herein can be conjugated to one or more therapeutic and / or diagnostic agents. The therapeutic agents are specifically selected from the group consisting of radionuclides, antiangiogenic agents, drugs, prodrugs, proapoptotic agents, interfering RNA, photoactive therapeutic agents, cytotoxic agents (which may be chemotherapeutic agents or toxins), and combinations thereof. Useful drugs may have pharmaceutical properties selected from the group consisting of antimitotic agents, antikinase agents, alkylating agents, antimetabolites, antibiotics, alkaloids, antiangiogenic agents, proapoptotic agents, and combinations thereof.

[0168] In preferred embodiments, the cytotoxic agent is selected from, but not limited to, a microtubule inhibitor, a meiosis inhibitor, an RNA polymerase inhibitor, a topoisomerase inhibitor, a DNA damaging agent, and a ribosome inhibitor.

[0169] Microtubule inhibitors, or tubulin inhibitors, such as auristatins, block tubulin assembly, resulting in G2 / M cell cycle arrest. Monomethyl auristatin F and monomethyl auristatin E, auristatin derivatives (MMAF, MMAE), are effective in the low nanomolar range (Gerber et al., 2009). Maytansinoids, or maytansine derivatives, are another class of tubulin inhibitors, such as emtansine (DM1) and ravtansine (DM4). Tubulysin and its analogs are a further class of tubulin inhibitors. Microtubule inhibitors (MTIs), such as taxanes, vinca alkaloids, and epothilones, stabilize or destabilize microtubules, thereby suppressing microtubule dynamics required for proper mitotic function, effectively blocking cell cycle progression and leading to apoptosis (Perez E., 2009).

[0170] Meiosis inhibitors may be, for example, cyclin-dependent kinase 2 (CDK2), such as flavopiridol and CY-202 (Malumbres M. et al. 2008). DNA damaging agents have the ability to be active throughout different cell cycle phases. Duocarmycins are cytotoxic DNA-alkylating compounds that bind to the minor groove of DNA. Anthracyclines and analogs, such as carminomycin, daunorubicin, and doxorubicin, are DNA-intercalating compounds that can be used as drug conjugates. Calicheamicin is a potent antitumor antibiotic that causes double-stranded DNA breaks and rapid cell death by binding to the minor groove of DNA. Calicheamicin is less dependent on cell cycle progression and may be useful against TICs with low proliferation rates (Gupta et al. 2009; Sapra et al. 2011). Alternatively, ozogamicin may be used. Another emerging category of DNA damaging agents is pyrrolobenzodiazepines (PBDs), which bind to distinct DNA sequences and cause lethal damage.

[0171] A potential new drug under investigation is α-amanitin, a picomolar range RNA polymerase II inhibitor derived from the mushroom, Amanita phalloides (Moldenhauer et al., 2012).

[0172] Other exemplary drugs that are useful include 5-fluorouracil, aplidine, azaribine, anastrozole, anthracyclines, bendamustine, bleomycin, bortezomib, bryostatin-1, busulfan, calicheamicin, camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, CELEBREX® (celecoxib), chlorambucil, cisplatin (CDDP), Cox-2 inhibitors, irinotecan (CPT-11), SN-3 8, carboplatin, cladribine, camptothecan, cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin, daunorubicin, doxorubicin, 2-pyrrolinodoxorubicin (2P-DOX), cyano-morpholinodoxorubicin, doxorubicin glucuronide, epirubicin glucuronide, estramustine, epipodophyllotoxin, estrogen receptor binding agents, etoposide (VP16), etoposide glucuronide, etoposide phosphate, flocc FudR, 3',5'-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide, farnesyl-protein transferase inhibitors, gemcitabine, hydroxyurea, idarubicin, ifosfamide, L-asparaginase, lenalidomide, leucovorin, lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, naphthalene These include, but are not limited to, berbine, nitrosoureas, plicomycin, procarbazine, paclitaxel, pentostatin, PSI-341, raloxifene, semustine, streptozocin, tamoxifen, Taxol®, temozolomide (aqueous form of DTIC), transplatinum, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracil mustard, vinorelbine, vinblastine, vincristine, and vinca alkaloids.

[0173] Useful toxins may include ricin, abrin, alpha toxin, saporin, ribonucleases (RNases) such as onconase, DNase I, Staphylococcal enterotoxin A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas aeruginosa exotoxin, and Pseudomonas aeruginosa endotoxin.

[0174] Radioisotopes (radioisotopes, radionuclides) useful for treating diseased tissue include: 11 C. 13 N, 15 O. 18 F, 32 P, 33 P, 47 Sc, 59 Fe, 64 Cu, 67 Cu, 67 Ga, 75 Se, 77 As, 89 Sr, 89 Zr, 90 Y, 99 mTc, 99 Mo, 103 Pd, 105 Rh, 109 Pd, 111 Ag, 111 In, 123 I, 124 I, 125 I, 131 I, 140 La, 142 Pr, 143 Pr, 149 Tb, 149 Pm, 153 Sm, 159 Gd, 161 Tb, 165 Dy, 166 Dy, 166 Ho, 169 Yb, 169 Er, 175 Yb, 177 Lu, 186 Re, 188 Re, 189 Re, 192 Ir, 193 mPt, 195 mPt, 194 Ir, 198 Au,199 Au, 211 At, 211 Pb. 212 Pb, 212 Bi, 213 Bi, 211 At, 223 Ra, 225 Ac and 227 These include, but are not limited to, Th.

[0175] Particularly useful therapeutic radionuclides include: 32 P, 33 P, 47 Sc, 64 Cu, 67 Cu, 67 Ga, 90 Y, 111 Ag, 111 In, 125 I, 131 I, 142 Pr, 153 Sm, 161 Tb, 166 Dy, 166 Ho, 177 Lu, 186 Re, 188 Re, 189 Re, 212 Pb, 212 Bi, 213 Bi, 211 At, 223 Ra and 225 Includes, but is not limited to, Ac.

[0176] Particularly useful diagnostic / detection radionuclides include: 18 F, 52 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 86 Y, 89 Zr, 94 mTc, 94 Tc, 99 mTc, 111 In, 123 I, 124 I, 125 I, 131 I, 154~158Gd, 32 P, 90 Y, 188 Re, and 175 These include, but are not limited to, Lu.

[0177] Therapeutic radionuclides preferably have decay energies in the range of 20 to 6,000 keV, preferably 60 to 200 keV for Auger emitters, 100 to 2,500 keV for beta emitters, and 4,000 to 6,000 keV for alpha emitters. Maximum decay energies for useful beta particle-emitting nuclides are preferably 20 to 5,000 keV, more preferably 100 to 4,000 keV, and most preferably 500 to 2,500 keV. Radionuclides that decay substantially by Auger-emitting particles are also preferred. For example: 58 Co, 67 Ga, 80 mBr, 99 mTc, 103 mRh, 109 Pt, 111 In, 119 Sb, 125 I, 161 Ho, 189 mOs and 192 Ir. Useful beta particle-emitting nuclides preferably have decay energies of <1,000 keV, more preferably <100 keV, and most preferably <70 keV. Also preferred are radionuclides that decay substantially by producing alpha particles. Such radionuclides include: 152 Dy, 211 At, 212 Bi, 223 Ra, 219 Rn, 215 Po, 211 Bi, 225 Ac, 221 Fr, 217 At, 213 Bi and 255 Useful alpha particle-emitting radionuclides include, but are not limited to, Fm. Decay energies of useful alpha particle-emitting radionuclides are preferably 2,000 to 10,000 keV, more preferably 3,000 to 8,000 keV, and most preferably 4,000 to 7,000 keV.

[0178] Specifically, the radionuclides are: 67 Ga, 89 Zr, 111 In, 124 I, 131 I, 177 Lu, and 225 Ac. Therapeutic agents may also include photoactive agents or dyes. Fluorescent compositions, such as fluorochromes, and other chromogens or dyes, such as porphyrins, that are sensitive to visible light, have been used to detect and treat lesions by directing the appropriate light toward the lesion. In therapy, this is called photoradiation, phototherapy, or photodynamic therapy.

[0179] In certain embodiments, anti-angiogenic agents such as angiostatin, baculostatin, canstatin, maspin, tissue inhibitor of metalloproteinase, 2-methoxyestradiol, carboxyamidotriazole, CM101, marimastat, pentosan polysulfate, herbimycin A, PNU145156E, linomide, thalidomide, pentoxifylline, genistein, TNP-470, endostatin, paclitaxel, accutin, angiostatin, cidofovir, vincristine, bleomycin, AGM-1470, or minocycline may be useful.

[0180] Therapeutic agent can comprise oligonucleotide such as siRNA.Those skilled in the art will understand that any siRNA or interfering RNA species can be combined with targetable construct for delivery to target tissue.Many siRNA species for a wide variety of targets are known in the art, and any such known siRNA can be utilized in the claimed method and composition.

[0181] Known potentially useful siRNA species include those specific for IKK-gamma VEGF, Flt-1, and Flk-1 / KDR, Bcl2 and EGFR, CDC20, transducin (beta)-like 3; KRAS; carbonic anhydrase II; complement component 3; interleukin-1 receptor-associated kinase 4 (IRAK4); survivin; superoxide dismutase 1; MET proto-oncogene; amyloid beta precursor protein (APP); IGF-1R; ICAM1; complement factor B; p53, and apolipoprotein B.

[0182] The diagnostic agent is preferably selected from the group consisting of a radionuclide, a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasound contrast agent, and a photoactive agent. Such diagnostic agents are well known, and any such known diagnostic agent can be used. Non-limiting examples of diagnostic agents include radionuclides, e.g., 18 F, 52 Fe, 110 In, 111 In, 177 Lu, 52 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 86 Y, 90 Y, 89 Zr, 94m Tc, 94 Tc, 99m Tc, 120 I, 123 I, 124 I, 125 I, 131 I, 154-158 Gd, 32 P, 11 C. 13 N, 15 O. 186 Re, 188 Re, 51 Mn, 52m Mn, 55 Co, 72 As, 75 Br, 76 Br, 82m Rb, 83Sr, or other gamma-, beta-, or positron-emitters may be included.

[0183] The three most commonly used PET radionuclides are: 18 F, 68 Ga and 89 It is Zr. Synthetic peptides may also be useful herein. For example, IMP-288 and IMP-449 are Tyr-D-Lys-D-Glu-D-Lys (SEQ ID NO: 86) tetrapeptides in which both lysine residues are substituted with HSG moieties via their ε-amino groups. IMP-449 is conjugated with NOTA: 1,4,7-triazacyclononane-N,N',N''-triacetic acid.

[0184] IMP-288 is a DOTA-conjugated D-Tyr-D-Lys-D-Glu-D-Lys-NH2 (SEQ ID NO: 87) tetrapeptide in which both lysine residues are derivatized with HSG moieties through their ε-amino groups.

[0185] IMP-453, IMP402, IMP457, or IMP498 as described in US9352036B2 may also be used. Useful paramagnetic ions may include chromium(III), manganese(II), iron(III), iron(II), cobalt(II), nickel(II), copper(II), neodymium(III), samarium(III), ytterbium(III), gadolinium(III), vanadium(II), terbium(III), dysprosium(III), holmium(III), or erbium(III). Metallic contrast agents may include lanthanum(III), gold(III), lead(II), or bismuth(III).

[0186] The ultrasound contrast agent may include a liposome, such as a gas-filled liposome. The radiopaque diagnostic agent may be selected from compounds, barium compounds, gallium compounds, and thallium compounds. A wide variety of fluorescent labels are known in the art, including, but not limited to, fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescamine. Useful chemiluminescent labels may include luminol, isoluminol, aromatic acridinium ester, imidazole, acridinium salt, europium(III), or oxalate ester.

[0187] The HSG moiety can have a variety of structures and is selected based not only on the availability of antibodies or fragments that bind to the HSG moiety with high affinity, but also on its rapid in vivo clearance when used in pretargeting methods. Hydrophobic agents are optimal for eliciting a strong immune response, while hydrophilic agents are preferred for rapid in vivo clearance. Therefore, a balance between hydrophobic and hydrophilic properties is established. This can be achieved, in part, by using hydrophilic chelators to offset the inherent hydrophobicity of many organic moieties. Alternatively, subunits of the targetable construct with opposite solution properties may be selected, such as peptides containing amino acids that are partly hydrophobic and partly hydrophilic. In addition to peptides, carbohydrates can also be used.

[0188] Pretargeting is a multi-step process originally developed to solve the slow blood clearance of direct targeting antibodies, which contributes to undesirable toxicity to normal tissues.By pretargeting, diagnostic or therapeutic agents such as radionuclides are bound to delivery small molecules that are removed from the blood within minutes.According to an embodiment of the present invention, the delivery small molecule is the HSG moiety described herein.The pretargeting bispecific antibody of the present invention, which has an HSG moiety and one or more binding sites for target tumor antigens, is first administered, and free antibody is removed from circulation, and then the targetable construct is administered.

[0189] A pretargeting method for treating or diagnosing a disease or disorder in a subject can be provided by: (1) administering to the subject a bispecific antibody; (2) optionally administering to the subject a clearing composition, allowing the composition to clear the antibody from circulation; and (3) administering to the subject an HSG moiety described herein containing one or more chelated or chemically bound therapeutic or diagnostic agents.

[0190] The bispecific antibodies described herein can be used to treat or detect malignant tumors in a pretargeting method, in which the antibody can be first administered to a subject, and the bispecific antibody can be allowed to bind to the target antigen and be allowed to clear the bound antibody from the circulation.

[0191] In some cases, a clearing composition may be administered after the antibody has been administered and the clearing composition has removed the antibody from the circulation. Clearing compositions may include, but are not limited to, antibodies against avidin, galactose, pretargeting antibodies, and Fc-antigen fusions. The ABDEG (antibody that enhances IgG degradation) clearing approach involves administering an antigen-specific IgG antibody followed by an unrelated IgG-YTE (an antibody with enhanced FcRn binding due to the M252Y / S254T / T256E mutations in the Fc domain) that enhances the degradation / clearance of free antigen-specific antibodies from the circulation by binding more strongly to FcRn after the antigen-specific mAb has localized to the tumor (Nazarova L. et al., 2020).

[0192] A targetable construct, such as the HSG moiety described above, labeled with or linked to a diagnostic agent, such as a radionuclide, can then be administered to the subject, allowing it to bind to the bispecific antibody and localize to diseased cells or tissues.

[0193] The slow blood clearance of directly radiolabeled antibodies and delayed tumor uptake in solid tumors result in continuous high radiation exposure to healthy tissues and organs. In vivo pretargeting radioimmunotherapy (PRAIT) using the antibodies of the present invention can overcome these limitations. PRAIT aims to improve the therapeutic index (tumor-to-normal tissue ratio) by delivering an increased absorbed dose to the tumor compared to directly radiolabeled antibodies or antibody fragments. PRAIT requires the administration of a bispecific antibody described herein that recognizes HSG and a tumor target, followed by the administration of a radiolabeled HSG hapten moiety. The administration of the radiolabeled HSG hapten moiety can be several days later, for example, 2, 3, 4, 5, 6, or 7 days later. This technique allows a significant amount of the antibody of the present invention to accumulate in or on the surface of tumors or malignant cells and is largely removed from the circulation, while the radiolabeled HSG hapten moiety binds to the antibody accumulated in or on the surface of tumors or malignant cells, while the unbound radioactive HSG hapten is removed from the circulation via the kidney within a few hours, thus minimizing exposure of normal organs to radioactivity.

[0194] For targeted tissue radionuclide therapy, for example, HSG hapten IMP-288 (described in US2005 / 0025709) and 177 Lu may be used. For treatment purposes, pretargeting approach can also be used as described above. In this case, HSG part comprises one or more of the above-mentioned therapeutic agents. Specifically, HSG hapten IMP-453 can be used, which is directly linked to therapeutic agent.

[0195] A "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0196] According to the present invention, the antibodies described herein may be used in the preparation of a medicament. Also encompassed herein are pharmaceutical compositions containing the antibodies of the present invention. The term "pharmaceutical composition" refers to a preparation of an active ingredient contained therein in a form that allows the biological activity of the active ingredient to be effective, together with pharmaceutical excipients such as carriers, and that does not contain additional ingredients that would be unacceptably toxic to the subject to which the formulation is administered.

[0197] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation other than the active ingredient that is non-toxic to a subject. Some examples of pharmaceutically acceptable carriers are water, saline, phosphate buffered saline, amino acids such as glycine or histidine, glucose, glycerol, ethanol, etc., and combinations thereof. In many cases, it is preferable to include an isotonic agent, such as a sugar, a polyalcohol such as mannitol, sorbitol, or sodium chloride, in the composition. Additional examples of pharmaceutically acceptable substances are wetting agents or minor amounts of auxiliary substances, such as wetting agents or emulsifying agents, preservatives, or buffers, which improve the shelf life or effectiveness of the antibody.

[0198] As used herein, "treatment," "treat," or "treating" refers to a clinical intervention to alter the natural course of the disease in the individual being treated, and can be performed prophylactically or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, alleviation of any direct or indirect pathological consequences, prevention of metastasis, slowing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, the antibodies of the invention are used to delay disease onset or slow disease progression.

[0199] The bispecific anti-tumor antigen / anti-HSG antibodies of the present invention and pharmaceutical compositions comprising same may be administered to a subject in a first step, and the HSG moiety is administered in a second step, said HSG moiety being bound to the antibody.

[0200] The pharmaceutical compositions of the present invention may be in a variety of forms, including liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injection and infusion solutions), dispersions or suspensions, tablets, pills, and powders. The preferred form depends on the intended method of administration and therapeutic or diagnostic application. Typical preferred compositions are in the form of injections or infusions, such as compositions similar to those used for passive immunization in humans. A preferred method of administration is parenteral (e.g., intravenous, intraarterial, intralymphatic, or intrathecal). In a preferred embodiment, the antibody is administered by intravenous infusion or injection. In another preferred embodiment, the antibody is administered by intravenous injection. As will be appreciated by those skilled in the art, the route and / or method of administration will vary depending on the desired results.

[0201] Administration of the bispecific anti-tumor antigen / anti-HSG antibody and labeled or conjugated HSG moiety of the present invention can be accomplished by administering the bsAb antibody at a time prior to administration of the labeled or conjugated HSG moiety. The dosage and timing of the reagents can be readily devised by those skilled in the art and will depend on the specific properties of the reagents used. In certain embodiments, the bispecific anti-tumor antigen / anti-HSG antibody is administered first, followed by administration of the labeled or conjugated HSG moiety after a sufficient time has passed for the bsAb of the present invention to target the diseased tissue, specifically 24-72 hours, or in alternative embodiments, 48-96 hours. When the bispecific anti-tumor antigen / anti-HSG antibody of the present invention is administered, particularly via a pretargeting method, the dosage of the antibody administered to a human will vary depending on factors such as the patient's age, weight, height, sex, general medical condition, and previous medical history. Typically, it is desirable to provide the recipient with a dose of bispecific antibody ranging from about 1 mg to 400 mg as a single intravenous infusion, although lower or higher doses may be administered depending on the circumstances. Typically, it is desirable to provide a recipient with a dosage of 1 to 200 mg, more preferably 1 to 70 mg, and most preferably 1 to 20 mg, although higher or lower doses may be used. The dosage of a therapeutic bispecific antibody may be higher, for example, 1 to 200 mg, 1 to 100 mg, 100 to 1000 mg, 100 to 500 mg, or 200 to 750 mg. The dosage for each subject depends on conditions such as body weight or the type of disease. However, the dosage can be determined by one of ordinary skill in the art with reference to general knowledge.

[0202] Generally, the dosage of the label or conjugated HSG moiety to be administered varies depending on factors such as patient's age, weight, height, sex, general medical condition and previous medical history.Preferably, the saturation dose of label or conjugated HSG moiety is administered to the patient.For the administration of radiolabeled molecule, dosage can be measured by millicurie.

[0203] Anti-tumor antigen / anti-HSG The antibodies described herein may be administered once, or in alternative embodiments, multiple times, each time followed by administration of a labeled or conjugated HSG moiety for diagnosis and / or treatment of malignant tumors.

[0204] In a preferred embodiment, the anti-tumor antigen / anti-HSG antibodies of the present invention are useful in the treatment of cancer. The term "cancer", as used herein, refers to malignant tumors, particularly proliferative diseases, particularly solid cancers such as colorectal cancer, ovarian cancer, pancreatic cancer, lung cancer, melanoma, squamous cell carcinoma (SCC) (e.g., of the head and neck, esophagus, and oral cavity), hepatocellular carcinoma, colorectal adenocarcinoma, renal cancer, medullary thyroid carcinoma, papillary thyroid carcinoma, astrocytoma, neuroblastoma, Ewing's sarcoma, non-Hodgkin's lymphoma, B-cell acute and chronic lymphocytic leukemia, Burkitt's lymphoma, Hodgkin's lymphoma, hairy cell leukemia, acute and chronic myeloid leukemia, T-cell lymphoma and leukemia, multiple myeloma, glioma, Waldenstrom's macroglobulinemia, melanoma, sarcoma, glioma, skin cancer, cervical cancer, endometrial cancer, breast cancer, prostate cancer, gastric cancer, and malignant seminoma (including refractory forms of any of the above cancers, or a combination of one or more of the above cancers).

[0205] Hyperproliferative disorders such as cancer, cancerous diseases that can be treated or detected by the anti-tumor antigen / anti-HSG antibodies of the present invention can affect any tissue or organ, including, but not limited to, brain, squamous cell, bladder, head, neck, liver, ovary, esophageal, nasopharyngeal, or thyroid cancer, melanoma, lymphoma, leukemia or multiple myeloma, oral cavity, gastrointestinal tract, colon, colorectum, stomach, pancreas, pulmonary tract, lung, breast, ovary, prostate, uterus, endometrium, cervix, bladder, pancreas, bone, liver, gallbladder, kidney, skin, and testes.

[0206] In particular, the anti-tumor antigen / anti-HSG antibodies of the present invention are useful in treating hematological and solid tumors, such as colorectal, ovarian, breast, prostate, pancreatic, and lung cancers.

[0207] In a particular embodiment, an antibody highly suitable for the treatment of cancerous diseases, in particular for the treatment of solid tumors, is a bispecific anti-tumor antigen / anti-HSG antibody comprising a binding site that specifically recognizes HSG, - a light chain variable (VL) domain comprising SEQ ID NO: 17 or SEQ ID NO: 19, in particular having glutamine at position 100 (Q100) and isoleucine at position 106 (I106) according to the Kabat numbering, or a sequence having at least 95% identity to SEQ ID NO: 17 or SEQ ID NO: 19, and - a heavy chain variable (VH) domain comprising a sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, and 10, in particular having an arginine at position 19 (R19) according to the Kabat numbering, or a sequence having at least 95% identity to SEQ ID NOs: 4, 6, 8, and 10; Including, - optionally, the VL and VH domains are linked via a linker sequence, and the binding site that specifically recognizes a tumor antigen is (a1) a light chain variable domain comprising SEQ ID NO: 45 with one or more amino acid substitutions M30L, F49Y, A51G, P80S, W93F, or (a2) a light chain variable domain having at least 95% sequence identity to SEQ ID NO: 45 with the conserved tyrosine at position 36 and one or more amino acid substitutions M30L, F49Y, A51G, P80S, W93F; and (b1) a heavy chain variable domain comprising SEQ ID NO: 46, or (b2) a heavy chain variable domain comprising SEQ ID NO: 46 with the amino acid substitutions L5Q and / or W97Y; or (b3) a heavy chain variable domain having at least 95% sequence identity to SEQ ID NO: 46 with the amino acid substitutions L5Q and / or W97Y. Including, It is a bispecific anti-tumor antigen / anti-HSG antibody, with amino acid positions numbered according to Kabat.

[0208] In a further particular embodiment, an antibody highly suitable for the treatment of cancerous diseases, in particular for the treatment of solid tumors, is a bispecific anti-tumor antigen / anti-HSG antibody comprising a binding site that specifically recognizes HSG, - a light chain variable (VL) domain comprising SEQ ID NO: 17 or SEQ ID NO: 19, in particular having glutamine at position 100 (Q100) and isoleucine at position 106 (I106) according to the Kabat numbering, or a sequence having at least 95% identity to SEQ ID NO: 17 or SEQ ID NO: 19, and - a heavy chain variable (VH) domain comprising a sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, and 10, in particular with an arginine at position 19 (R19), or a sequence having at least 95% identity to SEQ ID NOs: 4, 6, 8, and 10; Including, Optionally, the VL and VH domains are linked via a linker sequence, and the binding site that specifically recognizes a tumor antigen is a light chain variable domain comprising SEQ ID NO: 27, or a light chain variable domain comprising SEQ ID NO: 27 and further comprising the amino acid substitutions M30L and / or P80S, and - a heavy chain variable domain comprising SEQ ID NO: 46, specifically with the amino acid substitutions L5Q and / or W97Y wherein the amino acid positions are numbered according to Kabat.

[0209] In a further particular embodiment, the antibody is a bispecific anti-tumor antigen / anti-HSG antibody comprising a binding site that specifically recognizes HSG, - a light chain variable (VL) domain comprising any one of SEQ ID NOs: 17, 19, 89, 90 and 91, and a heavy chain variable (VH) domain comprising a sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, and 10 and a bispecific anti-tumor antigen / anti-HSG antibody comprising:

[0210] In yet an alternative embodiment, the antibody is a bispecific anti-tumor antigen / anti-HSG antibody comprising a binding site that specifically recognizes HSG, - a light chain variable (VL) domain comprising a sequence selected from the group consisting of SEQ ID NOs: 17, 19, 89, 90, and 91, and - a heavy chain variable (VH) domain comprising a sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, and 10, with one or two additional amino acid substitutions and with an arginine at position 19 (R19) according to the Kabat numbering; and a bispecific anti-tumor antigen / anti-HSG antibody further comprising:

[0211] In yet an alternative embodiment, the antibody is a bispecific anti-tumor antigen / anti-HSG antibody comprising a binding site that specifically recognizes HSG, - a light chain variable (VL) domain comprising a sequence selected from the group consisting of SEQ ID NOs: 17, 19, 89, 90, and 91, which has one or two additional amino acid substitutions and has a glutamic acid at position 105 (E105) and an isoleucine at position 106 (I106) according to the Kabat numbering, and a heavy chain variable (VH) domain comprising a sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, and 10 and a bispecific anti-tumor antigen / anti-HSG antibody further comprising:

[0212] In yet an alternative embodiment, the antibody is a bispecific anti-tumor antigen / anti-HSG antibody comprising a binding site that specifically recognizes HSG, A light chain variable (VL) domain comprising a sequence selected from the group consisting of SEQ ID NOs: 17, 19, 89, 90, and 91, with the proviso that glutamic acid at position -105 (E105) and isoleucine at position 106 (I106) are conserved, and comprising one or two additional amino acid substitutions; and - a heavy chain variable (VH) domain comprising a sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, and 10, with one or two additional amino acid substitutions and with an arginine at position 19 (R19) according to the Kabat numbering; and a bispecific anti-tumor antigen / anti-HSG antibody further comprising:

[0213] Optionally, the sequence also includes glutamine at position 100 according to the Kabat numbering (Q100). Also provided herein are nucleic acids encoding the antibodies of the invention.

[0214] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or in a chromosomal location that is different from its natural chromosomal location.

[0215] "Isolated nucleic acid encoding an anti-tumor antigen / anti-HSG antibody" refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecules in a single vector or separate vectors, and including such nucleic acid molecules present in one or more locations in a host cell.

[0216] "Lack of substantial cross-reactivity" means that a molecule (e.g., an antibody) does not recognize or specifically bind to an antigen (e.g., an antigen closely related to the target antigen) other than the molecule's actual target antigen, specifically reduced MIF, particularly when compared to its target antigen. For example, an antibody may bind to an antigen other than the actual target antigen at less than about 10% to less than about 5%, or may bind to an antigen other than the actual target antigen at less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1%, preferably less than about 2%, 1%, or 0.5%, and most preferably less than about 0.2% or 0.1%. Binding can be determined by any method known in the art, including, but not limited to, ELISA or surface plasmon resonance.

[0217] Recombinant production of the antibodies of the invention preferably uses an expression system, including, for example, an expression construct or vector containing a nucleotide sequence encoding the antibody format. The term "expression system" refers to a nucleic acid molecule containing desired coding and control sequences operably linked such that a host transformed or transfected with these sequences can produce the encoded protein. To effect transformation, the expression system may be included in a vector; however, the associated DNA may also then be integrated into the host chromosome. Alternatively, the expression system may be used for in vitro transcription / translation.

[0218] As used herein, an "expression vector" is defined as a DNA sequence required for the transcription of cloned recombinant nucleotide sequences, i.e., recombinant genes, and the translation of their mRNAs, in a suitable host organism. An expression vector contains an expression cassette and typically also contains an origin of autonomous replication or a genome integration site in a host cell, one or more selectable markers (e.g., amino acid synthesis genes, or genes that confer resistance to antibiotics such as zeocin, kanamycin, G418, or hygromycin), several restriction enzyme cleavage sites, a suitable promoter sequence, and a transcription terminator, with these components operably linked. As used herein, the terms "plasmid" and "vector" include autonomously replicating nucleotide sequences and genome-integrating nucleotide sequences.

[0219] Specifically, the term refers to a vehicle by which a DNA or RNA sequence (e.g., a foreign gene), such as a nucleotide sequence encoding an antibody format of the invention, can be introduced into a host cell in order to transform the host and promote expression (e.g., transcription and translation) of the introduced sequence. Plasmids are preferred vectors of the present invention.

[0220] A vector typically contains the DNA of a transmissible agent into which foreign DNA has been inserted. A common method for inserting one segment of DNA into another segment of DNA requires the use of enzymes called restriction enzymes, which cut the DNA at specific sites (specific groups of nucleotides) called restriction sites.

[0221] A "cassette" refers to a DNA coding sequence or segment of DNA that encodes an expression product that can be inserted into a vector at a defined restriction site. The cassette restriction sites are designed to ensure insertion of the cassette in the proper reading frame. Generally, foreign DNA is inserted at one or more restriction sites in the vector DNA and then carried by the vector along with the transmissible vector DNA into a host cell. A DNA segment or sequence into which DNA has been inserted or added, such as an expression vector, can also be referred to as a "DNA construct." A common type of vector is the "plasmid." Plasmids are generally self-contained molecules of double-stranded DNA that can readily accept additional (foreign) DNA and can be easily introduced into suitable host cells. Vectors of the present invention often contain coding DNA and expression control sequences, such as promoter DNA, and have one or more restriction sites suitable for inserting foreign DNA. Coding DNA is a DNA sequence that encodes a specific amino acid sequence of a particular polypeptide or protein, such as the antibody format of the present invention. Promoter DNA is a DNA sequence that initiates, regulates, or otherwise mediates or controls expression of coding DNA. The promoter DNA and coding DNA may be from the same gene or from different genes, and may be from the same or different organisms. Recombinant cloning vectors of the invention often contain one or more replication systems for cloning or expression, one or more markers for selection in the host (e.g., antibiotic resistance), and one or more expression cassettes.

[0222] The procedures used to ligate DNA sequences (e.g., providing or encoding elements of the invention and / or proteins of interest, promoters, terminators, and additional sequences, respectively) and insert them into appropriate vectors containing the information necessary for integration or host replication are well known to those skilled in the art and are described, for example, by Sambrook et al., 2012.

[0223] Also encompassed herein is the production of the antibodies of the invention using the host cells. A host cell is understood in particular to be a cell, recombinant cell or cell line which has been transfected with an expression construct such as a vector according to the invention.

[0224] The term "host cell line," as used herein, refers to an established clone of a particular cell type that has acquired the ability to grow over an extended period of time. The term host cell line refers to a cell line used to express endogenous or recombinant genes to produce a polypeptide, such as a recombinant antibody format of the present invention.

[0225] A "production host cell" or "production cell" is generally understood to be a cell line or cell culture ready for cultivation in a bioreactor to obtain the product of the production process, the recombinant antibody format of the invention. The host cell type according to the present invention may be any prokaryotic or eukaryotic cell.

[0226] The term "recombinant," as used herein, is intended to mean "prepared by genetic engineering" or "the result of genetic engineering," e.g., specifically using heterologous sequences incorporated into a recombinant vector or recombinant host cell.

[0227] The antibodies of the present invention can be produced using any known and well-established expression system and recombinant cell culture technology, for example, by expression in bacterial hosts (prokaryotic systems) or eukaryotic systems such as yeast, fungi, insect cells, or mammalian cells. The antibody molecules of the present invention can be produced in transgenic organisms, for example, goats, plants, or transgenic mice (modified mouse strains carrying large fragments of human immunoglobulin loci and deficient in mouse antibody production). Antibodies can also be produced by chemical synthesis.

[0228] According to particular embodiments, the host cells are production cell lines of cells selected from the group consisting of CHO, PerC6, CAP, HEK, HeLa, NS0, SP2 / 0 hybridoma and Jurkat. More particularly, the host cells are derived from CHO cells.

[0229] The host cells of the present invention are specifically cultured or maintained in serum-free culture (eg, containing other components such as plasma proteins, hormones, and growth factors as a replacement for serum). It is most preferred if the host cells are established, adapted and entirely cultured under serum-free conditions and optionally in a medium that is free of any proteins / peptides of animal origin.

[0230] The anti-tumor antigen / anti-HSG antibody of the invention can be recovered from the culture medium using standard protein purification methods. The present invention further encompasses the following embodiments: 1. A bispecific anti-tumor antigen / anti-HSG antibody comprising a binding site that specifically recognizes a tumor antigen and a binding site that specifically recognizes HSG, - a light chain variable (VL) domain comprising SEQ ID NO: 17 or SEQ ID NO: 19, which has glutamine at position 100 (Q100) and isoleucine at position 106 (I106) according to the Kabat numbering, or a sequence having at least 95% identity to SEQ ID NO: 17 or 19, and - a heavy chain variable (VH) domain comprising a sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, and 10 with an arginine at position 19 (R19) according to the Kabat numbering, or a sequence having at least 95% identity to SEQ ID NOs: 4, 6, 8, and 10; A bispecific anti-tumor antigen / anti-HSG antibody comprising: 2. The antibody of embodiment 1, comprising a single-chain variable fragment (scFv) that specifically recognizes an HSG of a formula selected from the group consisting of VH-linker-VL-linker, VL-linker-VH-linker, wherein the linker comprises SEQ ID NO: 39 or SEQ ID NO: 42. 3. An antibody described in embodiment 1, comprising VL and VH domains that specifically recognize HSG, wherein the VL domain is linked at its C-terminus to a CH1 cross comprising SEQ ID NO: 30, and the VH domain is linked at its C-terminus to a CL cross comprising SEQ ID NO: 38. 4. An antibody according to any one of embodiments 1 to 3, comprising an Fc region having SEQ ID NO: 31, which comprises asymmetric mutations in each CH3 domain that allow heterodimerization of two CHs from different antibodies, specifically one CH comprising "knob" mutations T366W and S354C according to the EU numbering index, and one CH comprising "hole" mutations T366S, L368A, Y407V, and Y349C. 5. The antibody of any one of embodiments 1 to 4, comprising a variant Fc region with reduced or eliminated effector function and / or FcRn binding. 6. The antibody of any one of embodiments 1 to 5, comprising a variant Fc region having an amino acid substitution at any one or more of positions E233, L234, L235, G236, G237, P238, I253, D265, S267, H268, N297, S298, T299, H310, E318, L328, P329, A330, P331, H435 of SEQ ID NO: 31 according to the EU numbering index, and optionally an aglycosylated Fc region. 7. An antibody described in any one of embodiments 1 to 3, comprising two constant heavy chain domains with hinge and Fc domains comprising SEQ ID NO: 35 and SEQ ID NO: 32. 8. An antibody described in any one of embodiments 1 to 3, comprising two constant heavy chain regions having hinge and Fc domains comprising SEQ ID NO: 36 and SEQ ID NO: 33. 9. The antibody of any one of embodiments 1 to 8, wherein the anti-tumor antigen is selected from the group consisting of oxMIF, mesothelin (MSLN), and folate receptor alpha (FRα). 10. An antibody described in any one of embodiments 1 to 9, wherein the anti-tumor antigen is oxMIF. 11. The binding site that specifically recognizes oxMIF is - a light chain variable domain comprising SEQ ID NO: 27, or a light chain variable domain comprising SEQ ID NO: 27 and further comprising the amino acid substitutions M30L and / or P80S, and - a heavy chain variable domain comprising SEQ ID NO: 46, specifically with the amino acid substitutions L5Q and / or W97Y wherein the amino acid positions are numbered according to Kabat. 12. The antibody according to any one of embodiments 1 to 11, which is selected from the group consisting of Fab-scFv-Fc, CrossMab, (scFv)2-Fc, scFv / scFv-Fc, Fab / (scFv)2-Fc, Fab / Fab-scFv-Fc (= IgG-central scFv), Fab / Fab-crossFab-Fc, Fab / crossFab-Fc, and IgG-scFv, IgG-(scFv)2. 13. The antibody of any one of embodiments 1 to 12 for use in treating or detecting a malignant tumor, wherein the antibody is administered to a subject in a first step and an HSG moiety is administered in a second step, and the HSG moiety binds to the antibody. 14. The antibody for use of embodiment 13, wherein the HSG moiety is conjugated to or labeled with one or more diagnostic and / or therapeutic agents, in particular the HSG moiety comprises one or more HSG haptens, one or more diagnostic and / or therapeutic agents, and optionally a chelator. 15. The antibody of any one of embodiments 1 to 12, wherein the antibody is bound to an HSG moiety conjugated to one or more diagnostic and / or therapeutic agents or labeled with one or more diagnostic and / or therapeutic agents, specifically wherein the HSG moiety comprises one or more HSG haptens, one or more diagnostic and / or therapeutic agents, and optionally a chelator. 16. The antibody for use according to embodiment 13 or 14, or the antibody according to embodiment 15, wherein the therapeutic agent is selected from a radionuclide, a chemotherapeutic agent, and a cytokine; and the diagnostic agent is a radionuclide. 17. Chelating agents bind to radionuclides, specifically DOTA, DTPA, deferoxamine B (DFO), and DFO * 17. The antibody of embodiment 16, selected from: 18. A radionuclide is 11 C. 13 N, 15 O. 18 F, 32 P, 33 P, 47 Sc, 59 Fe, 64 Cu, 67 Cu, 67 Ga, 75 Se, 77 As, 89 Sr, 89 Zr, 90 Y, 99 mTc, 99 Mo, 103 Pd, 105 Rh, 109 Pd, 111 Ag, 111 In, 123 I, 124 I, 125 I, 131 I, 140 La, 142 Pr, 143 Pr, 149 Tb, 149 Pm, 153 Sm, 159 Gd, 161 Tb, 165 Dy, 166 Dy,166 Ho, 169 Yb, 169 Er, 175 Yb, 177 Lu, 186 Re, 188 Re, 189 Re, 192 Ir, 193 mPt, 195 mPt, 194 Ir, 198 Au, 199 Au, 211 At, 211 Pb. 212 Pb, 212 Bi, 213 Bi, 211 At, 223 Ra, 225 Ac, and 227 18. The antibody of embodiment 16 or 17, selected from the group consisting of Th. 19. The antibody of any one of embodiments 1 to 12 for use in preparing a medicament. 20. A pharmaceutical composition comprising the antibody of any one of embodiments 1 to 12 together with a pharmaceutical excipient. 21. The pharmaceutical composition of embodiment 20, wherein the pharmaceutical composition is formulated for intravenous administration. 22. A pharmaceutical composition according to embodiment 20 or 21, for use in the treatment of patients suffering from cancer, particularly in the treatment of tumors, solid tumors, more particularly in the treatment of colorectal cancer, ovarian cancer, breast cancer, prostate cancer, pancreatic cancer and lung cancer. 23. An isolated nucleic acid encoding the antibody of any one of embodiments 1 to 12. 24. An expression vector comprising the nucleic acid of embodiment 23. 25. A method for diagnosing cancer in a subject in vivo, in which an antibody described in any one of embodiments 1 to 12 or an antibody described in any one of embodiments 15 to 18 is used to detect tumor cells. 26. A method for diagnosing cancer in vitro, in which the antibody according to any one of embodiments 1 to 12 or the antibody according to any one of embodiments 15 to 18 is used to detect tumor cells in a sample; 27. A method for treating cancer using an antibody described in any one of embodiments 1 to 12, or a pharmaceutical composition described in embodiment 20 or 21. [Example]

[0231] The examples described herein are illustrative of the present invention. Many changes and modifications can be made to the procedures described and illustrated herein without departing from the scope of the present invention. Therefore, it is understood that the examples are illustrative only and do not limit the scope of the present invention.

[0232] Example 1: Schematic of an exemplary anti-tumor antigen x anti-HSG bispecific mAb and amino acid sequence of an anti-oxMIF x anti-HSG bispecific mAb Figure 1 shows the results of Fab-scFv-Fc, CrossMab (CH1-C L Figure 1 provides a schematic diagram of anti-target X × anti-HSG bispecific mAbs with Fab-scFv-Fc, crossMab (CH1-CL crossover), and IgG-central scFv formats. Left: Fab-scFv-Fc, center: CrossMab (CH1-CL crossover), right: IgG-central scFv

[0233] [Table 1]

[0234] [Table 2-1]

[0235] [Table 2-2]

[0236] [Table 3-1]

[0237] [Table 3-2]

[0238] [Table 3-3]

[0239] [Table 4]

[0240] [Table 5]

[0241] [Table 6]

[0242] C0181 Polypeptide 1 (LC oxMIF) SEQ ID NO: 47 DIQMTQSPSSLSASVGDRVTITCRSSQRIMTYLNWYQQKPGKAPKLLIYVGSHSQSGVPSRFRGSGSETDFTLTISGLQPEDSATYYCQQSFFTPLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Polypeptide 2 (HC oxMIF) SEQ ID NO: 48 EVQLQESGGGLVQPGGSLRLSCAASGFTFSIYSMNWVRQAPGKGLEWVSSIGSSGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGSQYLYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPAEAPEAGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Polypeptide3(HSG HC) sequence number 49 EVQLVESGGDLVQPGGSLRLSCAASGFTFSIYTMSWVRQAPGKGLEWVATLSGDGDDIYYPDSVKGRFTISRDNAKNNLYLQMNSLRSADTALYYCARVRLGDWDFDVWGQGTTVTVSSGGSGGSGGSGGSGGSDVVMTQSPSSLAVSLGERVTINCKSSQSLFNSRTKNYLGWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQSEDVAVYYCTQVYYLCTFGQGTKLEIKGGGSDKTHTCPPCPPAEAPEAGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG C0182 Polypeptide 1 (LC oxMIF) SEQ ID NO: 47 Polypeptide 2 (HC oxMIF) SEQ ID NO: 48 Polypeptide 3 (HC HSG) SEQ ID NO: 50 EVQLVESGGDLVQPGGSLRLSCAASGFTFSIYTMSWVRQAPGKGLEWVATLSGDGDDIYYPDSVKGRFTISRDNAKNNLYLQMNSLRSADTALYYCARVRLGDWDFDVWGQGTTVTVSS GGSGGSGGSGGSGGSDIVMTQSPSSLAVSLGERATITCKSSQSLFNSRTRKNYLGWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQSEDLAVYYCTQVYYLCTFG QGTKLEIKGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG C0186 Polypeptide 1 (LC oxMIF) SEQ ID NO: 47 Polypeptide 2 (HC oxMIF) SEQ ID NO: 48 Polypeptide 3 (HC HSG) SEQ ID NO: 51 QVQLVESGGDLVKPGGSLRLSCAASGFTFSIYTMSWLRQTPEKRLEWVSTLSGDGDIYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCARVRLGDWDFDVWGQGTLVTVSS GGSGGSGGSGGSGGSDVVMTQSPSSLAVSLGERVTINCKSSQSLFNSRTRKNYLGWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQSEDVAVYYCTQVYYLCTFG QGTKLEIKGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG C0192 Polypeptide 1 (LC oxMIF) SEQ ID NO: 47 Polypeptide 2 (HC oxMIF) SEQ ID NO: 48 Polypeptide 3 (HC HSG) SEQ ID NO: 52 EVQLVESGGGLVKPGGSLRLSCAASGFTFSIYTMSWLRQTPEKRLEWVSTLSGDGDDIYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVRLGDWDFDVWGQGTTVSVSS GGSGGSGGSGGSGGSDIVMTQSPSSLAVSLGERATITCKSSQSLFNSRTRKNYLGWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQSEDLAVYYCTQVYYLCTFG QGTKLEIKGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG oxMIF epitope EPCALCS SEQ ID NO:53 C0239 Polypeptide 1 (LC oxMIF) SEQ ID NO: 47 Polypeptide 2 (HC oxMIF) SEQ ID NO: 54 EVQLQESGGGLVQPGGSLRLSCAASGTFSIYSMNWVRQAPGKGLEWVSSIGSSGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGSQYLYGMDVWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLAQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG Polypeptide 3 (HC HSG) SEQ ID NO: 55 EVQLVESGGDLVQPGGSLRLSCAASGFTFSIYTMSWVRQAPGKGLEWVATLSGDGDDIYYPDSVKGRFTISRDNAKNNLYLQMNSLRSADTALYYCARVRLGDWDFDVWGQGT TVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLAQDWLNGKEYKCKVSNKALAPIE KTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG Polypeptide 4 (LC HSG) SEQ ID NO: 56 DVVMTQSPSSLAVSLGERVTINCKSSQSLFNSRTRKNYLGWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQSEDVAVYYCTQVYYLCTFGQGTK LEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC C0240 Polypeptide 1 (LC oxMIF) SEQ ID NO: 47 Polypeptide 2 (HC oxMIF) SEQ ID NO: 54 Polypeptide 3 (HC HSG) SEQ ID NO: 55 Polypeptide 4 (LC HSG) SEQ ID NO: 57 DIVMTQSPSSLAVSLGERATITCKSSQSLFNSRTRKNYLGWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQSEDLAVYYCTQVYYLCTFGQGTK LEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC Figure 1 shows exemplary Fab-scFv-Fc, CrossMab, and IgG-central scFv formats of bispecific anti-tumor-targeted X x anti-HSG mAbs. Tables I and II show the anti-HSG VH (Table I) and anti-HSG VL (Table II) sequences and their respective closest human germlines. Table III shows the anti-oxMIF mAb sequences, mAb constant regions, and linkers. Table IV shows anti-oxMIF x anti-HSG humanized variants in Fab-scFv-Fc format. Table V shows anti-oxMIF x anti-HSG humanized variants in Crossmab format.

[0243] Example 2: In silico immunogenicity (humanization score) of newly humanized anti-HSG sequences versus VH and VL variants 1-5 of hz679 To generate novel humanized VH and VL sequences, the CDRs of the murine anti-HSG antibody mo679 (from US 20090246131 A1) were identified using a combination of the IMGT and Kabat numbering systems, which allows for optimal preservation of the CDR loop structure. A database of human IgG1 / Igk sequences was searched for comparison with the murine VH and VL regions of mo679 using the IgBLAST search algorithm (NCBI), and candidate human VH and VL domains were selected from the top 200 BLAST results. These were narrowed down to several candidates based on a combination of framework homology that preserved key framework residues and canonical loop structure. The murine CDRs of mo679 were grafted onto a human framework, defined as the "acceptor" framework, resulting in five newly humanized VH variants (VH1-VH5, see Table I) and eight newly humanized VL variants (VL1, VL2, VL3, VL4, VL5, VL3.1, VL4.1, VL5.1, see Table II).

[0244] Determination of antibody humanization degree: The variable (V) regions of the newly humanized variants (VH1-VH5 and VL1-VL5, VL3.1, VL4.1, VL5.1) and the previously humanized variable (V) regions of anti-HSG antibody hz679 (from US 2009 / 0240037 A1) were analyzed to determine 1) their percentage identity to the closest human germline and 2) their degree of humanization according to the WHO definition of a humanized antibody using the Immunogenetics Information System® (IMGT®) DomainGapAlign tool, https: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi.

[0245] Results and ConclusionsAs can be seen from Tables VII and VIII, all five newly humanized VH variants (VH1-5) and four of the newly humanized VL variants (VL1, VL2, VL5, and VL5.1) show higher percent identity to their respective closest human germlines when compared to the previously humanized anti-HSG antibody hz679 and its closest human germline (Table VII). Furthermore, two newly humanized VH variants, VH1 and VH2, and four newly humanized VL variants, VL1, VL2, VL5, and VL5.1, are defined as fully human according to the WHO definition of a humanized antibody (Table VIII). The newly humanized VH variants, VH3 and VH4, are equally close to human and mouse, and VH5 is equally close to human, mouse, and monkey, whereas the newly humanized VL variants, VL3, VL3.1, VL4, and VL4.1, are most closely related to monkey (Table VIII). In contrast, the previously humanized anti-HSG antibody hz679 is still closer to mouse according to the WHO definition of a humanized antibody (Table VIII). It is well known in the art that the closer an antibody's sequence is to its closest human germline, the less immunogenic the antibody will be when administered to humans (Gao SH et al., 2013). Therefore, increasing the humanization scores of newly humanized anti-HSG VH and VL variants reduces the immunogenic potential of antibodies containing newly humanized variable domain sequences.

[0246] [Table 7]

[0247] [Table 8]

[0248] Example 3: Production and manufacturing characteristics of anti-oxMIF x anti-HSG bispecific antibodies material and methodTo generate the novel anti-oxMIF x anti-HSG bispecific Fab-scFv-Fc mAb, the newly humanized VH and VL sequences were linked to scFv (VH-(GGS)5-VL) (SEQ ID NO: 88) and fused to an FcγR-silencing human IgG1 "hole" Fc via a linker containing a truncated hinge region (GGGGSDKTHTCPPCP, SEQ ID NO: 58), resulting in an anti-HSG scFv-Fc "hole" heavy chain. The VH and VL of the previously humanized anti-HSG antibody hz679 were used as a reference. A plasmid containing the synthetic anti-HSG scFv-Fc heavy chain (HC) was cotransfected into ExpiCHO-S cells with plasmids encoding the respective anti-oxMIF antibody chains (LC and HC, FcγR-silencing human IgG1 "knob" constant domains) at a ratio of 3:2:1 (anti-oxMIF LC:anti-oxMIF HC:anti-HSG scFv-Fc). This yielded the anti-oxMIF × anti-HSG bispecific antibodies C0176-C0200 (containing the newly humanized anti-HSG sequence) and C0132 (containing the previously humanized anti-HSG sequence of hz679) upon transient expression using the MAX Titer protocol (Thermo Fisher Scientific) (Table IV).

[0249] To generate the anti-oxMIF × anti-HSG bispecific CrossMab, we cloned the newly humanized anti-HSG VH sequences (VH1–VH3) into the constant region of an FcγR / FcRn-silenced human IgG1 “hole” HC (CL). カッパクロス The newly humanized anti-HSG VL sequences (VL1-VL4) were cloned in-frame with the CH1 region of human IgG1 (CH1-CH2-CH3) to obtain anti-HSG HC. クロス) to obtain the anti-HSG light chain. The VH and VL of the previously humanized anti-HSG antibody hz679 were used as a reference. Plasmids containing the synthetic anti-HSG heavy chain (HC) and light chain (LC) were cotransfected with plasmids encoding the respective anti-oxMIF antibody chains (LC and HC, FcγR / FcRn-silencing human IgG1 "knob" constant domains) at a ratio of 2:1:1:2 (anti-oxMIF LC:anti-oxMIF HC:anti-HSG HC:anti-HSG LC). This resulted in the anti-oxMIF x anti-HSG crossmab C0238–C0252 (containing the newly humanized anti-HSG sequence) and C0255 (containing the previously humanized anti-HSG sequence from hz679) (Table V).

[0250] Heterodimerization of two different heavy chains was achieved using the knob-into-hole (KIH) technique (anti-oxMIF HC "knob": T366W and S354C mutations for disulfide bond stabilization; anti-HSG HC "hole": T366S / L368A / Y407V and Y349C mutations for disulfide bond stabilization). Additionally, all heavy chains contain the L234A / L235A ("LALA") mutations, which minimize binding to Fcγ receptor-bearing immune cells and complement. The bispecific mAb in the CrossMab format further contains the mutations H310A / H435Q, which modulate binding to human FcRn, optimizing the half-life of the mAb for pretargeting in humans. The positions of the mutations are numbered according to the EU numbering system.

[0251] ExpiCHO-S cultures were harvested 12 days posttransfection, and mAbs were purified from cell culture supernatants by Protein A affinity chromatography (MabSelect Prism A HiTrap column, Cytiva). MAbs were further purified by preparative CEX using a Poros XS column and formulated in 10 mM sodium acetate / acetic acid, 200 mM NaCl, pH 5.8.

[0252] Mab expression titers were calculated as mg of Protein A-purified mAb per liter of harvested cell culture supernatant (mg / L). The relative abundance (monomer purity) of correctly assembled bispecific mAbs was assessed by analytical size-exclusion chromatography (SEC) of Protein A-purified samples using an ENrich 650 column (Bio-Rad) and analytical cation-exchange chromatography (CEX) using a Poros XS column (Cytiva).

[0253] MAb purity and the extent of anti-HSG heavy chain (scFv-Fc) cleavage were assessed by reducing NuPAGE™ 4-12% SDS-PAGE (Thermo Fisher Scientific, 3 μg / lane) followed by InstantBlue® Coomassie staining (Abcam). Band intensity (BI) was quantified by ImageStudioLite version 5.2 software (LI-COR), and cleavage was calculated using the formula: (BI anti-HSG HC / BI anti-oxMIF LC). * Calculated according to 100.

[0254] Results and ConclusionsAmong 25 Fab-scFv-Fc mAbs coexpressed at a 25 ml scale, mAbs C0176, C0181, C0182, C0186, and C0192, in which the newly humanized VL1 and VL2 variants of anti-HSG were paired with one of the newly humanized VH1-VH4 variants, demonstrated the best manufacturing characteristics, as defined by a combination of their mAb expression titer, monomer purity, and extent of anti-HSG scFv-Fc heavy chain cleavage (candidates with arrows pointing up in Table IX—→↑ / ↑). Mass spectrometry analysis revealed that cleavage of the anti-HSG scFv-Fc heavy chain occurred between amino acid residues E105 and L106 (Kabat numbering) in the "KLELK" motif (SEQ ID NO: 59) of FRW4 of the anti-HSG VL (see Example 4). Surprisingly, antibodies (C0178-0180, C0183-0185, C0188-0190, C0193-0195, C0198-0200) containing VL sequences comprising mutant variable domains VL3-5 that share the same FR4 region (FGAGTKLELK, SEQ ID NO: 60) as the previously humanized anti-HSG mAb hz679 (C0132) showed significantly enhanced cleavage of anti-HSG scFv HC (Figure 2, exemplified by C0178-C0180).

[0255] Therefore, the top five mAbs C0176, C0181, C0182, C0186, and C0192 were co-reexpressed at a larger scale (200 ml) with reference mAb C0132, which has a sequence derived from the previously humanized anti-HSG mAb hz679, to allow for a direct comparison of production characteristics. As can be seen from Table X, both the expression titers and monomer purity of the newly humanized bispecific mAbs C0176, C0181, C0182, C0186, and C0192 were significantly improved compared to C0132, which has the previously humanized anti-HSG sequence of hz679. In summary, bispecific Fab-scFv-Fc antibodies containing the newly humanized anti-HSG variable regions VL1-2 combined with VH1-4 exhibited advantageous properties compared to the previously humanized anti-HSG mAb hz679.

[0256] Anti-oxMIF x anti-HSG bispecific CrossMabs C0238–C0252 were co-expressed at a 100 ml scale with reference CrossMab C0255, which contains the previously humanized anti-HSG sequence of hz679, to allow direct comparison. CrossMabs were purified as previously described. As evident from Table XI, CrossMabs C0238, C0239, C0240, C0241, C0245, and C0250, which pair newly humanized VH variants (VH1–3) with newly humanized VL variants (VL1–2), exhibit improved manufacturing properties over reference C0255. This improvement is evidenced by a combination of improved expression titers and improved monomer purity by SEC and CEX (candidates with up-arrowed candidates in Table XI - →↑ / ↑). In summary, again, the bispecific Crossmab containing the newly humanized anti-HSG variable regions VL1-2 combined with VH1-3 showed advantageous properties compared to the reference C0255 containing the previously humanized anti-HSG mAb hz679.

[0257] [Table 9]

[0258] Figure 2 shows the evaluation of the purity of mAbs C0132 and C0176-C0180 and the extent of cleavage of the anti-HSG scFv-Fc heavy chain by SDS-PAGE and Coomassie staining. A total of 3 μg of Protein A-purified mAb was separated by NuPAGE™ 4-12% SDS-PAGE under reducing conditions ("red"). Spectra Multicolor Broad Range Protein Ladder was used as a standard. Arrows indicate the anti-oxMIF LC and cleaved anti-HSG heavy chain.

[0259] [Table 10]

[0260] [Table 11]

[0261] Example 4: Evaluation of anti-HSG scFv-Fc heavy chain cleavage in anti-oxMIF x anti-HSG bispecific mAb C0132 by mass spectrometry As described in Example 3, cleavage of the anti-HSG scFv-Fc heavy chain was observed to various degrees for all bispecific Fab-scFv-Fc anti-oxMIF × anti-HSG mAbs. Surprisingly, antibodies (C0178-0180, C0183-0185, C0188-0190, C0193-195, C0198-0200) containing VL sequences including mutant variable domains VL3-5, which share the same FR4 region (FGAGTKLELK; SEQ ID NO: 60) as the previously humanized anti-HSG mAb hz679 (C0132), showed significantly enhanced cleavage of the anti-HSG scFv HC (see Table IX and Figure 2). This was much less pronounced for variants containing the newly humanized VL1 and VL2 variants compared to C0132. In this example, Protein A and CEX purified C0132 fractions containing large amounts of cleaved anti-HSG HC were subjected to mass spectrometry to characterize the cleavage site in more detail.

[0262] material and method C0132 was diluted in 0.1% formic acid (FA) and purified by ultra-high performance liquid chromatography (ULC) using a reversed-phase column (MAbPac RP 4 μm 2.1 × 50 mm, Thermo Scientific). U ltra P performance L iquid C The separation was performed using a Waters ACQUITY Premier UPLC system. The eluent was 0.1% FA water and 0.1% FA acetonitrile. Mass spectrometry was performed using a compact quadrupole time-of-flight ( C ompact Q uadrupol T time of F The data were analyzed using a QTOF mass spectrometer (Bruker Daltonik).L iquid C Chromatography E lectro S pray I onization- M ass S Spectrometry (LC-ESI-MS) spectra were summed, deconvoluted and smoothed using the software DataAnalysis (Bruker Daltonik).

[0263] Results and Conclusions Figure 3 shows the intact mass spectrum of the C0132 mAb. Figure 3 reveals two major peaks at 126882.3 and 100701.9 Da, corresponding to the intact antibody and the truncated HC, respectively. The molecular mass of 100701.9 Da can only be obtained from the anti-HSG scFv-Fc HC with truncation of amino acids 1-244. This indicates that the truncation is at the "KL" of FRW4 of the anti-HSG VL present in the newly humanized variant containing VL3-5 and the previously humanized hz679 mAb. EL Amino acid residues in the "K" motif E 105 and L 106 (Kabat numbering). In contrast, Fab-scFv-Fc mAbs C0176, C0181, C0182, C0186 and C0192, which show only minor cleavage of anti-HSG scFv-Fc, all have "KL" in their FRW4. EI The VL variants VL1 or VL2 contain a novel humanized VL variant with a "K" motif (SEQ ID NO: 61).

[0264] Figure 3 shows the deconvoluted mass spectrum of C0132 mAb. The two major peaks correspond to the intact antibody (126882.3 Da) and the anti-HSG scFv-Fc heavy chain truncated antibody (100701.9 Da).

[0265] Example 5: Stability under different storage conditions of the newly humanized anti-oxMIF x anti-HSG bispecific Fab-scFv-Fc mAbs C0176, C0181, C0182, C0186, and C0192 compared to a reference antibody with the VH and VL sequences of the previously humanized anti-HSG Ab hz679 material and method Antibody stability was assessed by SEC on day 0 and after 83 days of storage at -80°C (Figure 4A) or 4°C (Figure 4B). For SEC, samples were diluted to 1 mg / ml in running buffer (0.1 M phosphate buffer, 0.2 M arginine, pH 6.8), and 100 μL samples were applied to an Enrich 650 (Bio-Rad) gel filtration column at a flow rate of 1.25 mL / min. Protein peaks were monitored using absorbance at 280 nm, and spectra were analyzed using the ChromLab software package (Bio-Rad). Peak areas were integrated using absorbance at 280 nm, and the relative areas of the different peaks were used to determine the abundance of aggregates, monomers, and fragments. To allow direct comparison of samples, % monomer values ​​were normalized to day 0 (= 100%) for each antibody.

[0266] Results and Conclusions As shown in Figure 4, bispecific antibodies containing the newly humanized anti-HSG sequence showed either improved (-80°C (Figure 4A): C0176, C0181, C0182, C0186, C0192; 4°C (Figure 4B): C0186 and C0192) or comparable stability (4°C (Figure 4B): C0181 and C0182) as evidenced by higher or similar monomer content compared to C0132 (containing the previously humanized anti-HSG sequence). In summary, again, the novel bispecific antibody containing the newly humanized VL variants VL1 and VL2 paired with the VH variants VH1-VH4 provided improved stability during long-term (83 days) storage compared to the reference C0132 containing the previously humanized anti-HSG sequence of hz679.

[0267] Figure 4 shows the stability of the newly humanized anti-oxMIF x anti-HSG bispecific Fab-scFv-Fc mAb compared to the previously humanized C0132 mAb containing the anti-HSG sequence. Stability was assessed by SEC before (day 0) and after 83 days of storage at -80°C (A) or 4°C (B). % monomer values ​​were normalized to day 0 (=100%) for each antibody to allow direct comparison of samples.

[0268] Example 6: Binding of bispecific anti-oxMIF x anti-HSG mAb to immobilized HSG material and method Streptavidin diluted to 1 μg / mL in PBS was immobilized in a 96-well Maxisorb flat-bottom plate (Thermo Fisher Scientific) for 4 hours at room temperature. After blocking with 2% fish gelatin (FG) in TBST overnight at 4°C and washing with TBST, the plate was incubated with 100 μL per well of Biotin-PEG4-dTyr-dLys(HSG)-dGlu-dLys(HSG)-NH2 (Eurogentec) at 0.1 μg / mL for 1.5 hours at room temperature. After washing with TBST, 100 μL of serial dilutions of bispecific anti-oxMIF × anti-HSG mAb or control mAb were added, and the plate was incubated with the mAb for 1.5 hours at room temperature. After washing with TBST, bound antibody was detected using a goat anti-human IgG (Fc-specific)-HRP conjugate and tetramethylbenzidine (TMB) as the substrate. The color reaction was stopped with 3M H2SO4 and the OD was measured at 450 nm. 50 Values ​​were determined by a four-parameter logistic fit using GraphPad Prism.

[0269] Results and Conclusions The binding of bispecific anti-oxMIF x anti-HSG mAb to immobilized HSG was measured over a wide concentration range. The results are shown in Figure 5. The binding curve (Figure 5A) and apparent K D Represents the calculated EC 50The values ​​indicate that all bispecific Fab-scFv-Fc mAbs (Figure 5B, C0176, C0181, C0182, C0186, and C0192) and CrossMabs (Figure 5C, C0238, C0239, C0240, C0241, C0245, and C0250) containing the newly humanized anti-HSG sequence exhibited improved or retained affinity (lower EC2) for HSG when compared to reference mAbs C0132 and C0255, which have the previously humanized anti-HSG sequence of hz679. 50 The values ​​clearly indicate that the ion exchange rate is either 0.01 or 0.1 (as is evident from the values).

[0270] Figure 5 shows the binding of bispecific anti-oxMIF x anti-HSG mAbs to immobilized HSG. OD values ​​at 450 nm (mean ± SEM, n = 3) were plotted against mAb concentration, and curve fitting was performed by a four-parameter logistic fit using GraphPad Prism. (A) Binding curves of Fab-scFv-Fc BsMAbs C0132, C0176, C0181, C0182, C0186, and C0192 to HSG; (B) EC of A. 50 Values ​​(mean ± SEM, n = 3); (C) EC for binding of CrossMabs (C0255, C0238, C0239, C0240, C0241, C0245, C0250) to HSG 50 Values ​​(mean ± SEM, n = 3). The dotted line indicates the EC of the reference bispecific anti-oxMIF × anti-HSG antibody. 50 Represents a value.

[0271] Example 7: Maintenance of binding to oxMIF of anti-oxMIF x anti-HSG bispecific antibodies with newly humanized anti-HSG sequence variants VH1-VH4 paired with VL1-VL2 material and methodRecombinant human MIF diluted in PBS at 1 μg / mL was immobilized on an ELISA plate overnight at 4°C (MIF converted to oxMIF according to Thiele et al., 2015). After blocking, serial dilutions of anti-oxMIF x anti-HSG bispecific antibody were added to the plate. Finally, bound antibody was detected using a goat anti-human IgG (Fc-specific)-HRP conjugate and tetramethylbenzidine (TMB) as a substrate. The color reaction was stopped with 3M H2SO4, and the OD was measured at 450 nm. The apparent K D Representing EC 50 Values ​​were determined by a four-parameter logistic fit using GraphPad Prism and are presented as mean (n=2 or 3)±SEM.

[0272] Results and Conclusions Binding of anti-oxMIF x anti-HSG bispecific mAb to immobilized MIF (oxMIF) was measured over a wide concentration range. Binding curves and calculated EC 50 The values ​​are shown in Figure 6. Figure 6 clearly shows that all anti-oxMIF × anti-HSG mAbs in both formats, i.e., Fab-scFv-Fc (Figure 6A, B) and CrossMab (Figure 6C), retained their low nanomolar affinity for oxMIF. The bivalent monospecific antibody C0008 (imalumab, the reference anti-oxMIF antibody) was used as a control. C0008 exhibited a stronger interaction with oxMIF compared to the monovalent anti-HSG / oxMIF bispecific antibody, demonstrating that avidity plays an important role in oxMIF binding in this setting. In summary, not using the newly humanized VH / VL sequence of the anti-HSG arm of the bsMab or choosing the bispecific mAb format (Fab-scFv-Fc vs. CrossMab) impairs mAb binding to oxMIF.

[0273] Figure 6 shows the maintenance of binding of anti-oxMIF x anti-HSG bispecific mAbs to immobilized MIF (oxMIF). Anti-oxMIF x anti-HSG Fab-scFv-Fc mAb (A, B) and anti-CrossMab (C) were bound to immobilized oxMIF and detected with goat anti-human IgG (Fc specific)-HRP conjugate. Fab-scFv-Fc bsAb (A) and EC 50 Binding curves of values ​​(B-C) are shown (mean ± SEM, n = 2-3). C0008 (imalumab) was used as the reference anti-oxMIF mAb.

[0274] Example 8: Evaluation of off-target binding of anti-oxMIF x anti-HSG bsMAb to A2780 MIF knockout cells material and method :A2780 MIF - / - A cell line was generated by CRISPR / Cas9 gene editing of the human MIF gene in the A2780 ovarian cancer cell line (ECACC / Sigma # 93112519). Briefly, the target gene sequence was analyzed, and the location of the target site was determined according to the general principles of designing a targeting guide RNA (gRNA) for the GenCRISPR™ system. The guide RNA (gRNA) was designed to specifically recognize the 5' region of the MIF gene (TTGGTGTTTACGATGAACATCGG, SEQ ID NO: 62), and the gRNA sequence was cloned into the PX459 (addgene) vector containing the Streptococcus pyogenes (S. pyogenes) Cas9 (SpCas9) nuclease. A2780 cells were transiently transfected by electroporation and seeded into 96-well plates by limiting dilution to generate isogenic single clones. A single isogenic clone in which the endogenous MIF gene was efficiently mutated, resulting in reduced (or ablated) expression of the MIF protein, was identified by Sanger sequencing screening. The final clone showed a 10-bp deletion at position +2 after the start codon of the human MIF gene. A2780 MIF - / - The absence of endogenous human MIF protein in the cell lines was confirmed by Western blotting using a polyclonal anti-human MIF antibody.

[0275] A2780 MIF - / - Cells were removed from the flasks using TrypLE™, washed with PBS, and stained with the fixable viability dye eFluor 780 (1:2000 dilution in PBS) for 15 minutes at 4° C. Cells were then washed with staining buffer (PBS + 5% FBS) and resuspended in staining buffer at 4×10 6 Resuspend at 2 x 10 cells / ml per well 5 Cells were seeded into 96-well U-bottom plates at 50 μL per well. Serial dilutions (2x stock) of bispecific anti-oxMIF x anti-HSG mAbs C0176, C0181, C0182, C0186, and C0192, or the control mAb rituximab (final concentrations of 75 nM, 37.5 nM, and 18 nM), were added at 50 μL per well. After 40 min of incubation at 4°C, cells were washed with staining buffer and resuspended in 100 μL of secondary antibody (goat anti-human IgG(H+L)-AlexaFluor 488 (AF488), 1:100 dilution). After 40 min of incubation at 4°C, cells were washed with staining buffer, resuspended in staining buffer, and analyzed on a Cytoflex-S flow cytometer (Beckman Coulter). After gating on live (eFluor 780-negative) cells, 10,000 events were acquired and data were analyzed using FlowJo. AF488 geometric mean fluorescence intensity (MFI) values ​​for live (eFluor 780-negative) cells were plotted against antibody concentration using GraphPad Prism.

[0276] Results and ConclusionsFigure 7 shows that bispecific anti-oxMIF × anti-HSG mAbs (C0176, C0181, C0186, C0182, C0192) with newly humanized anti-HSG sequences VL1 and VL2 paired with VH1–VH4 do not exhibit significant off-target binding beyond that of rituximab. This suggests a favorable safety profile, as rituximab has been used as a therapeutic antibody for many years. Rituximab has been shown to exhibit some surface hydrophobicity, leading to nonspecific binding to cells (Goyon AA et al., 2017; Jain T. et al., 2017). Here, rituximab was used as a control antibody targeting CD20, a B cell surface protein not present on A2780 cells.

[0277] Figure 7 shows the A2780 MIF knockout - / - Figure 1 shows the assessment of off-target binding of anti-oxMIF x anti-HSG bsMAbs to cells. A2780 MIF- / - cells were stained with serial dilutions of anti-oxMIF x anti-HSG bispecific mAbs C0176, C0181, C0182, C0186, and C0192, as well as the control therapeutic mAb rituximab. Binding was detected in live cells using an AF488-conjugated goat anti-human IgG (H+L) secondary antibody. Geometric mean fluorescence intensity (MFI) values ​​in the AF488 channel were plotted against mAb concentration in GraphPad Prism. The dotted line represents staining (MFI) of the secondary antibody alone.

[0278] Example 9: Thermal stability of the newly humanized anti-oxMIF x anti-HSG CrossMab The thermal stability of newly humanized anti-oxMIF × anti-HSG CrossMab (C0238, C0239, C0240, C0241, C0245, and C0250), containing the sequences of newly humanized VL variants VL1 and VL2 paired with VH variants VH1–VH3, was assessed in direct comparison with the reference anti-oxMIF × anti-HSG CrossMab C0255, which has the VH and VL sequences of the previously humanized anti-HSG Ab hz679.

[0279] material and method Antibody thermal stability was assessed by nanoscale differential scanning fluorimetry, nanoDSF (Figure 8). Purified antibody formulated at 0.5 mg / mL in 50 mM HEPES, 50 mM NaCl (pH 7.2) was analyzed in quadruplicate on a Prometheus NT.48 instrument (NanoTemper Technologies). A glass capillary was filled with 10 μL of antibody sample and placed in the sample holder, and the temperature was increased from 20°C to 95°C at a rate of 1°C / min. Simultaneously, fluorescence intensities at 330 nm and 350 nm were monitored (excitation wavelength 280 nm). Using the manufacturer's software, the ratio of emission intensities (350 nm / 330 nm) was plotted as a function of temperature, and its first derivative was calculated to determine the temperature of the inflection point of the first unfolding transition (T IP , °C) was determined.

[0280] Results and Conclusions As shown in Figure 8, compared to the C0255 CrossMab containing the previously humanized anti-HSG sequence, the CrossMabs containing the newly humanized anti-HSG sequences (C0238, C0240, C0241, C0245, and C0250) all exhibited superior stability to thermal unfolding, as evidenced by the higher temperatures at which the first unfolding transition occurred, a measure of overall stability. In summary, CrossMabs containing the newly humanized VL variants VL1 and VL2 paired with the VH variants VH1-VH3 provided improved thermal stability compared to the reference C0255 containing the previously humanized anti-HSG sequence of hz679.

[0281] Figure 8 shows the inflection temperature (T) of the first unfolding transition of the newly humanized anti-oxMIF x anti-HSG CrossMab compared to the C0255 CrossMab containing the previously humanized anti-HSG sequence. IP , °C). Data represent the mean (n=4 + / - SD).

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Claims

1. A bispecific anti-tumor antigen / anti-HSG antibody comprising a binding site that specifically recognizes at least one tumor antigen and a binding site that specifically recognizes HSG, a light chain variable (VL) domain comprising a sequence selected from the group consisting of SEQ ID NOs: 17, 19, 89, 90 and 91, or - a light chain variable (VL) domain comprising a sequence selected from the group consisting of SEQ ID NOs: 17, 19, 89, 90, and 91, which has one or two additional amino acid substitutions and which has a glutamine at position 100 (Q100), a glutamic acid at position 105 (E105), and an isoleucine at position 106 (I106) according to the Kabat numbering; and a heavy chain variable (VH) domain comprising a sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8 and 10, or a heavy chain variable (VH) domain comprising a sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, and 10, with one or two additional amino acid substitutions and with an arginine at position 19 (R19) according to the Kabat numbering; The bispecific anti-tumor antigen / anti-HSG antibody comprising:

2. The antibody of claim 1, comprising a single-chain variable fragment (scFv) that specifically recognizes HSG of a formula selected from the group consisting of VH-linker-VL-linker, VL-linker-VH-linker, wherein the linker comprises SEQ ID NO: 39 or SEQ ID NO:

42.

3. The antibody of claim 1, comprising VL and VH domains that specifically recognize HSG, wherein the VL domain is linked at its C-terminus to SEQ ID NO: 30 and the VH domain is linked at its C-terminus to SEQ ID NO:

38.

4. 4. The antibody of any one of claims 1 to 3, comprising an Fc region having SEQ ID NO: 31 comprising asymmetric mutations in each CH3 domain that allow heterodimerization of two CHs from different antibodies, specifically one CH comprising "knob" mutations T366W and S354C according to the EU numbering index, and one CH comprising "hole" mutations T366S, L368A, Y407V, and Y349C.

5. 5. The antibody of any one of claims 1 to 4, comprising a variant Fc Region with reduced or eliminated effector function and / or FcRn binding, specifically a variant Fc Region with amino acid substitutions at any one or more of positions E233, L234, L235, G236, G237, P238, 1253, D265, S267, H268, N297, S298, T299, H310, E318, L328, P329, A330, P331, H435 of SEQ ID NO: 31 according to the EU numbering index, and optionally an aglycosylated Fc Region.

6. The antibody of any one of claims 1 to 5, wherein the anti-tumor antigen is selected from the group consisting of oxMIF, mesothelin (MSLN), and folate receptor alpha (FRα), specifically, the anti-tumor antigen is oxMIF.

7. A binding site that specifically recognizes oxMIF is a light chain variable domain comprising SEQ ID NO: 27, or a light chain variable domain comprising SEQ ID NO: 27 and further comprising the amino acid substitutions M30L and / or P80S, and a heavy chain variable domain comprising SEQ ID NO: 46, in particular with the amino acid substitutions L5Q and / or W97Y 7. The antibody of claim 6, comprising:

8. The antibody according to any one of claims 1 to 7, which is selected from the group consisting of Fab-scFv-Fc, CrossMab, (scFv)2-Fc, scFv / scFv-Fc, Fab / (scFv)2-Fc, Fab / Fab-scFv-Fc (= IgG-central scFv), Fab / Fab-crossFab-Fc, Fab / crossFab-Fc, IgG-scFv, and IgG-(scFv)2.

9. 9. The antibody of any one of claims 1 to 8 for use in treating or detecting malignant tumors, wherein the antibody is administered to a subject in a first step and the HSG moiety is administered in a second step, and the HSG moiety binds to the antibody.

10. The antibody for use of claim 9, wherein the HSG portion is conjugated to or labeled with one or more diagnostic and / or therapeutic agents, specifically the HSG portion comprises one or more HSG haptens, one or more diagnostic and / or therapeutic agents, and a chelator.

11. 9. The antibody of any one of claims 1 to 8, wherein the antibody binds to an HSG moiety conjugated to one or more diagnostic and / or therapeutic agents or labeled with one or more diagnostic and / or therapeutic agents, particularly wherein the HSG moiety comprises one or more HSG haptens, one or more diagnostic and / or therapeutic agents, and a chelator, particularly wherein the therapeutic agent is a radionuclide or a cytotoxic agent; and wherein the diagnostic agent is a radionuclide.

12. Chelating agents bind to radionuclides, specifically DOTA, DTPA, deferoxamine B (DFO), and DFO ＊ In particular, the radionuclide is selected from １１ C. １３ N. １５ O. １８ F. ３２ P. ３３ P. ４７ Sc, ５９ Fe, ６４ Cu, ６７ Cu, ６７ Ga, ７５ Se, ７７ As, ８９ Sr, ８９ Zr, ９０ Y. ９９ mTc, ９９ Mo, １０３ Pd, １０５ Rh, １０９ Pd, １１１ Ag, １１１ In, １２３ I, １２４ I, １２５ I, １３１ I, １４０ La, １４２ Pr, １４３ Pr, １４９ Tb, １４９ Pm, １５３ Sm, １５９ Gd, １６１ Tb, １６５ Dy, １６６ Dy, １６６ Ho, １６９ Yb, １６９ Er, １７５ Yb, １７７ Lu, １８６ Re, １８８ Re, １８９ Re, １９２ Ir, １９３ mPt, １９５ mPt, １９４ Ir, １９８ Au, １９９ Au, ２１１ At, ２１１ Pb, ２１２ Pb, ２１２ Bi, ２１３ Bi, ２１１ At, ２２３ Ra, ２２５ Ac, and ２２７ The antibody of claim 11, wherein the antibody is selected from the group consisting of Th.

13. Use of an antibody according to any one of claims 1 to 8 in the preparation of a medicament.

14. A pharmaceutical composition comprising the antibody of any one of claims 1 to 8 together with a pharmaceutical excipient, in particular the pharmaceutical composition is formulated for intravenous administration.

15. 15. The pharmaceutical composition according to claim 14, for use in the treatment of patients suffering from cancer, in particular in the treatment of solid tumors, more in particular in the treatment of colorectal cancer, ovarian cancer, breast cancer, prostate cancer, pancreatic cancer and lung cancer.

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