Improved bispecific anti-tumor antigen / anti-HSG antibodies for pre-targeting of hyperproliferative disorders

Abstract

The present invention relates to a bispecific anti-tumor antigen / anti-HSG antibody having screened light and heavy chain variable domains, which is capable of improving the pre-targeting of tumors for the specific delivery of therapeutic or diagnostic agents. The antibodies have a significant affinity for tumor antigens and stay at desired sites for a sufficient time. Antibodies that do not bind to the antigen can be rapidly cleared from the body, minimizing exposure of normal tissue. A humanized anti-HSG antibody is bound to each of a group containing histamine-succinyl-glycine (HSG) and a tumor antigen with high affinity. The bispecific antibodies can be further Fc silenced, resulting in additional properties, such as reduced binding to Fc [gamma] R and FcRn, thereby modulating effector function and regulating half-life. These antibodies are useful in the diagnosis and treatment of subjects suffering from malignancies.

Description

TECHNICAL FIELD

[0001] The present invention relates to a bispecific anti-tumor antigen / anti-HSG antibody having screened light and heavy chain variable domains that improve the pre-targeting of malignant tumors, in particular tumors, more particularly solid tumors. Humanized anti-HSG (histamine-succinyl-glycine) binding sites bind molecules containing histamine-succinyl-glycine (HSG) groups with high affinity. The present invention also discloses methods of making bispecific anti-tumor antigen / anti-HSG antibodies comprising humanized anti-HSG variable domains. Due to the replacement of screened amino acids in the variable domains, the bispecific antibodies can further possess properties such as reduced aggregation propensity and reduced hydrophobicity. Anti-tumor antigen / anti-HSG antibodies can be used for the diagnosis and treatment of subjects with cancer. BACKGROUND

[0002] Tumor targeting with monoclonal antibodies is an attractive approach for selective cancer therapy. Radiolabeled antibodies have proven effective in patients with hematological malignancies, but the application of radioimmunotherapy (RIT) to solid tumors is challenging. Directly radiolabeled antibodies suffer from slow blood clearance, delayed tumor uptake, which leads to persistent radiation exposure of normal organs and bone marrow, and high background signals when used as diagnostic tools. To overcome these limitations, pre-targeting technology was developed. In pre-targeting, a non-radiolabeled humanized bispecific monoclonal antibody (bsMAb) is first injected intravenously. After the bsMAb localizes in the tumor and is cleared from the blood circulation, a radiolabeled hapten peptide is administered, which is rapidly captured by the bsMAb in the tumor, while the remainder is very rapidly cleared from the blood and excreted via the kidneys (Stawski R.S. et al., 2015). Pre-targeting reduces radiation exposure of radiation-sensitive normal tissues, such as the bone marrow, and other tissues (Stawski R.S. et al., 2015).

[0003] Schoffelen R. et al. (2013) and Rossi E.A. et al. (2006) describe a tri-Fab bispecific monoclonal antibody, TF2, which is bivalent for carcinoembryonic antigen (CEACAM-5, or CEA) and monovalent for the histamine-succinyl-glycine (HSG) hapten peptide. TF2 binds to the tumor-associated antigen CEACAM-5 expressed on the surface of colon tumor cells. Subsequently, the HSG hapten peptide IMP288, carrying a radionuclide, is administered, which binds to the anti-HSG arm on the bispecific monoclonal antibody (Schoffelen R. et al., 2013). In the phase I study NCT00860860, depending on the radionuclide used (111In or 90Y), the tumor-to-background ratio was 3.5 to 6.5 (Schoffelen R. et al., 2013). The tumor-to-background ratio is a measure for the specificity of the antibody. A higher tumor-to-background ratio indicates a higher specificity of the antibody. The tumor-to-background ratio is calculated by dividing the tumor uptake by the uptake in a non-tumor tissue. The non-tumor tissue is usually the liver, but can also be the muscle or the blood. The tumor-to-background ratio is a measure for the specificity of the antibody. A higher tumor-to-background ratio indicates a higher specificity of the antibody. 111 In or 177Leveraging the properties of TF2 and radiolabeled IMP288, radioimaging and / or radioimmunotherapy were performed on CEACAM-5-expressing tumor cells. This study demonstrated for the first time that pre-targeting with TF2 and radiolabeled IMP288 is feasible and safe in CEACAM-5-expressing colorectal cancer patients. Using this pre-targeting approach, tumors are specifically and rapidly targeted (Schoffelen R. et al., 2013).

[0004] The anti-HSG monoclonal antibody sequence in TF2 is derived from humanized anti-HSG monoclonal antibody clone 679, which was initially discovered in mouse hybridoma cells (Morel A. et al., 1990). Mouse anti-HSG monoclonal antibody 679 and humanized monoclonal antibody 679 are described in US20090246131 and US20090240037A1, respectively. In US 2009 / 0240037 A1, humanized antibody 679 is generated and detected as a single-chain variable fragment (scFv) form, which is an anti-CEACAM-5 × anti-HSG biantibody carrying a C-terminal 6×His tag. However, existing humanized HSG monoclonal antibodies still have several drawbacks. The monoclonal antibody remains highly similar to the mouse amino acid sequence, leading to unwanted immunogenicity. Furthermore, the production of Tri-Fab (anti-tumor antigen × anti-HSG) antibodies requires a special technique called the dock-and-lock method (DNL). DNL involves using a pair of distinct protein domains involved in the natural binding between cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) and kinase A-anchored protein (AKAP). By attaching the dimerization and docking domains of the PKA regulatory subunit and the anchoring domain (AD) of the interactive AKAP to a biological entity, a well-defined and stable complex can be readily formed when the resulting derivative is bound. Further attachment of non-antibody subunits to the Fab arm increases the risk of immunogenicity of the antibody. Furthermore, large-scale fabrication of DNL Tri-Fab is considered extremely challenging.

[0005] Macrophage migration inhibitory factor (MIF) was first described in 1966 (David, JR, 1966; Bloom, B.R. and Bennet, B., 1966). After MIF was cloned and recombinantly expressed, its biochemical properties and physiological functions were elucidated (Bernhagen et al., 1993; Bernhagen et al., 1994). Currently, MIF is widely accepted as a key regulator of innate immunity, playing a central role in inflammatory responses and cancer.

[0006] MIF is upregulated in a variety of human tumors (e.g., pancreatic, breast, prostate, colon, brain, skin, and lung-derived tumors) (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). Multiple studies have reported that MIF expression is closely associated with tumor aggressiveness and metastatic potential, suggesting that MIF can play an important role in disease severity and cell survival (Rendon et al., 2009). Recent data suggest that extracellular MIF can contribute to a more aggressive phenotype in tumors compared to intracellular MIF (Verjans et al., 2009). MIF contributes to the formation of a microenvironment that is conducive to tumor growth, angiogenesis, invasion, and metastasis. In addition to pro-inflammatory functions, MIF has anti-apoptotic and pro-proliferative effects, including inhibition of p53 (Hudson et al., 1999; Mitchell and Bucala, 2000) and activation of the central kinases ERK1 / 2 (Mitchell et al., 1999) and AKT (Lue et al., 2007). MIF has also been described as a pro-angiogenic factor that can promote neovascularization (Coleman et al., 2008) and tumor angiogenesis by stabilizing HIF-1a (Winner et al., 2007) and upregulating pro-angiogenic factors such as vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) (Ren et al., 2004). MIF also acts as a chemotactic factor, with the potential to recruit inflammatory cells in the tumor environment through the chemokine receptors CXCR2 and CXCR4 (Bernhagen et al., 2007; Rendon et al., 2007).

[0007] However, MIF is distinct from other cytokines and chemokines in that it is continuously expressed and present in the blood circulation of healthy subjects. Preformed MIF stores are present in the cytosolic pools of macrophages, T cells, and many other cells in the body, including the hypothalamic-pituitary-adrenal axis, and can be rapidly released upon stimulation without de novo synthesis (Bernhagen et al., 1993; Bacher et al., 1997; Fingerle-Rowson et al., 2003).

[0008] Due to its ubiquitous presence, MIF can be considered an inappropriate target for therapeutic intervention. However, MIF exists in two immunologically distinct conformers, namely reduced MIF (redMIF) and oxidized MIF (oxMIF) (Thiele M. et al., 2015). It was found that redMIF is the isoform of MIF that is abundantly expressed and can be detected in the cytosol and in the blood circulation of any subject. RedMIF appears to represent a latent, inactive storage form (Schinagl. A. et al., 2018).

[0009] In contrast, oxMIF appears to be the isoform that is physiologically relevant and disease-relevant and can be detected in tumor tissue, in particular in tumor tissue of patients with colorectal cancer, pancreatic cancer, ovarian cancer and lung cancer, which indicates that oxMIF is highly tumor-specific (Schinagl. A. et al., 2016), while it can also be detected in the blood circulation and in inflamed tissue of patients with inflammatory diseases (Thiele et al., 2015).

[0010] Antibodies targeting oxMIF show 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 anti-oxMIF / anti-CD3 bispecific antibodies.

[0012] WO2009 / 086920A1 describes the anti-oxMIF antibody Bax69 (Imalumab).

[0013] WO2022069712A1 describes anti-oxMIF antibodies with reduced aggregation propensity and reduced hydrophobicity.

[0014] Protein aggregation, in particular antibody aggregation, is frequently observed in multiple stages of bioprocessing, including protein expression, purification and storage. Antibody aggregation can impact the overall yield of a therapeutic protein manufacturing process and can affect the stability and immunogenicity of therapeutic antibodies.

[0015] Therefore, protein aggregation of antibodies remains a major challenge in their developability and is a major area of focus in antibody production. Antibody aggregation can be triggered by partial unfolding of its domains, leading to monomer-monomer association, followed by nucleation and aggregate growth. While the aggregation propensity of antibodies, as well as of antibody-based proteins, can be influenced by external experimental conditions, they are largely dependent on intrinsic antibody properties determined by sequence and structure.

[0016] For example, anti-aggregation can be achieved by stabilizing the native state (i.e., resisting unfolding) or reducing the tendency of proteins to aggregate in unfolded or partially folded states. One drawback of stabilizing the native state is that the protein may be exposed to environments that cause it to unfold. Generally, when a protein denatures or unfolds, amino acid residues that typically mediate intramolecular contacts within the protein are exposed. This exposure often makes the protein more susceptible to forming intermolecular contacts and aggregation. Unlike proteins resistant to unfolding, when a protein unfolds, if its aggregation tendency is reduced, it will simply refold into a biologically active, non-aggregated state after exposure to the aforementioned environments.

[0017] The anti-aggregation or aggregation tendency of antibodies and proteins containing antigen-binding domains is generally influenced by the most aggregate-prone domain contained therein and the strength of the interaction between that domain and surrounding domains (if present).

[0018] This is because once the domain unfolds, if it cannot refold, it may interact with other domains in the same or other proteins and form aggregates. Constant domains of antibodies typically do not aggregate and exhibit little variation. Therefore, in terms of aggregation potential and stability, the weakest domains in antibodies are often considered to be variable domains (e.g., heavy chain variable domains (V... H ) and / or light chain variable structural domains (V L (Ewert S. et al., 2003). In this regard, the easily aggregated V H or V L Incorporating domains into otherwise stable recombinant antibody products often imparts undesirable properties to new recombinant designs. Therefore, engineering variable domains to be anti-aggregating is most likely to make the entire protein containing that variable domain anti-aggregating. Various strategies for reducing variable domain aggregation have been proposed, such as rational design of anti-aggregating proteins, transplantation of complementarity-determining regions (CDRs), or introduction of disulfide bonds into the variable domain. Rational design of anti-aggregating proteins typically involves using computer simulations to predict the impact of point mutations on protein aggregation tendencies; however, simple computer simulations do not necessarily yield practical improvements in this regard.

[0019] It has been demonstrated that a decrease in aggregation propensity is accompanied by an increase in expression titer, which indicates that reducing protein aggregation throughout development is beneficial and can provide a more effective pathway for clinical studies. For therapeutic proteins, aggregates are a major risk factor for eliciting harmful immune responses in patients and can form through multiple mechanisms. Controlling aggregation can improve protein stability, manufacturability, attrition rate, safety, formulation, titer, immunogenicity, and solubility (Wei Li et al., 2016; Van der Kant R. et al., 2017).

[0020] Modulating antibody effector functions to suit specific therapeutic purposes is also increasingly gaining attention. Specifically, Fc-null or Fc-silenced antibodies can be a strategy to eliminate Fc effector functions, as strong immune effector functions generated through FcyR and complement interactions can sometimes adversely affect the mechanism of action of antibodies.

[0021] WO2023 / 031397A1 describes Fc-silenced anti-oxMIF antibodies comprising light and heavy chain variable domain amino acid substitutions.

[0022] Schinagl et al., published April 1, 2023, report on pre-targeted immunotherapy with anti-oxMIF / HSG bispecific antibodies.

[0023] Fc gamma receptors (FcyR) are a well-described family of proteins that include membrane-bound surface receptors, atypical intracellular receptors, and cytoplasmic glycoproteins. FcyR control humoral immunity and innate immunity, which are critical for mounting an appropriate response to infection and preventing chronic inflammation or autoimmune disease. For example, membrane-bound receptors are FcyRIIa, FcyRllb, FcyRllla, and FcyRIa receptors. Antibodies can modulate immune responses through interactions with FcyR.

[0024] On innate immune effector cells, activating and inhibitory FcyRs set thresholds for immune complex-activated cells. Important examples of effector responses regulated by FcyRs are phagocytosis, ADCC, and release of inflammatory mediators. On dendritic cells (DCs), FcyRs expressed in pairs can modulate cell maturation and antigen presentation, thereby indirectly controlling cellular immune responses. On B cells, inhibitory FcyRIIB is essential for the maintenance of humoral tolerance. It functions as a late checkpoint at the level of class-switched memory B cells, plasmablasts, or plasma cells. In addition, FcyRIIB plays an important role in regulating plasmablast homeostasis and survival. Antibody-FcyR interactions are influenced by a variety of factors that affect the expression level of activating and inhibitory FcyRs (e.g. cytokines), or alter the affinity of antibody-FcyR interactions (e.g. antibody glycosylation differences). Depending on the specific glycosylation pattern, IgG molecules can have enhanced pro-inflammatory or anti-inflammatory activity. Importantly, antibody glycosylation is regulated during the course of an immune response.

[0025] Atypical FcyRs are the neonatal Fc receptor (FcRn) and cytosolic glycoproteins such as the complement factor Clq protein. FcRn is expressed by endothelial cells that internalize serum components including soluble IgG from the blood stream by pinocytosis. Binding of IgG to FcRn is pH-dependent; the acidic pH in the lumen of endosomes (pH 6.0) enables IgG to bind to FcRn. Upon recycling back to the cell surface, IgG dissociates from FcRn at physiological pH (approximately pH 7.2) and is re-released back into the blood circulation, thereby escaping lysosomal degradation, extending the half-life of IgG. Thus, FcRn acts as a transcytosis receptor responsible for maintaining IgG and albumin levels in the blood circulation. It is known in the art that modifications to the Fc region can reduce or eliminate Fc-associated binding to FcRn, which is described, for example, by Kenanova V. et al. (2005) and Pyzik M. et al. (2019).

[0026] Likewise, there is an increasing interest in modulating the binding of IgG antibodies to the neonatal Fc receptor (FcRn) for the modulation of pharmacokinetics.

[0027] In particular, in view of the above, it has been and still is a challenge to adapt antibodies in general to respective fields of application. Therefore, there is still a need in the art for improved bispecific antibodies, in particular for bispecific antibodies for more efficient targeted delivery approaches, in particular pre-targeting, for the delivery of therapeutic and diagnostic agents. SUMMARY

[0028] It is an object of the present application to provide improved bispecific antibodies for pre-targeting tumors for the subsequent specific delivery of therapeutic or diagnostic agents.

[0029] The object of the present invention is achieved by the subject-matter of the present invention.

[0030] The antibody of the present invention selectively binds to a tumor antigen and the HSG hapten group. The antibody has a significant affinity for the tumor antigen and is able to stay at the desired tumor site for a sufficient time. Antibody not bound to the tumor antigen is cleared from the circulation, thereby minimizing exposure of normal tissues to the subsequently administered therapeutic or diagnostic agent.

[0031] Provided herein are bispecific anti-tumor antigen / anti-HSG antibodies comprising a binding site that specifically recognizes a tumor antigen and a binding site that specifically recognizes HSG, comprising: - a light chain variable (VL) domain comprising a sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 89, SEQ ID NO: 90, and SEQ ID NO: 91, or - a light chain variable (VL) domain comprising a sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 89, SEQ ID NO: 90, and SEQ ID NO: 91, which sequence has one or two additional amino acid substitutions and is glutamine (Q100), glutamic acid (E105), and isoleucine (1106) at positions 100, 105, and 106, respectively, according to Kabat numbering; and - a heavy chain variable (VH) domain comprising a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10, or - a heavy chain variable (VH) domain comprising a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10, which sequence has one or two additional amino acid substitutions and is arginine (R19) at position 19, according to Kabat numbering.

[0032] The bispecific anti-tumor antigen / anti-HSG antibodies of the present invention are very low immunogenic, in particular due to the modified anti-HSG VL and VH domains. In addition, the antibodies of the present invention have improved expression titers, high monomer purity, and reduced cleavage of the anti-HSG heavy chain, and are thus very suitable for scale-up manufacturing.

[0033] The antibodies of the present application have a high selective binding specificity for the tumor antigen and the HSG hapten and show a high storage stability. Targeting antibody modifications can reduce the binding to FcRn, thereby optimizing the half-life of the antibodies of the present application, in particular for pre-targeting.

[0034] According to one embodiment, the antibodies of the present application are in the Fab-scFv-Fc format.

[0035] According to one embodiment of the present application, the antibodies described herein comprise a single chain variable fragment (scFv) specifically recognizing HSG having a structural 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.

[0036] According to another embodiment, the antibodies have a CrossMab (CH1-CL crossover) format, i.e. a Fab carrying a CH1-CL domain crossover.

[0037] In particular, the antibodies of the present application comprise a VL and a VH domain specifically recognizing HSG, wherein the C-terminus of the VL domain is linked to a CH1-cross comprising SEQ ID NO: 30 and the C-terminus of the VH domain is linked to a CL-cross comprising SEQ ID NO: 38.

[0038] In another embodiment, the antibodies described herein comprise a Fc region comprising SEQ ID NO: 31, or a sequence having at least 95% (in particular 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 31, and comprising asymmetric mutations in each CH3 domain, such that two CH domains from different antibodies are heterodimerized, in particular, one CH domain comprises the "knob" mutations T366W and S354C and one CH domain comprises the "hole" mutations T366S, L368A, Y407V and Y349C, according to the EU numbering index.

[0039] In particular, the antibodies of the present application comprise a variant Fc region having reduced or ablated effector function and / or binding to FcRn.

[0040] In particular, the antibody of the application has a variant Fc region with an amino acid substitution in one or more of the positions E233, L234, L235, G236, G237, P238, I253, D265, S267, H268, N297, S298, T299, H310, E318, L328, P329, A330, P331, H435 according to EU numbering index, and optionally a aglycosylated Fc region.

[0041] In particular, the antibody of the application comprises two constant heavy chain domains, the two domains having a hinge and an Fc region comprising SEQ ID NO: 35 and SEQ ID NO: 32.

[0042] In another embodiment, the antibody of the application comprises two constant heavy chain regions, the two heavy chain regions having a hinge and an Fc domain comprising SEQ ID NO: 36 and one of SEQ ID NO: 33 or SEQ ID NO: 34.

[0043] In another embodiment of the application, the anti-tumor antigen binding of the antibody described herein is directed against oxMIF, mesothelin (MSLN) and folate receptor alpha (FRa), in particular the antibody binds to oxMIF.

[0044] In a particular embodiment of the application, the binding site specifically recognizing oxMIF comprises: - 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 the heavy chain variable domain comprising the amino acid substitutions L5Q and / or W97Y, wherein the amino acid positions are according to Kabat numbering.

[0045] According to a particular embodiment, the antibody described herein 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.

[0046] In another embodiment, the antibody is used for the treatment or detection of a malignant tumor, wherein the antibody is administered to the subject in a first step and the HSG group is administered in a second step, wherein the HSG group binds to the antibody.

[0047] More specifically, the HSG group is coupled or labeled with one or more diagnostic and / or therapeutic agents, even more specifically, the HSG group comprises one or more HSG haptens, one or more diagnostic and / or therapeutic agents and a chelator.

[0048] In another embodiment, the antibody described herein is bound to a HSG group, which is coupled or labeled with one or more diagnostic and / or therapeutic agents, specifically, the HSG group comprises one or more HSG haptens, one or more diagnostic and / or therapeutic agents and a chelator.

[0049] Specifically, the chelator coupled to the HSG group is bound to a radionuclide, and the chelator is specifically selected from the group consisting of DOTA, DTPA, deferoxamine B (DFO) and DFO*.

[0050] Specifically, the radionuclide is selected from the group consisting of 67 Ga, 89 Zr, 111 In, 124 I, 131 I, 177 Lu and 225 Ac.

[0051] In another embodiment of the application, the therapeutic agent is a radionuclide (radioisotope) or a cytotoxic agent, and the diagnostic agent is a radionuclide.

[0052] Also provided herein is the use of the antibody for the preparation of a medicament.

[0053] Also provided herein is a pharmaceutical composition comprising the antibody and a pharmaceutically acceptable excipient.

[0054] Specifically, the pharmaceutical composition provided herein is formulated for intravenous administration.

[0055] In another embodiment of the application, the pharmaceutical composition is used for the treatment of a patient suffering from cancer, specifically for the treatment of a tumor, a solid tumor, more specifically for the treatment of colorectal cancer, ovarian cancer, breast cancer, prostate cancer, pancreatic cancer and lung cancer.

[0056] Also provided herein is an isolated nucleic acid encoding the antibody described herein.

[0057] Also provided herein is an expression vector comprising the nucleic acid.

[0058] In another embodiment of the application, provided herein is a method of diagnosing cancer in a subject in vivo, wherein the antibody described herein is used to detect tumor cells.

[0059] In another embodiment of the application, provided herein is also a method of diagnosing cancer in vitro, wherein the antibody described herein is used to detect tumor cells in a sample.

[0060] Also provided herein is a method of treating cancer using the antibody described herein or the pharmaceutical composition described herein. BRIEF DESCRIPTION OF DRAWINGS

[0061] Figure 1 Schematic representation of anti-target X x anti-HSG bispecific monoclonal antibodies with Fab-scFv-Fc, CrossMab (CH1-CLcross) and IgG-central scFv (IgG-central scFv) format. Left: Fab-scFv-Fc; Middle: CrossMab (CH1-CLcross); Right: IgG-central scFv (IgG-central scFv). L cross) and IgG-central scFv (IgG-central scFv) format. Left: Fab-scFv-Fc; Middle: CrossMab (CH1-CLcross); Right: IgG-central scFv (IgG-central scFv).

[0062] Figure 2 Evaluation of purity of monoclonal antibodies C0132 and C0176-C0180 and extent of cleavage of anti-HSG scFv-Fc heavy chain by SDS-PAGE and Coomassie staining. A total of 3 pg of Protein A purified monoclonal antibodies were separated by NuPAGE™ 4-12% SDS-PAGE under reducing conditions (“red.”). Spectra Multichrome Wide Range Protein MW Standard was used as standard. Arrows indicate anti-oxMIF light chain (LC) and cleaved anti-HSG heavy chain.

[0063] Figure 3 Deconvoluted mass spectrum of monoclonal antibody C0132. Two main peaks correspond to the intact antibody (126882.3 Da) and the antibody with a truncated anti-HSG scFv-Fc heavy chain (100701.9 Da), respectively.

[0064] Figure 4: Stability comparison of novel humanized anti-oxMIF x anti-HSG bispecific Fab-scFv-Fc monoclonal antibodies to C0132 monoclonal antibody comprising a previously humanized anti-HSG sequence. Stability was assessed by size exclusion chromatography (SEC) before (day 0) and after 83 days of storage at -80°C (A) or 4°C (B). For ease of direct comparison of samples, monomer percentage (% monomer) values were normalized to day 0 (= 100%) for each antibody.

[0065] Figure 5 : Binding of bispecific anti-oxMIF x anti-HSG monoclonal antibodies to immobilized HSG. The optical density (OD) values at 450 nm (mean ± SEM, n = 3) were plotted against the monoclonal antibody concentration and curve fitting was performed by 4-parameter logistic fit using GraphPad Prism software. (A) Binding curves of Fab-scFv-Fc bispecific monoclonal antibodies (BsMAbs) C0132, C0176, C0181, C0182, C0186, C0192 to HSG; (B) EC 50 values (mean ± SEM, n = 3) of A; (C) EC 50 values (mean ± SEM, n = 3) of CrossMabs (C0255, C0238, C0239, C0240, C0241, C0245, C0250) binding to HSG. Dotted lines indicate the EC 50 values of the reference bispecific anti-oxMIF x anti-HSG antibodies.

[0066] Figure 6 : Retained binding of anti-oxMIF x anti-HSG bispecific monoclonal antibodies to immobilized MIF (oxMIF). Anti-oxMIF x anti-HSG Fab-scFv-Fc monoclonal antibodies (A, B) and anti-CrossMabs (C) were bound to immobilized oxMIF and detected by goat anti-human IgG (Fc-specific) -HRP conjugate. Binding curves (A) and EC 50 values (mean ± SEM, n = 2-3; B-C) of the Fab-scFv-Fc bispecific antibodies are shown. C0008 (imatinib mab) was used as a reference anti-oxMIF monoclonal antibody.

[0067] Figure 7 : Evaluation of non-target binding of anti-oxMIF x anti-HSG bispecific monoclonal antibodies (bsMAbs) to A2780 MIF knockout cells. A2780 MIF- / - cells were stained with serially diluted anti-oxMIF x anti-HSG bispecific monoclonal antibodies C0176, C0181, C0182, C0186, C0192 and the control therapeutic monoclonal antibody rituximab. Binding was detected on live cells with AF488-conjugated goat anti-human IgG (H+L) secondary antibody. Geometric mean fluorescence intensity (MFI) values of the AF488 channel were plotted against the monoclonal antibody concentration in GraphPad Prism software. Dotted lines indicate the MFI values of staining with secondary antibody only.

[0068] Figure 8 Thermal stability of novel humanized anti-oxMIF x anti-HSG CrossMabs. The thermal stability of novel humanized anti-oxMIF x anti-HSG CrossMabs C0238, C0239, C0240, C0241, C0245, C0250 and CrossMab C0255 with the sequence of the previously humanized anti-HSG antibody 679 was assessed by nano-differential scanning fluorimetry (nanoDSF). The monitored emission intensity ratio (350 nm / 330 nm) was plotted as a function of temperature, its first derivative was calculated and the inflection point temperature (T IP °C, mean ± SD, n = 4). DETAILED DESCRIPTION

[0069] Unless otherwise stated or defined, all terms used herein have the meaning commonly understood by a person of ordinary skill in the art to which the present application belongs. Reference can be made, for example, to standard handbooks, such as Sambrook et al., "Molecular Cloning: A Laboratory Manual" (4thedition), Vols. 1-3, Cold Spring Harbor Laboratory Press (2012); Krebs et al., "Lewin's Genes XI", Jones & Bartlett Learning, (2017), and Murphy & Weaver, "Janeway's Immunobiology" (9thedition, or newer version), Taylor & Francis Inc, 2017.

[0070] The subject matter of the claims specifically relates to artificial products or methods employing or producing these artificial products, which products can be variants of natural (wild-type) products. Although there can be some degree of sequence identity with the natural structure, it is expressly understood that the materials, methods and uses of the present application, e.g., the isolated nucleic acid sequences, amino acid sequences, fusion constructs, expression constructs, transformed host cells and modified proteins, are "artificial" or synthetic and are therefore not to be considered as a result of the "laws of nature".

[0071] As used herein, the terms "comprise", "contain", "have", and "include" and other similar forms are used interchangeably and are understood either to refer to a situation where one or more steps, features, structures, elements, members, parts, or components are present or to a situation where one or more steps, features, structures, elements, members, parts, or components are not present. "Consisting" is considered the most restrictive definition and excludes additional elements. Thus, "comprising" is broader than "consisting" and includes the definition of "consisting."

[0072] As used herein, the term "about" refers to a value that is + / - 5% of a given value.

[0073] As used herein and in the claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0074] As used herein, amino acids refer to the twenty naturally occurring amino acids encoded by sixty-one triplet codons. The 20 amino acids can be divided into amino acids with neutral charge, positive charge, and negative charge: neutral The amino acids and their corresponding three-letter, one-letter codes, and polarity are as follows: alanine (Ala, A; nonpolar, neutral), asparagine (Asn, N; polar, neutral), cysteine (Cys, C; nonpolar, neutral), glutamine (Gln, Q; polar, neutral), glycine (Gly, G; nonpolar, neutral), isoleucine (lie, I; nonpolar, neutral), leucine (Leu, L; nonpolar, neutral), methionine (Met, M; nonpolar, neutral), phenylalanine (Phe, F; nonpolar, neutral), proline (Pro, P; nonpolar, neutral), serine (Ser, S; polar, neutral), threonine (Thr, T; polar, neutral), tryptophan (Trp, W; nonpolar, neutral), tyrosine (Tyr, Y; polar, neutral), valine (Val, V; nonpolar, neutral), and histidine (His, H; polar, 10% positively charged, 90% neutral).

[0075] positively charged The amino acids with "polar, positive charge" are: arginine (Arg, R; polar, positively charged) and lysine (Lys, K; polar, positively charged).

[0076] negatively charged The amino acids with "polar, negative charge" are: aspartic acid (Asp, D; polar, negatively charged) and glutamic acid (Glu, E; polar, negatively charged).

[0077] ​​​The bispecific antibodies of the present invention comprise at least one binding site that specifically recognizes oxMIF and one binding site that specifically recognizes the HSG (histamine-succinyl-glycyl) hapten, in particular with high affinity for HSG and oxMIF.

[0078] The VL and VH domains of the HSG binding site of the bispecific anti-oxMIF / anti-HSG antibodies described herein are significantly modified compared to known anti-HSG variable domains. Although mouse anti-HSG monoclonal antibody 679 (mo679) and humanized monoclonal antibody 679 (hz679) are described in US20090240037A1 and US20090246131, respectively, further modifications to the sequences were required to provide the improved antibodies described herein. As shown below, the sequence modifications could not be made simply according to known techniques, but rather a careful and complex selection of the resulting domains was required to arrive at the particular VL and VH sequences that have advantages in manufacturing, stability and immunogenicity, while still showing high affinity for groups comprising the HSG hapten.

[0079] The anti-HSG binding site of the antibodies of the present invention contains a light chain variable (VL) domain comprising one of SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 89, SEQ ID NO: 90 or SEQ ID NO: 91, and a heavy chain variable (VH) domain comprising a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10.

[0080] In another embodiment, one or both of the VL and VH comprise a sequence having at least 95% (in particular 96%, 97%, 98%, 99%) sequence identity. Alternatively, one or both of the VL and VH comprise 1 or 2 additional amino acid substitutions. In this regard, in particular the following: The following amino acid positions of SEQ ID NO: 17 are conserved: E105, I106, and optionally Q100.

[0081] The following amino acid positions of SEQ ID NO: 19 are conserved: E105, I106, and optionally Q100.

[0082] The following amino acid positions of SEQ ID NO: 89 are conserved: E105, I106, and optionally Q100.

[0083] The following amino acid positions of SEQ ID NO: 90 are conserved: E105, I106, and optionally Q100. The following amino acid positions of SEQ ID NO: 90 are conserved: E105, I106, and optionally Q100.

[0084] The following amino acid positions of SEQ ID NO: 91 remain conserved: E105, I106, and optionally Q100.

[0085] The following amino acid positions of SEQ ID NO: 4 remain conserved: R19.

[0086] The following amino acid positions of SEQ ID NO: 6 remain conserved: R19.

[0087] The following amino acid positions of SEQ ID NO: 8 remain conserved: R19.

[0088] The following amino acid positions of SEQ ID NO: 10 remain conserved: R19. Numbering according to Kabat.

[0089] SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10 encompass all complementarity determining regions (CDRs) and framework regions of the bispecific anti-tumor antigen / anti-HSG antibodies described herein.

[0090] In a particular embodiment, the carboxy-terminal end of the VH is connected to the amino-terminal end of the VL via a peptide linker Gly-Gly-Ser (G2S), thereby forming a single chain variable fragment (scFv). In particular, the linker is (G2S) n wherein n is 1, 2, 3, 4 or 5, in particular 3, 4 or 5.

[0091] In a particular embodiment, the linker is SEQ ID NO: 39.

[0092] In particular, said linker is used for antibodies of the application in scFv-Fab-Fc and IgG-central scFv (IgG-central scFv) format.

[0093] The scFv can be directly connected to the respective constant region via a flexible linker, e.g. (G4S) n wherein n is 1, 2, 3, 4 or 5, in particular 1 to 3.

[0094] In a particular embodiment, the linker is SEQ ID NO: 42.

[0095] In particular, said flexible linker is used for antibodies of the application in scFv-Fab-Fc and IgG-central scFv (IgG-central scFv) format.

[0096] The flexible linker is in turn linked to the Fc region of the antibody heavy chain (e.g. SEQ ID NO: 32 or SEQ ID NO: 34) via the truncated hinge region of SEQ ID NO: 40, in particular for antibodies of the application in the scFv-Fab-Fc and IgG-central scFv IgG-central scFv) format.

[0097] The VH and VL are also linked to the respective antibody constant regions (e.g. CLKcross (SEQ ID NO: 38) and CH1cross (e.g. SEQ ID NO: 30), respectively, which are in turn linked to the Fc region of SEQ ID NO: 32 via the truncated hinge of SEQ ID NO: 40, or to the truncated hinge-Fc of SEQ ID NO: 33, in particular for antibodies of the application in the CrossMab (CH1-CL) format. According to a particular embodiment of the application, the antibody comprises 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 one specific embodiment, the bispecific antibodies described herein contain a variant Fc domain that has reduced or eliminated effector function and / or Fc Rn binding.

[0098] As used herein, "effector function" refers to biochemical events triggered after an antibody Fc region interacts with an Fc receptor or ligand. Effector functions include, but are not limited to, ADCC, ADCP, and CDC.

[0099] As used herein, "effector cell" refers to a cell in 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; can be from any organism, including but not limited to, humans, mice, rats, rabbits, and monkeys. Due to the amino acid substitutions at selected positions in the heavy chain constant region, specifically the Fc region, the effector function of the bispecific antibodies described herein is silenced according to the present application. Due to the reduction in complement and FcyR-mediated activities, the effector function of these antibodies is reduced or completely silenced, which can include a reduction or elimination of complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and / or antibody-dependent cellular phagocytosis (ADCP).

[0100] The term "Fc region" (or "fragment, crystallizable region") of "Fc" or "Fc domain" as used herein refers to a polypeptide that comprises the constant region of an antibody, but does not include the first constant region immunoglobulin domain (CHI domain), and in some cases, does not include part of the hinge. The Fc region refers to the C-terminal region of an antibody. The Fc region is comprised of two, specifically identical, protein fragments that originate from the second and third constant domains of the two heavy chains (A chain and B chain) of an antibody. The second and third constant domains are referred to as CH2 domain and CH3 domain, respectively. The CH2 domain comprises the CH2 domain sequence of the A chain and the CH2 domain sequence of the B chain. The CH3 domain comprises the CH3 domain sequence of the A chain and the CH3 domain sequence of the B chain. The Fc region as used herein includes the hinge region or a portion thereof.

[0101] The CH2 domain of a human IgG Fc region sequence typically extends from about amino acid position 231 to about amino acid position 340 according to EU numbering. The CH2 domain sequence is unique in that it does not pair tightly with another domain sequence. Rather, in an intact native IgG molecule, two N-linked branched carbohydrate chains are located between the two CH2 domain sequences.

[0102] In an Fc region sequence, the CH3 domain comprises the amino acid residues located C-terminal to the CH2 domain sequence (i.e., from about amino acid residue 341 to about amino acid residue 447 in IgG according to EU numbering).

[0103] A "functional Fc region" or "functional Fc domain" possesses the "effector functions" and FcRn binding of a native Fc region. Typical "effector functions" include Clq binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); etc. These effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using various art-known assays as well as the assays disclosed herein.

[0104] A "native Fc region" or "native Fc domain" comprises an amino acid sequence identical to that of an Fc region found in nature. Human Fc region native sequences include human IgGl Fc region native sequences (non-A and A allotype); human IgG2 Fc region native sequences, human IgG3 Fc region native sequences, and human IgG4 Fc region native sequences, as well as natural variants thereof.

[0105] A "variant Fc region" or "variant Fc domain" comprises an amino acid sequence that differs from a native Fc region sequence by "one or more amino acid substitutions". A variant Fc region sequence has at least one amino acid substitution compared to a native Fc region sequence or to 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 a native Fc region sequence or in the Fc region sequence of a parent polypeptide. In certain embodiments, a variant Fc region sequence herein has at least about 80% identity, most preferably at least about 90% identity, more preferably at least about 95% identity to a native Fc region sequence and / or to the Fc region sequence of a parent polypeptide.

[0106] In a particular embodiment, amino acid substitutions are made at any one or more of positions E233, L234, L235, G236, I253, G237, P238, D265, S267, H268, N297, S298, T299, H310, E318, L328, P329, A330, P331 and H435, with reference to IgG1 or SEQ ID NO: 31, according to EU numbering.

[0107] In another particular embodiment, amino acid substitutions are made at any one or all of positions L234, L235, H310 in the CH2 domain, and at position H435 in the CH3 domain, with reference 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 almost complete inhibition of FcyR interaction, thereby achieving almost complete or complete Fc silencing.

[0108] In the prior art, it is known that modifications to the Fc region can reduce or eliminate Fc-related effector functions, as described in Saunders K. (2019) and Liu R. et al. (2020).

[0109] In an alternative embodiment, amino acid substitutions are made at any one or all of positions L234F, H268Q, K274Q, Y296F, A327G, A330S, P331S in the CH2 domain, and at positions R355Q, K409R, Q419E, P445L in the CH3 domain.

[0110] In particular, the Fc-silent bispecific antibodies described herein comprise one or more of the following combinations of amino acid substitutions or deletions: i) L235, G237 and E318, in particular L235A, G237A and E318A; ii) L234, L235, in particular L234A, L235A; iii) S228, L235, in particular S228P, L235E; iv) G236, L328, in particular G236R, L328R; v) S298, T299, in particular S298G, T299A; vi) L234, L235, P331, in particular L234F, L235E, P331S; vii) H268, V309, A330, P331, in particular H268Q, V309L, A330S, P331S; viii) E233, L234, L235, G236, S267, in particular E233P, L234V, L235A, G236 deletion (G236del), S267K; ix) L234, L235, P329, in particular L234A, L235A, P329G; x) V234, G237, P238, H268, A330, P331, in particular V234A, G237A, P238S, H268A, A330S, P331S; xi) L234, L235, D265, in particular L234F, L235E, D265A; xii) D265, in particular D265A; xiii) G237, in particular G237A; xiv) E318, in particular E318A; xv) E233, in particular E233P; xvi) G236, L328, in particular G236R, L328R; xvii) L235, in particular L235E; xviii) L234, L235, P331, in particular L234Q, L235F, P331S; xix) L234, L235, G237, P238, H268, A330, P331, in particular L234A, L235A, G237A, P238S, H268A, A330S, P331S; xx) N297, in particular N297A, N297Q or N297G, thereby generating an aglycosylated antibody.

[0111] Glycosylation, including O-glycosylation and N-glycosylation, is a post-translational modification of antibodies (Abs) that is regulated by a variety of B-cell stimuli, including environmental factors (e.g., stress or disease), cytokine activity, and innate immune signaling receptors (e.g., Toll-like receptors). The glycosylation pattern of a parent antibody can be modified by methods well known in the art. In particular, O-linked glycosylation sites are located in the CH2 and hinge regions.

[0112] In particular, the bispecific antibodies described herein comprise one or more of the following combinations of amino acid substitutions: i) I253A; ii) H310A; iii) H435, in particular 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.

[0113] However, according to EU numbering, silencing and further mutations of the CH domains described herein can also be introduced at the corresponding positions in the Fc of wild-type IgG2, IgG3, or IgG4.

[0114] The term "aglycosylated" means that the Fc region is not glycosylated. The constant region of all human IgG isotypes is known to be glycosylated at the aspartic acid (Asp) residue at position 297, which forms part of the N-glycosylation motif Asp297-X298-Ser299 or Thr299, wherein X is any amino acid residue except proline. The sugar chain has a heptose core and a variable extension, e.g., fucose, galactose, and / or sialic acid. Thus, the antibodies of the present application can be aglycosylated by replacing Asp 297 in such constant region with another amino acid that cannot be glycosylated or cannot be enzymatically deglycosylated. Any other amino acid residue can be used, but most preferably alanine (Ala). Alternatively, glycosylation of Asp 297 is prevented by changing one of the other residues in the motif, e.g., replacing the residue at position 298 with proline (Pro), or replacing the residue at position 299 with any amino acid except serine (Ser) or threonine (Thr). The skilled person is well aware of techniques to perform such site-directed mutations, e.g., using commercially available site-directed mutagenesis kits.

[0115] The term "silent Fc" refers to an Fc region of an antibody in which effector function and / or binding to FcRn is reduced or eliminated as a result of amino acid substitution or modification of the glycosylation pattern (resulting in reduced or eliminated binding of the modified glycan to any FcyR (e.g., FcyRIIaH, FcyRIIaR, FcyRllb, FcyRlllaF, FcyRlllaV, and FcyRIa), and / or FcRn receptor and complement factor Clq protein). This reduction or elimination of binding results in a reduction or elimination of effector function and / or binding to FcRn normally mediated by a wild-type IgG Fc region.

[0116] The term "Fc null" can be used herein if the binding of the antibody to FcyR (e.g., any FcyRIIa, FcyRll, FcyRllla, and FcyRIa), and / or FcRn receptor and complement factor Clq protein is completely eliminated.

[0117] Fc silencing can be achieved substantially by combining L234 and L235 mutations, which are located close to the hinge region and when substituted with alanine, can reduce binding to FcyR. For example, L234A and L235A in combination with P329G can result in almost complete inhibition of the interaction of all FcyR subtypes with FcyR.

[0118] Fc-silent bispecific antibodies described herein have substantially reduced, silenced, negligible or eliminated binding affinity to FcyRs and C1q binding affinity compared to a parent polypeptide or a polypeptide comprising a native Fc region sequence, are antibodies with reduced FcyR binding activity and C1q binding activity. In certain embodiments, Fc-silent bispecific antibodies have substantially reduced, silenced, negligible or eliminated binding affinity to FcR and C1q binding affinity compared to a parent polypeptide or a polypeptide comprising a native Fc region sequence, also have substantially reduced, silenced, negligible or eliminated ADCC, ADCP and CDC activity. Fc-silent bispecific antibodies described herein, which have reduced or undetectable binding to FcyRs, have lower binding affinity to all FcyRs compared to a parent polypeptide. An exemplary parent polypeptide can be the antibody Ibritumomab. Such variants with reduced binding to FcyRs can have little or no significant binding or no binding at all to FcyRs. In a particular embodiment, the variant has a binding rate to FcyRs of 0-20%, e.g., as measured by a change in equilibrium constant, compared to a native IgG Fc region. In an embodiment, the variant has a binding rate to FcyRs of 0-10% compared to a native IgG Fc region. In an embodiment, the variant has a binding rate to FcyRs of 0-5% compared to a native IgG Fc region. In an embodiment, the variant has a binding rate to FcyRs of 0-1% compared to a native IgG Fc region.

[0119] Antibodies described herein with silenced complement activity can be determined by a cell-based CDC assay and reduced or eliminated binding to C1q by methods such as surface plasmon resonance (SPR) or enzyme-linked immunosorbent assay (ELISA).

[0120] CDC activity is reduced or silenced at least 1.5-fold, specifically at least 2-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, more specifically at least 10-fold down compared to a reference (i.e., unmodified wild-type Fc).

[0121] ADCC or ADCP activity is reduced at least 2-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, more specifically potency is reduced at least 10-fold compared to a reference antibody (i.e., unmodified wild-type Fc).

[0122] In one embodiment, the binding of the anti-tumor antigen / anti-HSG antibody to FcRn is reduced due to amino acid substitutions at selected positions of the heavy chain constant region. The Fc-silent bispecific antibodies of the present application have reduced binding to FcRn, resulting in a shorter half-life in the circulation system and a faster clearance in vivo. The determination of FcRn binding and clearance / half-life in vivo can also be performed using methods known in the art (e.g., see Petkova, S.B. et al., 2006).

[0123] The Fc domain of the bispecific antibodies described herein has reduced binding to FcRn compared to an anti-tumor antigen / anti-HSG antibody comprising a wild-type IgG Fc region, preferably the Fc domain comprises one, two or three amino acid substitutions at any of positions I253, H310 and H435.

[0124] The bispecific antibodies described herein can also carry a L234A / L235A (“LALA”) mutation at the beginning of the heavy chain CH2 region. Introduction of the L234A / L235A mutation can almost completely eliminate the binding of a monoclonal antibody (mAb) to FcyR and significantly reduce its binding to complement (Lo M. et al., 2017; Wang X. et al., 2018; Zhou Q. et al., 2020).

[0125] Fc-silencing also helps to minimize potential adverse effects by binding of the antibodies described herein to Fcy receptor-bearing immune cells and complement during radioimaging, in particular when pre-targeting is performed.

[0126] The FcRn binding (i.e. affinity) is reduced at least 2-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, more specifically at least 10-fold compared to a reference antibody (i.e. unmodified wild-type antibody).

[0127] According to one embodiment, the antibodies described herein can bind two different heavy chains. Heterodimerization of two different heavy chains can be achieved by using construction techniques of, for example, heterodimeric Fc variants, in which the interactions promoting homodimer formation are replaced by interactions promoting heterodimer formation at the CH3 domain interface, by introducing asymmetric mutations in each CH3 domain, promoting the assembly of heavy chains from different antibodies. Thus, structure-based rational design and directed evolution approaches can be used to make variants that thermodynamically favor heterodimer over homodimer formation. Strategies known in the art are, for example, symmetric-asymmetric steric complementation design (e.g., KiH, HA-TF and ZW1); charge exchange (e.g., DD-KK); charge-steric complementation exchange plus additional long-range electrostatic interactions (e.g., EW-RVT); and isotype chain exchange (e.g., chain exchange engineered domain (SEED)).

[0128] Knob-into-hole (KiH) technology: One heavy chain carries in its CH3 region the "knob" mutations T366W and S354C for disulfide stabilization (Merchant A.M. et al., 1998), in particular the CH3 region carries an anti-tumor antigen binding site, e.g. an anti-oxMIF binding site. The other heavy chain carries in its CH3 region the "hole" mutations T366S / L368A / Y407V and Y349C for disulfide stabilization (Merchant A.M. et al., 1998), in particular the CH3 region carries an anti-HSG binding site.

[0129] According to a specific embodiment of the application, the antibody described herein comprises SEQ ID NO: 35 and SEQ ID NO: 32.

[0130] According to an embodiment of the application, the antibody described herein comprises SEQ ID NO: 36 and SEQ ID NO: 33 or SEQ ID NO: 36 and SEQ ID NO: 34.

[0131] The antibodies described herein can be used to detect and localize tumors expressing tumor-associated antigens or tumor-specific antigens on their cell surface.

[0132] The anti-tumor antigen binding site can specifically target and bind to any tumor antigen or tumor associated antigen, including but not limited to melanoma cell surface antigens, breast cancer cell surface antigens (e.g., CA15-3), lung cancer cell surface antigens, colorectal cancer cell surface antigens, gastric cancer cell surface antigens, pancreatic cancer cell surface antigens, glioma cell surface antigens, common sarcoma cell surface antigens, gastrointestinal cancer cell surface antigens, brain tumor cell surface antigens, esophageal cancer cell surface antigens, common epithelial cancer cell surface antigens, osteosarcoma cell surface antigens, fibrosarcoma cell surface antigens, bladder cancer cell surface antigens, prostate cancer cell surface antigens, renal cancer cell surface antigens, ovarian cancer cell surface antigens, testicular cancer cell surface antigens, endometrial cancer cell surface antigens, cervical cancer cell surface antigens, Hodgkin's disease cell surface antigens, lymphoma cell surface antigens, leukemia cell surface antigens, trophoblastic tumor cell surface antigens, tumor necrosis antigens, oxMIF. When the disease state is cancer, for example, many antigens are known in the art to be expressed by or associated with tumor cells, including but not limited to carbonic anhydrase IX, alpha-fetoprotein, alpha-actinin-4, A3, A33 antibody specific antigen, ART-4, B7, Ba733, BAGE, BrE3 antigen, CEA, tumor antigen CA-125, PSA, CAP-1, CASP-8 / m, CCL19, CCL21, CEA CEACAM5, CEACAM6, CEACAM8, c-met, CDK-4 / m, CDKN2A, CXCR4, CXCR7, CXCL12, HIF-1 alpha, colon-specific antigen-p (CSAp), Flt-1, Flt-3, folate receptor, G250 antigen, GAGE, gp100, GRO-beta, HLA-DR, HM1.24, human chorionic gonadotropin (HCG) and subunits thereof, HER2 / neu, HMGB-1, hypoxia-inducible factor (HIF-1), HSP70-2M, HST-2, Ia, IGF-1R, IFN-gamma, IFN-alpha, IFN-beta, IL-2, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-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), oxMIF, MAGE, MAGE-3, MART-1, MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B, folate receptor alpha (FRa),EGFR, Trop-2, MUC1, MUC2, MUC3, MUC4, MUC5, MUM-1 / 2, MUM-3, NCA66, NCA95, NCA90, pancreatic mucins, 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, angiogenesis markers, bcl-2, bcl-6, KRAS, cMET, oncogene markers and oncogene products, tumor necrosis antigens, TNF-alpha, TRAIL receptors (R1 and R2), NCA-90, NCA-95, Transmembrane Activator and CAML Interactor (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, CD90, CD137, CD177, CDC27, HLA-DR alpha chain, KIR, LSECtin or SLC44A2, B7, la, li, HM1.24, HLA-DR, tenascin, VEGF, PIGF, ED-B fibronectin, oncogenes, oncogene products (e.g., c-met or PLAGL2), IL-2, T101, TAG, etc.

[0133] According to the detailed description of the application, the anti-tumor antigen binding site of the antibody described herein can specifically recognize oxMIF.

[0134] oxMIF antibodies have been described in PCT / EP2021 / 077106 and PCT / EP2022 / 052463.

[0135] The oxMIF binding site is specific for the oxidized form of MIF (i.e. specifically, human oxMIF) and does not significantly cross-react with the reduced form of MIF. oxMIF is a disease- relevant structural isoform of MIF that can be specifically and predominantly detected in the circulation of subjects suffering from inflammatory diseases and in tumor tissue of cancer patients.

[0136] The oxMIF binding specificity can be determined by any assay suitable for determining selective binding to oxMIF, such as any competition assay for binding of a control antibody (e.g. imatinib) to oxMIF, or various assays known in the art and described herein.

[0137] The bispecific antibody of the present application comprises at least one binding site specifically recognizing oxMIF and, according to the specific embodiments of the present application, exhibits a reduced aggregation propensity and a reduced hydrophobicity due to the targeted amino acid substitution in the variable domains of the heavy and light chains, compared to the unmodified antibody lacking said amino acid substitution.

[0138] The reduction of the aggregation potential is due to the amino acid substitution at selected positions within the variable domains of the antibodies described herein.

[0139] The level of antibody aggregation can be measured using various 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) techniques (e.g. micro flow imaging (MFI) method) and Coulter counting (CC) method, differential scanning fluorimetry (DSF).

[0140] “Reduced hydrophobicity” and “reduced aggregation potential” as used herein refer to a reduction of the surface hydrophobicity and aggregation potential of the antibody compared to a reference antibody (e.g. the antibody imatinib published in Proposed INN List 111 (WHO Drug Information, Vol. 28, No. 2, 2014), but lacking the C-terminal lysine). The measurement 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).

[0141] In one embodiment, the oxMIF binding site of the antibody of the application specifically comprises a light chain variable domain having SEQ ID NO: 45 and one or more (specifically 1, 2, 3, 4 or 5) amino acid substitution(s) at position M30, F49, A51, P80, W93, specifically M30L, F49Y, A51G, P80S, W93F, more specifically F49Y, A51G, W93F, according to the Kabat numbering, or a light chain variable domain having at least 95% (specifically at least 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 45 and which contains a tyrosine at position 36, further comprising at least one (specifically 1, 2, 3, 4 or 5) amino acid substitution at position M30, F49, A51, P80, W93, specifically M30L, F49Y, A51G, P80S and W93F, more specifically F49Y, A51G and W93F, according to the Kabat numbering, in combination with a heavy chain variable domain comprising SEQ ID NO: 46, or a heavy chain variable domain comprising SEQ ID NO: 46 and comprising an amino acid substitution at position L5 and / or W97 (specifically L5Q and / or W97Y), or a heavy chain variable domain having 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 46 and comprising an amino acid substitution at position L5 and / or W97 (specifically L5Q and / or W97Y).

[0142] According to one particular embodiment, the amino acid W93 is substituted by F, Y or H.

[0143] In another embodiment, the anti-oxMIF antibody of the application having reduced aggregation propensity and hydrophobicity specifically comprises a heavy chain variable domain comprising SEQ ID NO: 46 and comprising an amino acid substitution at position W97 (specifically W97Y), or an amino acid substitution at position L5 (specifically L5Q), or an amino acid substitution at both positions L5 and W97 (specifically L5Q and W97Y), or a heavy chain variable domain having 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 46 and comprising the amino acid substitution W97Y (optionally in combination with L5Q).

[0144] According to one particular embodiment, the amino acid W97 is substituted by F, Y or H.

[0145] In one preferred embodiment, the anti-oxMIF antibody of the application having reduced aggregation propensity and hydrophobicity specifically comprises an amino acid substitution at both positions W93 and W97.

[0146] The tyrosine at position 36 of the light chain is specifically left unmodified to maintain the binding properties of the antibodies described herein. Any modification of this amino acid position can result in impaired binding properties with adverse effects.

[0147] In one particular embodiment, the antibodies described herein comprise: - a heavy chain variable domain comprising SEQ ID NO: 46, in particular with the amino acid substitutions L5Q and / or W97Y, - a heavy chain variable domain comprising SEQ ID NO: 46, in particular with the amino acid substitutions L5Q and / or W97Y, wherein the amino acid positions are according to Kabat numbering.

[0148] The term "antibody" as used herein is in the broadest sense to encompass a polypeptide or protein consisting of or comprising antibody domains, which are to be understood as constant and / or variable domains of immunoglobulin heavy and / or light chains, with or without linker sequences. The term also encompasses fusion proteins, e.g. fusions with immunotoxins or antibody conjugates, e.g. antibody drug conjugates binding to HSG and oxMIF.

[0149] Antibody domains can be native structures or modified by mutagenesis or derivatization, e.g. to alter antigen binding properties or any other properties, e.g. stability or functional properties, e.g. binding to Fc receptors (e.g. FcRn and / or Fc-gamma receptors). A polypeptide sequence is considered to be an antibody domain if it comprises a beta barrel structure consisting of at least two beta strands of an antibody domain structure connected by loop sequences.

[0150] It is to be understood that the term "antibody" includes antigen binding derivatives, variants and fragments thereof. Derivatives or variants refer to any combination of one or more antibody domains or antibodies described herein with / and fusion proteins, wherein any domain of an antibody described herein can be fused to any site of another protein or proteins (e.g. other antibodies or antibody formats, e.g. binding structures comprising CDR loops, receptor polypeptides, also including ligands, scaffold proteins, enzymes, tags, toxins, etc.).

[0151] The term "antibody" particularly refers to a polypeptide or protein having binding properties to a target tumor antigen, in particular oxMIF and HSG.

[0152] The terms "antibody fragment, antigen-binding fragment, antigen-binding variant or antibody variant" are used interchangeably herein to refer to a molecule other than an intact antibody that comprises an antigen-binding portion of an intact antibody that is capable of binding an antigen bound by the intact antibody, also including multispecific antibodies formed from antibody fragments or variants, and further comprising variant Fc regions as described herein. Examples of antigen portions include, but are not limited to, Fv, Fab, Fab', Fab'-SH, single-chain antibody molecules (e.g., scFv), diabodies, cross-Fab fragments, linear antibodies. According to the present application, the antibody fragment or variant is fused to a silent Fc portion or a silent Fc domain via a hinge region and / or a linker, in particular a hinge region preceding a linker, such as (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.

[0153] Furthermore, the antibody fragment comprises a single chain polypeptide having the properties of a VH domain, i.e. being capable of assembling with a VL domain into a functional antigen binding site, thereby providing the antigen binding properties of a full length antibody, or a single chain polypeptide having the properties of a VL domain, i.e. being capable of assembling with a VH domain into a functional antigen binding site, thereby providing the antigen binding properties of a full length antibody. The antibody fragment described herein also includes a silent Fc domain comprising one or more loop structure regions comprising an antigen binding region, such as Fcab TM , or a full length antibody format comprising an IgG structure, wherein the silent Fc region has been substituted by an Fcab TM comprising a second different antigen binding site.

[0154] Herein, "Fab fragment or Fab" refers to an antibody fragment comprising a light chain fragment comprising a VL domain and a light chain constant region (CL), and a VH domain and a heavy chain first constant region (CH1). The antibody of the present application can comprise at least one Fab fragment, wherein the variable regions or the constant regions of the heavy and light chains can be exchanged. Due to the exchange of the variable regions or the constant regions, the Fab fragment is also referred to as "cross-Fab fragment" or "crossover Fab fragment". The crossover Fab molecule consists of two different chain compositions, and both are comprised in the antibody of the present application: the variable regions of the Fab heavy chain and the light chain can be exchanged, i.e. the crossover Fab molecule comprises one peptide chain. According to the present application, the Fab fragment is fused to a silent Fc portion or a silent Fc domain via a hinge region.

[0155] As used herein, a "Fab arm" refers to a Fab fragment fused to a silent Fc portion or a silent Fc domain via a hinge region.

[0156] The term "functional variant" or "functionally active variant" also includes naturally occurring allelic variants, as well as mutants or any other non-naturally occurring variants. As is known in the art, allelic variants (also known as homologues) are alternative forms of a nucleic acid or peptide, characterized by one or more substitutions, deletions or additions of nucleotides or amino acids, respectively, which do not substantially alter the biologic function of the nucleic acid or peptide. In particular, a functional variant can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 substitutions, deletions and / or additions of amino acid residues, or combinations thereof, which are conservative modifications and which do not alter the antigen binding properties. In particular, a functional variant as described herein comprises no more than or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 substitutions, deletions and / or additions of amino acids which are conservative modifications and which do not alter the antibody function. In particular, a functionally active variant as described herein comprises up to 15, preferably up to 10 or 5 substitutions, deletions and / or additions of amino acids which are conservative modifications and which do not alter the antibody function.

[0157] Functional variants can be obtained by altering the polypeptide or nucleotide sequence, e.g., by one or more point mutations, wherein such sequence alterations can maintain or improve the function of the unaltered polypeptide or nucleotide sequence when used in combination with the present application. Such sequence alterations include, but are not limited to, (conservative) substitutions, additions, deletions, mutations and insertions. Conservative substitutions refer to the substitution of a member of one family for another member of the same family, based on side-chain and chemical properties. Examples of such families include amino acids with basic side chains, acidic side chains, uncharged polar side chains, nonpolar side chains, beta sheet formers and the like.

[0158] According to one particular embodiment, the antibodies described herein can comprise one or more tags for purification and / or detection, including but not limited to affinity tags, solubility-enhancing tags and monitoring tags.

[0159] In particular, the affinity tag is selected from the group consisting of a polyhistidine tag, a polyarginine tag, an antibody peptide substrate, a chitin binding domain, an RNAse S peptide, protein A, beta-galactosidase, a FLAG tag, a Strep II tag, a streptavidin binding peptide (SBP) tag, a calmodulin binding peptide (CBP), glutathione S-transferase (GST), maltose binding protein (MBP), an S tag, an HA tag, and a c-Myc tag, in particular the tag is a His tag comprising one or more H, for example a tag of six histidines.

[0160] "Fusion", "linkage" or "coupling" means that the components (e.g. the Fab molecule and the Fc domain subunit) are directly linked by a peptide bond or linked by one or more peptide linkers.

[0161] Herein, the term "linker" in the context of the linkage between the HSG hapten and the chelator and / or therapeutic / diagnostic agent refers to a peptide linker, preferably a peptide of an amino acid sequence of a length of 2, 3, 4, 5, 6, 7 or more amino acids, preferably of a length of 3-15, more preferably of a length of 3-5 amino acids. Specific linkers are described herein, for example SEQ ID NO: 39 and SEQ ID NO: 42, which vary in length.

[0162] The term "immunoglobulin" refers to a protein having the structure of a natural antibody. For example, an IgG class immunoglobulin is a heterotetrameric glycoprotein of about 150,000 daltons, comprising two light chains and two heavy chains that are interconnected by disulfide bonds. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CHI, CH2, and CH3), also called a heavy chain constant region. Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a light chain constant region (CL), also called a light chain constant region. An IgG class immunoglobulin is essentially composed of two Fab molecules and an Fc domain, which are connected by an immunoglobulin hinge region. The heavy chains of an immunoglobulin can be assigned to one of five types, namely alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG), or mu (IgM), some of which can be further divided into subtypes, for example gamma 1 (IgG1), gamma 2 (IgG2), gamma 3 (IgG3), gamma 4 (IgG4), alpha 1 (IgA1), and alpha 2 (IgA2). The light chains of an immunoglobulin can be assigned to one of two types, namely kappa (K) and lambda (l).

[0163] The term "chimeric antibody" refers to an antibody in which a portion of the heavy and / or light chain is of a specific origin or species, while the remainder of the heavy and / or light chain is of a different origin or species, usually prepared by recombinant DNA technology. A chimeric antibody can comprise a rabbit or murine variable region with a human constant region. Chimeric antibodies are the products of expressed immunoglobulin genes that comprise DNA segments from both an immunoglobulin variable domain and an immunoglobulin constant domain. Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques, which are well known in the art (Morrison, S.L. et al., 1984).

[0164] A "human antibody" has an amino acid sequence corresponding to an antibody produced by a human or human cell, or derived from a human antibody library or other human antibody-encoding sequences from a non-human source. Human antibodies as defined herein expressly exclude humanized antibodies comprising non-human antigen binding residues. As with chimeric and humanized antibodies, the term "human antibody" as used herein also includes antibodies in which the constant region has been modified, e.g., by "class switching," i.e., alteration or mutation of the Fc portion (e.g., from IgGl to IgG4 and / or IgGl / IgG4 mutations).

[0165] The term "recombinant human antibody" as used herein is intended to include all human antibodies that are prepared, expressed, generated, or isolated

[0166] A "human consensus framework" refers to a framework which is derived from the most commonly occurring amino acid residues in a group of human immunoglobulin VL or VH framework sequences. Generally, the group of human immunoglobulin VL or VH sequences is selected from VH or VL sequences of a particular subgroup or subgroup of subgroups of variable domain sequences. Generally, the sequences of the subgroup are described in Kabat et al. (1991).

[0167] A "humanized" antibody refers to an antibody which contains both amino acid residues from non-human hypervariable regions (HVRs) and amino acid residues from human framework regions (FRs) and which has been humanized. In certain embodiments, a humanized antibody will comprise substantially all of at least one (and typically two) variable domains, in which all or nearly all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or a significant portion of the FRs correspond to those of a human antibody. A humanized antibody optionally also will comprise at least a portion of an antibody constant region derived from a human antibody. In particular, the humanized antibody forms contemplated herein, the constant region has been additionally modified or changed relative to the original antibody to produce new properties, i.e., reduced or eliminated Clq binding and / or Fc receptor (FcR) binding.

[0168] Methods and details of humanization of anti-HSG antibodies are described herein, particularly in the Examples.

[0169] The term "bispecific" as used herein refers to at least a binding reaction with an anti-tumor antigen (e.g., oxMIF antigen) and an additional antigen (HSG hapten). A bispecific antibody specifically can comprise at least two sites with specific binding properties, wherein the antibody can recognize two different target antigens, i.e., at least one tumor antigen and HSG. A bispecific antibody format comprises two binding sites, wherein each binding site is capable of specifically binding a tumor antigen and HSG. Alternatively, another exemplary bispecific format can comprise more than two binding sites, e.g., 3, 4, 5 or more binding sites, wherein one or more binding sites bind one or more of the same or different tumor antigens and one or more binding sites are capable of specifically binding HSG.

[0170] A "bispecific antibody" according to the present application refers to an antibody having two different binding specificities. Bispecific antibodies can be prepared as full-length antibodies as described herein, or as antibody fragments still comprising a silent Fc region. IgG Fc heterodimers can be designed by modifying the CH3 domain interface, with different mutations on each domain, such that engineered Fc fragments carrying pairs of CH3 variants preferentially form heterodimers rather than homodimers (Ha J-H. et al. 2016). Examples of bispecific antibody formats include, but are not limited to, bispecific IgG (BsIgG), IgG appended with additional antigen binding groups, 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, and the like, as described in Spiess C. et al. (2015) and Brinkmann U. and Kontermann R.E. (2017).

[0171] In another embodiment encompassing the bispecific antibodies described herein, the term "IgG-scFv" refers to a bispecific antibody designed by fusing one scFv to a monospecific immunoglobulin G (IgG) to have a dual specificity. According to one embodiment of the present application, the bispecific antibody is Fc region silent, i.e. it comprises a wild-type human IgG variant Fc region having one or more amino acid substitutions or glycosylation modifications described herein. The specificity of the IgG can be directed against a tumor antigen, while the specificity of the scFv can be directed against the HSG hapten, or vice versa. Furthermore, the scFv can be attached at the amino- or carboxy-terminus of one light or heavy chain, thereby generating various types of IgG-scFv bispecific antibodies (BsAb): (i) IgG(H)-scFv, i.e. the scFv is attached at the C-terminus of the full-length IgG HC; (ii) scFv-(H)IgG, which is identical to IgG(H)-scFv, except that the scFv is attached at the N-terminus of the HC; (iii) IgG(L)-scFv or (iv) scFv-(L)IgG, i.e. the scFv is attached at the C- or N-terminus of the IgG light chain, forming IgG(L)-scFv or scFv-(L)IgG, respectively. In particular, the molecular weight of the IgG-scFv ranges from 165 kDa to 185 kDa, in particular about 175 kDa.

[0172] In a particular embodiment, the term "Fab / bs(scFv)2-Fc" refers to a bispecific antibody which is an IgG wherein one Fab arm is replaced by bs(scFv)2 and the second IgG arm is retained.

[0173] In a particular embodiment, the term "Fab / scFv-Fc" refers to a bispecific antibody which is an IgG wherein one Fab arm is replaced by scFv and the second IgG arm is retained. Antibody C0181 described herein and illustrated in schematic form in Figure 1 Figure 2 is a non-limiting example of a Fab / scFv-Fc.

[0174] In a particular embodiment, the term "Fab / Fab-scFv-Fc (IgG-central scFv (IgG- central scFv))" refers to a bispecific antibody which is an IgG wherein one Fab arm is replaced by Fab-scFv and the second IgG arm is retained.

[0175] The term "CrossMab" (wherein Mab refers to monoclonal antibody) refers to a bispecific antibody format derived from separate parental antibodies. Heavy chain mispairing is avoided by applying the "knobs-into-holes" (KIH) approach. Since the bispecific antibody is generated by antibody domain exchange, light chain mispairing is avoided, i.e. the variable or constant domains (CL and CHI) of one Fab arm are exchanged between light and heavy chain. This "crossover" preserves antigen binding affinity and retains two different arms to avoid light chain mispairing. Examples of CrossMabs include, but are not limited to, exchanging Fabs, VH-VL and CHI-CL in different regions. In CrossMab Fabs, the complete VH-CHI and VL-CL regions are exchanged; in CrossMab VH-VL formats, only the VH and VL regions are exchanged; in CrossMab CHI-CL1 formats, the CHI and CL regions of the bispecific antibody are exchanged. Specifically, CrossMabs have a molecular weight of about 150 kDa.

[0176] The term "antigen" as used herein is used interchangeably with "target" or "target antigen" and refers to the entire target molecule or a fragment of this molecule which is recognized by the antibody binding site. Specifically, a substructure of an antigen (e.g. a polypeptide or carbohydrate structure, often referred to as an "epitope", e.g. a B cell epitope or a T cell epitope) has immunological relevance and is recognized by such a binding site.

[0177] The term "epitope" as used herein refers specifically to a molecular structure which can constitute the complete specific binding partner for the binding site of the antibody format according to the present application or which can constitute a part of the specific binding partner. Epitopes can be constituted by carbohydrates, peptide structures, fatty acids, organic, biochemical or inorganic substances or derivatives thereof and any combination thereof. If the epitope is comprised in a peptide structure (e.g. a peptide, polypeptide or protein), it usually comprises at least 3 amino acids, specifically 5 to 40 amino acids, more specifically less than 10 amino acids, specifically 4 to 10 amino acids. Epitopes can be linear epitopes or conformational epitopes. Linear epitopes are constituted by a single fragment of the primary sequence of a polypeptide or carbohydrate chain. Linear epitopes can be contiguous or overlapping. Conformational epitopes are constituted by amino acids or carbohydrates which come together when a polypeptide folds into a tertiary structure and which are not necessarily adjacent in the linear sequence. An exemplary oxMIF epitope can be the EPCALCS sequence (SEQ ID NO: 53) located in the central region of oxMIF.

[0178] The term "antigen binding domain", "binding domain" or "binding site" refers to the part of an antigen binding moiety comprising the region which specifically binds to and is complementary to a part or the whole of an antigen. When the antigen is large, the antigen binding molecule can only bind to a specific part of the antigen, which is referred to as an epitope. The antigen binding domain can be provided by, for example, one or more antibody variable domains (also referred to as antibody variable regions). Preferably, the antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0179] The term "binding site" as used herein with respect to the antibodies according to the present application refers to the molecular structure which is capable of interacting in a binding event with an antigen. Typically, the binding site is located within the complementarity determining regions (CDRs) of the antibody, also referred to herein as "CDR binding sites", which are specific regions of varying structure that are responsible for the ability of the antibody to bind different antigens. These varying structures can be derived from a natural antibody repertoire (e.g. a mouse or human antibody repertoire) or can be generated by recombinant or synthetic methods, for example by mutagenesis and specifically by randomization techniques. These regions include mutated CDR regions, loop regions of antibody variable domains, specifically CDR loops, for example CDR1, CDR2 and CDR3 loops in VL and / or VH antibody domains. The antibody formats used according to the present application typically comprise one or more CDR binding sites, each of which is directed against a specific antigen.

[0180] The oxMIF binding site of the antibodies described herein is specific for the oxidized form of MIF, i.e., for oxMIF of animals, in particular mammals, including but not limited to mice, rats, monkeys, and humans, in particular for human oxMIF, but does not exhibit significant cross-reactivity to the reduced form of MIF. In one embodiment, the humanized or human anti-oxMIF binding site comprises one or more (e.g., all three) light chain complementarity determining regions of a humanized or human anti-oxMIF binding domain described herein.

[0181] The term "specificity" herein refers to the ability to determine a binding reaction with a cognate ligand of interest in a heterogeneous series of molecules. Here, the binding reaction is at least to oxMIF antigen. Thus, an antibody that specifically binds its particular target under the specified conditions (e.g., immunoassay conditions) does not bind in significant amounts to other molecules present in the sample, in particular does not exhibit significant cross-reactivity to the reduced form of MIF.

[0182] A particular binding site typically does not cross-react with other targets. However, a binding site can specifically bind to one or more epitopes, isoforms, or variants of a target, or cross-react with other related target antigens (e.g., homologs or analogs).

[0183] A particular binding means binding with selectivity in terms of target identity, high, medium, or low binding affinity or avidity, as the case can be. Selective binding is typically achieved if the binding constant or binding kinetics to a target tumor antigen (e.g., oxMIF) or HSG hapten is at least 10-fold different, preferably at least 100-fold different, more preferably at least 1000-fold different, from the binding constant or binding kinetics to a non-target antigen.

[0184] The term "valency" herein denotes the particular number of binding sites in an antibody molecule. Thus, the terms "bivalent," "tetravalent," and "hexavalent" denote the presence of two, four, and six binding sites, respectively, in an antibody molecule.

[0185] The term "monovalent" herein with respect to a binding site of an antibody means that the molecule comprises only one binding site for a target antigen. Thus, the term "valency" is understood to mean the number of binding sites for the same target antigen, which can specifically bind to the same or different epitopes of the antigen.

[0186] The antibodies of the present application are understood to comprise monovalent, bivalent, tetravalent, or multivalent binding sites that specifically bind to oxMIF and HSG.

[0187] The term "hypervariable region" or "HVR" as used herein refers to each of the regions of an antibody variable domain which are highly divergent in sequence and / or form structurally defined loops ("hypervariable loops") that are involved in binding of the antibody to an antigen. In general, native four-chain antibodies comprise six HVRs; three in the VH (HI, H2, H3), and three in the VL (LI, L2, L3). The HVRs generally comprise amino acid residues from the hypervariable loops and / or "complementarity determining regions" (CDRs), which are the regions of highest sequence variability and / or involved in antigen recognition (Kabat et al., 1991). Hypervariable regions (HVRs) are also referred to as complementarity determining regions (CDRs), and the two terms are used interchangeably herein when referring to the portions of the variable region that comprise the antigen binding regions. The precise extent of the HVRs can vary, depending on the sequence and length of the CDRs. Those skilled in the art will be able to determine what residues are included in a particular CDR without undue experimentation.

[0188] Kabat defines a numbering system for variable region sequences that is applicable to any antibody. The "Kabat numbering" system can be unambiguously applied to any variable region sequence without recourse to experimental data other than the sequence itself. For a given antibody, the Kabat number for a residue can be determined by aligning its sequence with the "standard" Kabat numbered sequence in the region of homology. Herein, "Kabat numbering" refers to the numbering system set forth in Kabat et al., 1983, U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest." Unless otherwise specified, the numbering of particular amino acid residue positions in an antibody variable region follows the Kabat numbering system. The numbering of constant regions follows the EU index of substitution.

[0189] CDRs also include "specificity determining residues" or "SDRs," which are residues within CDRs that contact antigens, in regions referred to as abbreviated-CDRs or a-CDRs. Unless otherwise specified, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered according to the numbering system described above by Kabat et al. Determination of CDRs can also be made according to IMGT (Lefranc MP, 1997). IMGT has its own definition for framework regions (named FR-IMGT) and CDRs (named CDR-IMGT). The IMGT numbering method numbers residues consecutively from 1 to 128 based on alignment of germline V sequences.

[0190] CDRs (or SDRs) can also be further defined according to MacCallum RM et al. (1996). This study analyzed antigen-contacting residues and combined this with the site shapes available from antibody Fv and Fab crystal structures in the protein database. This study gave an antigen-contacting propensity for each antibody residue, allowing the definition of CDRs from observed antigen contacts. CDR residues located in the center of the binding site were more often contacting; some non-central CDR residues only contacted large antigens. Residues within CDRs that did not contact coincided with residues identified by Chothia and colleagues (Chothia C et al., 1987) that played an important role in defining the "canonical" conformation.

[0191] "Point mutation" specifically refers to engineering of a polynucleotide such that the expressed amino acid sequence has one or more (non-contiguous) single or pairs of amino acids substituted, exchanged, deleted, or inserted with different amino acids as compared to the un-engineered amino acid sequence. Preferably, a point mutation refers to an exchange between amino acids of the same polarity and / or charge.

[0192] "Percent (%) sequence identity" with respect to a polypeptide sequence 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 specific polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Suitable parameters for measuring alignment can be determined by those skilled in the art, including any algorithm needed to achieve maximum alignment over the full length of the sequences being compared.

[0193] According to the present application, the sequence identity of the variable region or constant region sequence is at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the corresponding sequence described herein.

[0194] As described above, the bispecific antibodies described herein comprise a first binding site for a tumor antigen or tumor-associated antigen, and a second binding site for a HSG (histidine succinylglycyl) hapten, which is specifically located on the HSG group. In another embodiment, the antibody can comprise two, three, or more HSG hapten binding sites, and one or more binding sites for a target antigen associated with a disease or condition.

[0195] The HSG group can have a variety of structures and preferably comprises a peptide containing only two amino acid residues, more preferably a peptide containing two to ten residues, and can be coupled to other groups, such as chelators. The HSG group is preferably a low molecular weight molecule, preferably less than 50,000 daltons, more preferably less than about 20,000 daltons, 10,000 daltons or 5,000 daltons in molecular weight, including chelates and chelated metals, and optionally diagnostic and / or therapeutic agents. In a preferred embodiment, the HSG group 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-tetraazacyclododecane tetraacetic acid. The HSG group can also include non-natural amino acids (such as D-amino acids) in the peptide backbone structure to improve the stability of the peptide in vivo.

[0196] Some specific embodiments of HSG groups 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 IDNO: 78); -(Tscg-Cys)-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(DOTA)-NH2 (SEQ IDNO: 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-NH2 (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). Peptides used as targeting constructs are conveniently synthesized on an automated peptide synthesizer using standard techniques of solid support and repeated orthogonal deprotection and coupling. Free amino groups in the peptide that are subsequently used for chelator coupling are advantageously blocked with standard protecting groups (e.g., Boc groups), while the N-terminal residue can be acetylated (Ac-) to improve serum stability. Such protecting groups are well known to those skilled in the art. See, Greene and Wuts, Protective Groups in Organic Synthesis, 1999 (John Wiley and Sons, New York). In preferred embodiments, the HSG group can comprise one or more hydrophilic chelating groups that are capable of binding metal ions and also help to ensure rapid clearance in vivo. The chelator can be selected for its particular metal binding properties and can be readily interchanged.

[0197] Particularly useful metal-chelator combinations include 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs. Macrocyclic chelators such as NOTA (1,4,7-triazacyclononane-N,N',N"-triacetic acid), DOTA, TETA (p-bromoacetamidobenzyl- tetraethylenethamine tetraacetic acid), and NETA can also be used with a variety of metals. DTPA and DOTA type chelators containing hard base chelating functionality (e.g., carboxylate or amine groups) are most effective for chelating hard acid cations, particularly Group IIa and IIIa metal cations. Such metal-chelator complexes can be made very stable by adjusting the ring size to accommodate the target metal. Other cyclic chelators, such as macrocyclic polyethers, are also attractive for stabilizing bound nuclides. Porphyrin chelators can be used with a variety of metal complexes. Chelators such as thiosemicarbazonylglyoxylcysteine (Tscg-Cys) and thiosemicarbazinyl-acetylcysteine (Tsca-Cys) can be effective for binding soft acid cations of technetium (Tc), rhenium (Re), bismuth (Bi), and other transition metals, lanthanides, and actinides that bind tightly to soft base ligands. It can be useful to link more than one type of chelator to a peptide. In one embodiment, two different hard or soft acid chelators can be incorporated into a targetable construct, for example, using different chelating ring sizes so as to preferentially bind two different hard or soft acid cations due to differences in cation size, chelating ring geometry, and preferred coordination structures of the cations. This would allow two different metals to be incorporated into the HSG group, ultimately to be captured by the bispecific antibodies of the application.

[0198] The HSG group can be labeled or coupled to one or more diagnostic and / or therapeutic agents.

[0199] In particular, it can comprise one or more HSG hapten, one or more therapeutic / diagnostic agent, and one or more chelator.

[0200] Some useful non-limiting examples are cyclic chelators or bifunctional chelators thereof, such as diethylenetriaminepentaacetic acid (DTPA) and 1,4,7,10-tetraazacyclododecane-N,N',N",N -Tetraacetic acid, ca-DTPA, ibca-DTPA, 1B4M-DTPA, lys-DTPA, vinyl DTPA, glu-DTPA, p-SCN-bn-DOTA, DOTA-NHS-ester, deferoxamine B or derivatives thereof; or linear chelating agents or bifunctional chelating agents thereof, such as p-SCN-Bn-DTPA, HOPO and CHX-A"-DTPA, ethylenediaminetetraacetic acid (EDTA), DTPA, EDTMP, NOTA, TETA, DOTMP, N2S2, N3S, HEDP. Other suitable examples of chelating agents include DOTA derivatives, such as benzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (p-SCN-Bz-DOTA), and DTPA derivatives, such as benzyl-diethylenetriaminepentaacetic acid (p-SCN-Bz-DTPA), the former being a cyclic chelating agent and the latter a linear chelating agent.

[0201] Other examples of deferoxamine (DFO) and its derivatives include, but are not limited to, p-NCS-Bz-DFO, DFOSq, DFO*, oxoDFO*, DFO*Sq, DFO*-NCS, and DFO*pPhe-NCS.

[0202] Specifically, a DFO can have the following structure: .

[0203] Specifically, DFO* can have the following structure: .

[0204] The peptide backbone can be directly linked to DOTA, or it can be linked through a cleavable or non-cleavable linker.

[0205] In some embodiments, the HSG group disclosed herein may be linked to one or more therapeutic agents and / or diagnostic agents. The therapeutic agents are specifically selected from the group consisting of radionuclides, anti-angiogenic agents, drugs, prodrugs, apoptosis-promoting agents, interfering RNA, photosensitizing agents, cytotoxic agents (which may be chemotherapeutic drugs or toxins), and combinations thereof. The drugs used may possess pharmacological properties selected from the group consisting of anti-mitotic, anti-kinase, alkylating, anti-metabolic, antibiotic, alkaloid, anti-angiogenic, and apoptosis-promoting agents, and combinations thereof.

[0206] In a preferred embodiment, the cytotoxic drug is selected from (but not limited to) microtubule inhibitors, meiosis inhibitors, RNA polymerase inhibitors, topoisomerase inhibitors, DNA damaging agents, and ribosome inhibitors.

[0207] Microtubule inhibitors or tubulin inhibitors (e.g. auristatins) can block tubulin assembly, resulting in cell cycle arrest at the G2 / M phase. The auristatin derivatives monomethyl auristatin F and monomethyl auristatin E (MMAF, MMAE) are effective at low nanomolar concentrations (Gerber et al., 2009). Maytansinoids or maytansine derivatives are another class of tubulin inhibitors, e.g. trastuzumab emtansine (DM1) and ravtansine (DM4). Tubulysins and analogs thereof are another class of tubulin inhibitors. Microtubule inhibitors (MTIs) (e.g. taxanes, vinca alkaloids and epothilones) can stabilize or destabilize microtubule stability, thereby inhibiting the dynamic changes in microtubules required for normal mitotic function, effectively blocking cell cycle progression, leading to apoptosis (Perez E., 2009).

[0208] Meiotic inhibitors can be, for example, cyclin-dependent kinase 2 (CDK2) inhibitors, e.g. flavopiridol, CY-202 (Malumbres M. et al., 2008). DNA damaging agents are active at different cell cycle stages. Duocarmycins are cytotoxic DNA alkylating compounds that bind to the minor groove of DNA. Anthracyclines and analogs thereof (e.g. carminomycin, daunorubicin and doxorubicin) are compounds that can intercalate into DNA and can be used as drug conjugates. Calicheamicin is a potent antitumor antibiotic that binds to the minor groove of DNA, causing double-stranded breaks and rapid cell death with low dependence on cell cycle progression, and can therefore have potential efficacy on tumor initiating cells (TICs) with low proliferation rates (Gupta et al., 2009; Sapra et al., 2011). Alternatively, ozogamycin can also be used. Another class of novel DNA damaging agents are pyrrolobenzodiazepines (PBDs) that bind to discrete DNA sequences, causing lethal damage.

[0209] One potential new drug under investigation is a-amanitin, a picomolar inhibitor of RNA polymerase II derived from the mushroom toxin A. phalloides (Moldenhauer et al., 2012). Amanita phalloides

[0210] ​Examples of other agents used include, but are not limited to, 5-fluorouracil, aplidin, azaribine, anastrozole, anthracyclines, bendamustine, bleomycin, bortezomib, brequinar-1, busulfan, calicheamicin, camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, celebrex® (celecoxib), chloroambucil, cisplatin (CDDP), Cox-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin, cladribine, camptothecins, cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin, daunorubicin, doxorubicin, 2-pyrrolinodoxorubicin (2P-DOX), cyanomorpholino doxorubicin, doxorubicin glucuronide, epirubicin glucuronide, estramustine, etoposide phosphate, estrogen receptor binding agents, etoposide (VP- 16), etoposide glucuronide, etoposide phosphate, floxuridine (FUdR), 3',5'-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide, farnesyl protein transferase inhibitors, gemcitabine, hydroxyurea, idarubicin, ifosfamide, L-asparaginase, lenalidomide, leucovorin calcium, lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mytomycin, mitotane, navelbine, nitrosoureas, plicamycin, procarbazine, paclitaxel, pentostatin, PSI-341, reloxafine, semustine, streptozocin, tamoxifen, taxol®, temozolomide (water soluble form of DTIC), transplatinum, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracil mustard, vinorelbine, vinblastine, vincristine, and vinca alkaloids.

[0211] Toxins used include ricin, abrin, alpha toxin, saporin, ribonuclease (RNase) (e.g., onconase), DNase I, Staphylococcus aureus enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas aeruginosa exotoxin and Pseudomonas aeruginosa endotoxin.

[0212] Radioisotopes (radio-nuclides, radio-nuclei) useful for treating diseased tissue include, but are not limited to 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, 89Sr, 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 Th.

[0213] Particularly useful therapeutic radionuclides include, but are not limited to 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 Ac.

[0214] Particularly useful diagnostic / detection radionuclides include, but are not limited to, those used for testing. 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, 154-158 Gd, 32 P, 90 Y、 188 Re and 175 Lu.

[0215] Therapeutic radionuclides preferably have a decay energy of 20 to 6000 keV, preferably 60 to 200 keV for argon electron emitters, preferably 100 to 2500 keV for beta particle emitters, and preferably 4000 to 6000 keV for alpha particle emitters. The maximum decay energy of useful beta particle emitting radionuclides is preferably 20 to 5000 keV, more preferably 100 to 4000 keV, and most preferably 500 to 2500 keV. Also preferably are radionuclides that release argon electrons during their primary decay, for example, 58 Co、 67 Ga、 80 mBr、 99 mTc, 103 mRh、 109 Pt, 111 In、 119 Sb、 125 I, 161 Ho、 189 mOs and192 The decay energy of useful beta particle emitting nuclides is preferably less than 1000 keV, more preferably less than 100 keV, most preferably less than 70 keV. Also preferred are radio nuclides that primarily decay by producing alpha particles, such radio nuclides include, but are not limited to: 152 Dy, 211 At, 212 Bi, 223 Ra, 219 Rn, 215 Po, 211 Bi, 225 Ac, 221 Fr, 217 At, 213 Bi and 255 Fm. The decay energy of useful alpha particle emitting radio nuclides is preferably 2000 to 10000 keV, more preferably 3000 to 8000 keV, most preferably 4000 to 7000 keV.

[0216] In particular, the radio nuclide is selected from the group consisting of 67 Ga, 89 Zr, 111 In, 124 I, 131 I, 177 Lu and 225 Ac.

[0217] The therapeutic agent can also include a photosensitizer or dye. Fluorescent compositions (e.g., fluorescein) and other chromophores or dyes (e.g., porphyrins that are sensitive to visible light) have been used to detect and treat lesions by directing appropriate light to the site of the lesion. In therapy, this is referred to as photoradiation therapy, phototherapy, or photodynamic therapy.

[0218] In certain embodiments, an anti-angiogenic agent can be useful, such as angiostatin, baculovirus inhibitor, cabozantinib, maspin, tissue metalloproteinase inhibitor, 2-methoxyestradiol, carboxyamidotriazole, CM101, marimastat, pentosan polysulfate, herbimycin A, PNU145156E, linomide, thalidomide, pentoxifylline, genistein, TNP-470, endostatin, paclitaxel, acitretin, angiostatin, cidofovir, vincristine, bleomycin, AGM-1470, or minocycline.

[0219] The therapeutic agent can comprise an oligonucleotide, such as a small interfering RNA (siRNA). Those skilled in the art will recognize that any siRNA or interfering RNA species can be attached to a targeting construct for delivery to a targeted tissue. Numerous siRNA species are known in the art for a variety of targets, and any such known siRNA can be used in the claimed methods and compositions.

[0220] Known siRNA species of potential use include siRNAs specific for IKK-gamma, VEGF, Flt-1, Flk-1 / KDR, Bcl2 and EGFR, CDC20, Discoidin (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.

[0221] The diagnostic agent is preferably selected from the group consisting of a radionuclide, a radiocontrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasound contrast agent, and a photosensitizer. Such diagnostic agents are well known, and any such known diagnostic agent can be used. Non-limiting examples of diagnostic agents include radionuclides such as 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, 72As、 75 Br、 76 Br、 82m Rb、 83 Sr or other gamma, beta or positron emitters.

[0222] The three most commonly used positron emission tomography (PET) radionuclides are 18 F、 68 Ga and 89 Zr.

[0223] Synthetic peptides can also be useful herein. For example, IMP-288 and IMP-449 are the Tyr-D-Lys-D-Glu-D-Lys (SEQ ID NO: 86) tetrapeptide, in which both lysine residues are substituted with their epsilon-amino groups with HSG groups. IMP-449 is conjugated to NOTA (1,4,7-triazacyclononane-N,N',N"-triacetic acid).

[0224] IMP-288 is a D-Tyr-D-Lys-D-Glu-D-Lys-NH2 (SEQ ID NO: 87) tetrapeptide conjugated to DOTA, in which both lysine residues are derivatized with their epsilon-amino groups with HSG groups.

[0225] IMP-453, IMP402, IMP457 or IMP498 described in US9352036B2 can also be used.

[0226] Useful paramagnetic ions 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). Metal contrast agents can include lanthanum (III), gold (III), lead (II), or bismuth (III).

[0227] Ultrasound contrast agents can include liposomes, such as gas-filled liposomes. Radiopaque diagnostic agents can be selected from the group consisting of compounds, barium compounds, gallium compounds, and thallium compounds. A variety of fluorescent markers are known in the art, including but not limited to fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phenylenediamine, and fluorescamine. Useful chemiluminescent markers can include luminol, isoluminol, acridinium ester, imidazole, acridium salt, europium (III), or oxalate.

[0228] The HSG group can have a variety of structures, selected not only on the basis of being able to obtain an antibody or fragment that binds with high affinity to the HSG group, but also on the basis of being able to be rapidly cleared in vivo when used in a pretargeting method. Hydrophobic reagents are best at eliciting a strong immune response, while hydrophilic reagents are more amenable to rapid clearance in vivo. Thus, a balance between hydrophobicity and hydrophilicity needs to be struck. This can be achieved in part by using hydrophilic chelators to offset the inherent hydrophobicity of many organic groups. In addition, targeting construct subunits can be selected that have opposite solution properties, e.g., peptides comprising amino acids, some of which are hydrophobic and others of which are hydrophilic. In addition to peptides, carbohydrates can also be used.

[0229] Pretargeting is a multi-step procedure that was originally developed to address the problem of slow blood clearance of directly targeted antibodies, which leads to undesirable toxicity to normal tissues. With pretargeting technology, a diagnostic or therapeutic agent (e.g., a radionuclide) is attached to a small delivery molecule that can be cleared from the blood in minutes. According to one embodiment of the application, the small delivery molecule is an HSG group as described herein. The pretargeting bispecific antibody of the application has one or more HSG group binding sites and one target tumor antigen binding site, and is administered first, with the free antibody being cleared from the circulation before the targetable construct is administered.

[0230] A pretargeting method of treating or diagnosing a disease or condition in a subject can be provided by: (1) administering a bispecific antibody to the subject; (2) optionally administering a clearing composition to the subject, and allowing the composition to clear the antibody from the circulation; and (3) administering to the subject an HSG group as described herein, which contains one or more chelated or chemically bound therapeutic or diagnostic agents.

[0231] The bispecific antibodies described herein can be used in a pretargeting method of malignant tumor treatment or detection, in which the antibody can be administered first to a subject. Sufficient time can be allowed for the bispecific antibody to bind to the target antigen, and for unbound antibody to be cleared from the circulation.

[0232] Optionally, a clearing composition can be administered after administration of the antibody, which clears the antibody from circulation. The clearing composition can be, but is not limited to, avidin, galactose, an antibody against the pre-targeting antibody, and Fc-antigen fusions. The ABDEG (antibody-enhanced degradation) clearing method involves administering an antigen-specific IgG antibody first, and after the antigen-specific monoclonal antibody localizes in the tumor, administering an irrelevant IgG-YTE (i.e., an antibody with enhanced binding to FcRn through M252Y / S254T / T256E mutations in the Fc region), which enhances the degradation / clearance from circulation of the free antigen-specific antibody by binding more strongly to FcRn (Nazarova L. et al., 2020).

[0233] Then, the subject can be administered a diagnostic agent (e.g., a radionuclide) labeled or linked to the targetable construct (e.g., the HSG group described above) bound to the bispecific antibody and localized at the diseased cell or tissue.

[0234] In solid tumors, the slow blood clearance and delayed tumor uptake of directly radiolabeled antibodies can result in prolonged exposure of healthy tissues and organs to high doses of radiation. In vivo pre-targeted radioimmunotherapy (PRAIT) using the antibodies of the present application can overcome these limitations. PRAIT aims to improve the therapeutic index (tumor to normal tissue ratio) by delivering a higher absorbed dose to the tumor compared to directly radiolabeled antibodies or antibody fragments. PRAIT involves administration of the bispecific antibody described herein that recognizes HSG and a tumor target followed by administration of a radiolabeled HSG hapten group. Administration of the radiolabeled HSG hapten group can be after several days, e.g., 2, 3, 4, 5, 6, or 7 days. With this technique, large amounts of the antibodies of the present application can accumulate inside or on the surface of the tumor or on the surface of malignant cells and be largely cleared from circulation, and the radiolabeled HSG hapten group binds to the antibodies accumulated inside or on the surface of the tumor or on the surface of malignant cells, while unbound radioactive HSG haptens are cleared from circulation by the kidneys within a few hours. This results in minimal radiation exposure of normal organs.

[0235] For targeted radionuclide therapy, for example, the HSG hapten IMP-288 (described in, e.g., US 2005 / 0025709) and 177 Lu as the radionuclide.

[0236] The pre-targeting approach described above can also be used for therapeutic purposes. In this case, the HSG group comprises one or more of the therapeutic agents described above. Specifically, the HSG hapten IMP-453, which is directly coupled to a therapeutic agent, can be used.

[0237] A "subject" refers to a mammal. Mammals include, but are not limited to, domesticated mammals such as 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.

[0238] According to the present application, the antibodies described herein can be used in the manufacture of a medicament.

[0239] Further included herein are pharmaceutical compositions comprising the antibodies of the present application.

[0240] A "pharmaceutical composition" refers to a preparation with a drug excipient (e.g., carrier) formulated so as to render the active ingredient contained therein efficacious for the intended use. The composition can be formulated in any manner suitable for the intended use.

[0241] A "pharmaceutically acceptable carrier" refers to a component of a pharmaceutical formulation other than the active ingredient that is nontoxic to the subject. Some examples of pharmaceutically acceptable carriers include water, physiological saline, phosphate buffered saline, amino acids (e.g., glycine or histidine), glucose, glycerol, ethanol, and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Other examples of pharmaceutically acceptable substances include wetting or minor auxiliary substances such as wetting or emulsors, preservatives, or buffers, which enhance the shelf life or effectiveness of the antibody.

[0242] The terms "treatment," "treat," or "treating," as used herein refer to clinical intervention with the intent to alter the natural course of a disease of an individual receiving treatment, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or reoccurrence of the disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In certain embodiments, the antibodies of the present application are used to delay development of a disease or to slow the progression of a disease.

[0243] The bispecific anti-tumor antigen / anti-HSG antibody of the present application and the pharmaceutical composition comprising the same can be administered to a subject in two steps, the first step administering the bispecific anti-tumor antigen / anti-HSG antibody and the second step administering the HSG moiety, wherein the HSG moiety binds to the antibody.

[0244] The pharmaceutical compositions of the present application can have various forms, such as liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, and powders. The preferred form depends on the intended mode of administration and the therapeutic or diagnostic application. A typical preferred composition is an injectable or infusible solution, such as a composition similar to that used for passive immunization of humans. The preferred mode of administration is parenteral (e.g., intravenous, intra-arterial, intra-lymphatic, or intrathecal injection). In a preferred embodiment, the antibody is administered by intravenous infusion or injection. In another preferred embodiment, the antibody is administered by intravenous injection. The route and / or mode of administration will vary depending upon the desired results, as will be apparent to those skilled in the art.

[0245] The administration of the bispecific anti-tumor antigen / anti-HSG antibody of the present application with a labeled or conjugated HSG moiety can be achieved by administering the bispecific antibody (bsAb) first at some time prior to administration of the labeled or conjugated HSG moiety. The dosage and timing of administration of the agents can be readily designed by one skilled in the art and depends on the specific nature of the agents used. In a particular embodiment, the bispecific anti-tumor antigen / anti-HSG antibody is administered first, and then the labeled or conjugated HSG moiety is administered after a sufficient time, specifically 24-72 hours, or in another embodiment 48-96 hours, after the bispecific antibody of the present application has targeted the diseased tissue. When the bispecific anti-tumor antigen / anti-HSG antibody of the present application is administered, particularly in the pre-targeting technique, the dosage of the antibody administered to a human will vary depending on such factors as the patient's age, weight, height, sex, general medical condition, and prior medical history. Generally, it is desirable to administer to the subject a dosage of the bispecific antibody in the range of about 1 mg to 400 mg as a single intravenous infusion, although lower or higher dosages can be administered depending on the circumstances. Generally, it is desirable to administer to the subject a dosage of 1 to 200 mg, more preferably 1 to 70 mg, most preferably 1 to 20 mg, although higher or lower dosages can be administered. The dosage of the therapeutic bispecific antibody can be higher, e.g., 1 to 200 mg, 1 to 100 mg, 100 to 1000 mg, 100 to 500 mg, 200 to 750 mg. The dosage for each subject depends on the circumstances, e.g., body weight or type of disease. However, the dosage can be determined by one skilled in the art with reference to general knowledge.

[0246] Generally, the dosage of the labeled or conjugated HSG moiety administered will vary depending on such factors as the patient's age, weight, height, sex, general medical condition, and prior medical history. Preferably, a saturating dose of the labeled or conjugated HSG moiety is administered to the patient. For administration of radiolabeled molecules, the dosage can be measured in millicuries.

[0247] The anti-tumor antigen / anti-HSG antibodies described herein can be administered once, or, in another embodiment, multiple times, each time followed by administration of a labeled or conjugated HSG group for the diagnosis and / or treatment of a malignant tumor.

[0248] In a preferred embodiment, the anti-tumor antigen / anti-HSG antibodies of the application are useful for cancer therapy.

[0249] As used herein, "cancer" refers to a malignant tumor, in particular a proliferative disease, in particular a solid cancer, such as colorectal cancer, ovarian cancer, pancreatic cancer, lung cancer, melanoma, squamous cell carcinoma (SCC) (e.g. 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 myelogenous 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 germ cell tumors, including refractory forms of any of the above, or a combination of one or more of the above cancers.

[0250] The anti-tumor antigen / anti-HSG antibodies of the application can treat or detect a hyperproliferative disorder, such as a cancerous disease or cancer, which can involve any tissue or organ, including but not limited to brain, squamous cell, bladder, head, neck, liver, ovarian, esophageal, nasopharyngeal or thyroid cancer, melanoma, lymphoma, leukemia or multiple myeloma, oral cavity, gastrointestinal tract, colon, colorectal, stomach, pancreas, lung airways, lung, breast, ovary, prostate, uterus, endometrium, cervix, bladder, pancreas, bone, liver, gall bladder, kidney, skin and testis.

[0251] In particular, the anti-tumor antigen / anti-HSG antibodies of the application can be used for the treatment of hematological and solid tumors, such as colorectal cancer, ovarian cancer, breast cancer, prostate cancer, pancreatic cancer and lung cancer.

[0252] In a particular embodiment, the antibodies that are well suited for the treatment of cancerous diseases, in particular for the treatment of solid tumors, are bispecific anti-tumor antigen / anti-HSG antibodies comprising a binding site that specifically recognizes HSG comprising: - a light chain variable (VL) domain comprising SEQ ID NO: 17 or SEQ ID NO: 19, or a sequence having at least 95% identity with SEQ ID NO: 17 or SEQ ID NO: 19, in particular a glutamine (Q100) at position 100 and an isoleucine (1106) at position 106 according to the Kabat numbering; and - a heavy chain variable (VH) domain comprising a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10, or a sequence having at least 95% identity to SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10, in particular an arginine (R19) at position 19 according to Kabat numbering; and - optionally, the VL and VH domains are connected by a linker sequence; and a binding site specifically recognizing a tumor antigen, the binding site comprising: (a1) a light chain variable domain comprising SEQ ID NO: 45 with one or more of the 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, having a conserved tyrosine at position 36, and having one or more of the 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 and having 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 and having the amino acid substitutions L5Q and / or W97Y, wherein the amino acid positions are numbered according to Kabat numbering.

[0253] In another particular embodiment, the antibody very suitable for the treatment of a cancerous disease, in particular for the treatment of a solid tumor, is a bispecific anti-tumor antigen / anti-HSG antibody comprising a binding site specifically recognizing HSG, the binding site comprising: - a light chain variable (VL) domain comprising SEQ ID NO: 17 or SEQ ID NO: 19, or a sequence having at least 95% identity to SEQ ID NO: 17 or SEQ ID NO: 19, in particular a glutamine (Q100) at position 100 and an isoleucine (1106) at position 106 according to Kabat numbering; and - a heavy chain variable (VH) domain comprising a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10, or a sequence having at least 95% identity to SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10, in particular, an arginine (R19) at position 19; and - optionally, the VL and VH domains are connected by a linker sequence; and a binding site specifically recognizing a tumor antigen, the binding site comprising: - 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, wherein the amino acid positions are numbered according to Kabat.

[0254] In another particular embodiment, the antibody is a bispecific anti-tumor antigen / anti-HSG antibody comprising a binding site specifically recognizing HSG, the binding site comprising: - a light chain variable (VL) domain comprising any one of SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 89, SEQ ID NO: 90 and SEQ ID NO: 91; and - a heavy chain variable (VH) domain comprising a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10.

[0255] In another alternative embodiment, the antibody is a bispecific anti-tumor antigen / anti-HSG antibody comprising a binding site specifically recognizing HSG, the binding site further comprising: - a light chain variable (VL) domain comprising a sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 89, SEQ ID NO: 90 and SEQ ID NO: 91; and - a heavy chain variable (VH) domain comprising a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10, having one or two additional amino acid substitutions, and being an arginine (R19) at position 19 according to Kabat numbering.

[0256] In another alternative embodiment, the antibody is a bispecific anti-tumor antigen / anti-HSG antibody comprising a binding site that specifically recognizes HSG, which further comprises: - a light chain variable (VL) domain comprising a sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 89, SEQ ID NO: 90 and SEQ ID NO: 91, having one or two additional amino acid substitutions, and being a glutamic acid (E105) at position 105 and an isoleucine (1106) at position 106 according to Kabat numbering; and - a heavy chain variable (VH) domain comprising a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10.

[0257] In another alternative embodiment, the antibody is a bispecific anti-tumor antigen / anti-HSG antibody comprising a binding site that specifically recognizes HSG, which further comprises: - a light chain variable (VL) domain comprising a sequence selected from the group consisting of SEQ ID NO: SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 89, SEQ ID NO: 90 and SEQ ID NO: 91, and further having 1 or 2 amino acid substitutions, with the proviso that the glutamic acid (E105) at position 105 and the isoleucine (1106) at position 106 are preserved; and - a heavy chain variable (VH) domain comprising a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10, having one or two additional amino acid substitutions, and being an arginine (R19) at position 19 according to Kabat numbering.

[0258] Optionally, the sequence further comprises a glutamine (Q100) at position 100 according to Kabat numbering.

[0259] Also provided herein are nucleic acids encoding the antibodies of the application.

[0260] An "isolated" nucleic acid is a nucleic acid molecule that has been removed from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that typically comprise the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally, or is located in a different chromosomal location from where it naturally occurs.

[0261] An "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 such nucleic acid molecules present in one or more locations in a host cell.

[0262] "Substantially no cross-reactivity" means that a molecule (e.g., an antibody) does not recognize or specifically bind to an antigen other than the actual target antigen of the molecule (e.g., an antigen closely related to the target antigen), specifically, reduced form MIF, as compared to the target antigen. For example, the binding rate of an antibody to an antigen other than the actual target antigen can be less than about 10% to less than about 5%, or the amount of binding to an antigen other than the actual target antigen is no more 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%, 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.

[0263] Recombinant production of the antibodies of the present application preferably employs expression systems, e.g., including expression constructs or vectors that contain nucleotide sequences encoding the antibody form.

[0264] An "expression system" refers to a nucleic acid molecule that comprises a desired coding sequence and operably linked control sequences, such that a host transformed or transfected with these sequences is capable of producing the encoded protein. To effect the transformation, the expression system can be included in a vector; however, the relevant DNA can be integrated into the host chromosome at a later date. Alternatively, the expression system can be used in an in vitro transcription / translation system.

[0265] An "expression vector" as used herein is defined as a cloned recombinant nucleotide sequence (i.e., a recombinant gene) and the DNA sequences required for its transcription and translation of its mRNA in a suitable host organism. An expression vector comprises an expression cassette and typically also comprises an origin of autonomous replication or a genomic integration site in a host cell, one or more selectable markers (e.g., an amino acid synthesis gene or a gene conferring resistance to an antibiotic, such as a bleomycin, kanamycin, G418, or hygromycin resistance gene), a plurality of restriction enzyme cleavage sites, a suitable promoter sequence, and a transcription terminator, which components are operably linked together. "Plasmid" and "vector" as used herein include both autonomously replicating nucleotide sequences and genome-integrated nucleotide sequences.

[0266] In particular, 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 application) can be introduced into a host cell, thereby transforming the host and promoting expression (e.g., transcription and translation) of the introduced sequence. Plasmids are preferred vectors of the application.

[0267] Vectors typically comprise DNA into which a transmissible agent is inserted. A common method for inserting a piece of DNA into another piece of DNA involves the use of restriction enzymes, which cut DNA at specific sites (specific sets of nucleotides) called restriction sites.

[0268] A "cassette" refers to a DNA coding sequence or a DNA fragment that encodes an expression product, which can be inserted into a vector at specific restriction sites. The restriction sites of a cassette are designed to ensure that the cassette is inserted in the correct reading frame. Typically, foreign DNA is inserted into one or more restriction sites of a vector DNA, which is then carried by the vector into a host cell along with the transmissible vector DNA. A DNA fragment or sequence having inserted or added DNA (e.g., an expression vector) can also be referred to as a "DNA construct." One common type of vector is a "plasmid," which is typically a self-contained double-stranded DNA molecule that readily accepts additional (foreign) DNA and can be readily introduced into a suitable host cell. Vectors of the application typically comprise coding DNA and expression control sequences (e.g., promoter DNA) and have one or more restriction sites suitable for insertion of foreign DNA. Coding DNA is a DNA sequence that encodes a specific amino acid sequence of a particular polypeptide or protein (e.g., an antibody format of the application). Promoter DNA is a DNA sequence that initiates, regulates, or otherwise mediates or controls expression of coding DNA. Promoter DNA and coding DNA can be from the same or different genes and can be from the same or different organisms. Recombinant cloning vectors of the application typically include one or more replication systems for cloning or expression, one or more selection markers (e.g., antibiotic resistance) for use in hosts, and one or more expression cassettes.

[0269] Methods for joining DNA sequences (e.g., providing or encoding factors and / or target proteins of the application, promoters, terminators, and other sequences) and inserting them into suitable vectors containing the information required for integration or host replication are well known to those of skill in the art, such as described by Sambrook et al. (2012).

[0270] Also included herein is the use of host cells to produce antibodies of the application.

[0271] Host cells specifically refer to cells, recombinant cells, or cell lines that are transfected with an expression construct (e.g., a vector of the application).

[0272] As used herein, "host cell line" refers to an established clone of a particular cell type that has acquired long-term proliferative ability. Host cell line refers to a cell line used to express endogenous or recombinant genes to produce polypeptides, such as the recombinant antibody formats of the present application.

[0273] "Production host cell" or "production cell" is generally understood to be a cell line or cell culture that can be directly used for cultivation in a bioreactor to obtain a production process product, i.e. the recombinant antibody formats of the present application. The host cell type according to the present application can be any prokaryotic or eukaryotic cell.

[0274] The term "recombinant" as used herein refers to "produced by genetic engineering" or "result of genetic engineering", for example, specifically to the use of a heterologous sequence incorporated into a recombinant vector or a recombinant host cell.

[0275] The antibodies of the present application can be produced using any known and well-established expression systems and recombinant cell culture techniques, for example, by expression in a bacterial host (prokaryotic system) or in a eukaryotic system (e.g. yeast, fungi, insect cells or mammalian cells). The antibody molecules of the present application can be produced in transgenic organisms, such as goats, plants or transgenic mice (a genetically engineered strain of mice that carries large fragments of human immunoglobulin loci and is deficient in mouse antibody production). Antibodies can also be produced by chemical synthesis.

[0276] According to one particular embodiment, the host cell is a production cell line of a cell selected from the group consisting of CHO, PerC6, CAP, HEK, HeLa, NSO, SP2 / 0 hybridoma cells and Jurkat cells. More particularly, the host cell is taken from a CHO cell.

[0277] The host cells of the present application are specifically cultivated or maintained in serum-free media, for example, the media comprises plasma proteins, hormones and other components such as growth factors as a replacement for serum.

[0278] It is most desirable when the host cells are in serum-free conditions throughout the establishment, adaptation and full cultivation process, and optionally cultivated in a medium that does not contain any animal-derived proteins / peptides.

[0279] The anti-tumor antigen / anti-HSG antibodies of the present application can be recovered from the culture medium using standard protein purification methods.

[0280] The present application also includes 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, comprising: - a light chain variable (VL) domain comprising SEQ ID NO: 17 or SEQ ID NO: 19, or a sequence having at least 95% identity to SEQ ID NO: 17 or SEQ ID NO: 19, according to the Kabat numbering, position 100 is glutamine (Q100) and position 106 is isoleucine (1106); and - a heavy chain variable (VH) domain comprising a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10, or a sequence having at least 95% identity to SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10, according to the Kabat numbering, position 19 is arginine (R19).

[0281] 2. The antibody of embodiment 1 comprising a single chain variable fragment (scFv) specifically recognizing HSG having a structural 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.

[0282] 3. The antibody of embodiment 1 comprising a VL and a VH domain specifically recognizing HSG, wherein the C-terminus of the VL domain is cross-linked to a CH1 comprising SEQ ID NO: 30 and the C-terminus of the VH domain is cross-linked to a CL comprising SEQ ID NO: 38.

[0283] 4. The antibody of any one of embodiments 1 to 3 comprising a Fc region having SEQ ID NO: 31, the Fc region comprising asymmetric mutations in each CH3 domain enabling heterodimerization of two CH domains from different antibodies, in particular, the one CH domain comprises the "knobs" mutations T366W and S354C and the other CH domain comprises the "cleft" mutations T366S, L368A, Y407V and Y349C, according to the EU numbering index.

[0284] 5. The antibody of any one of embodiments 1 to 4 comprising a variant Fc region having reduced or ablated effector function and / or FcRn binding.

[0285] 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 according to EU numbering index, and optionally an aglycosylated Fc region.

[0286] 7. The antibody of any one of embodiments 1 to 3, comprising two constant heavy chain domains with a hinge region and an Fc domain comprising SEQ ID NO: 35 and SEQ ID NO: 32.

[0287] 8. The antibody of any one of embodiments 1 to 3, comprising two constant heavy chain regions with a hinge region and an Fc domain comprising SEQ ID NO: 36 and SEQ ID NO: 33.

[0288] 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 (FRa).

[0289] 10. The antibody of any one of embodiments 1 to 9, wherein the anti-tumor antigen is oxMIF.

[0290] 11. The antibody of embodiment 10, wherein the binding site specifically recognizing oxMIF comprises: - 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, wherein the amino acid positions are according to Kabat numbering.

[0291] 12. The antibody of any one of embodiments 1 to 11, 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.

[0292] 13. The antibody of any one of embodiments 1 to 12 for use in the treatment or detection of a malignant tumor, wherein the antibody is administered to a subject in a first step and the HSG group is administered in a second step, wherein the HSG group is bound to the antibody.

[0293] 14. The antibody for use of embodiment 13, wherein the HSG group is coupled or labeled with one or more diagnostic agents and / or therapeutic agents, in particular the HSG group comprises one or more HSG haptens, one or more diagnostic agents and / or therapeutic agents and optionally one chelator.

[0294] 15. The antibody of any one of embodiments 1 to 12, wherein the antibody is bound to a HSG group, which is coupled or labeled with one or more diagnostic agents and / or therapeutic agents, in particular the HSG group comprises one or more HSG haptens, one or more diagnostic agents and / or therapeutic agents and optionally one chelator.

[0295] 16. The antibody for use of embodiment 13 or 14, or the antibody of embodiment 15, wherein the therapeutic agent is selected from the group consisting of radionuclides, chemotherapeutic agents and cytokines; and the diagnostic agent is a radionuclide.

[0296] 17. The antibody of embodiment 16, wherein the chelator binds a radionuclide, in particular the chelator is selected from the group consisting of DOTA, DTPA, deferoxamine B (DFO) and DFO*.

[0297] 18. The antibody of embodiment 16 or 17, wherein the radionuclide is selected from the group consisting of 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, 124I、 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 Th.

[0298] 19. Use of the antibody of any one of embodiments 1 to 12 in the manufacture of a medicament.

[0299] 20. A pharmaceutical composition comprising the antibody of any one of embodiments 1 to 12 and a pharmaceutically acceptable excipient.

[0300] 21. The pharmaceutical composition of embodiment 20, wherein the pharmaceutical composition is formulated for intravenous administration.

[0301] 22. The pharmaceutical composition of embodiment 20 or 21 for use in the treatment of a patient suffering from cancer, in particular for use in the treatment of a tumor, a solid tumor, more particularly for use in the treatment of colorectal cancer, ovarian cancer, breast cancer, prostate cancer, pancreatic cancer and lung cancer.

[0302] 23. An isolated nucleic acid encoding the antibody of any one of embodiments 1 to 12.

[0303] 24. An expression vector comprising the nucleic acid of embodiment 23.

[0304] 25. A method of diagnosing cancer in a subject, wherein the antibody of any one of embodiments 1 to 12 or the antibody of any one of embodiments 15 to 18 is used to detect tumor cells.

[0305] 26. A method of diagnosing cancer in vitro, wherein the antibody of any one of embodiments 1 to 12 or the antibody of any one of embodiments 15 to 18 is used to detect tumor cells in a sample.

[0306] 27. A method of treating cancer using the antibody of any one of embodiments 1 to 12 or the pharmaceutical composition of embodiments 20 or 21.

[0307] Examples The examples described herein are intended to be illustrative of the present application. Numerous modifications and variations of the techniques described herein and illustrated in the figures are possible without departing from the scope of the present application. It is therefore to be understood that the examples are merely illustrative and do not limit the scope of the present application.

[0308] Example 1: Schematic representation of exemplary anti-tumor antigen x anti-HSG bispecific monoclonal antibodies, and amino acid sequences of anti-oxMIF x anti-HSG bispecific monoclonal antibodies.

[0309] Figure 1 Schematic representation of anti-target X x anti-HSG bispecific monoclonal antibodies are provided, which have Fab-scFv-Fc, CrossMab (CH1-CLcrossover), and IgG-central scFv (IgG-central scFv) formats. Left: Fab-scFv-Fc; Middle: CrossMab (CH1-CLcrossover); Right: IgG-central scFv (IgG-central scFv). L crossover) and IgG-central scFv (IgG-central scFv) formats. Left: Fab-scFv-Fc; Middle: CrossMab (CH1-CLcrossover); Right: IgG-central scFv (IgG-central scFv).

[0310] Table I. Anti-HSG heavy chain variable region (VH) sequences and their closest human germline sequences.

[0311]

[0312] *US2009 / 0246131A1; **US2009 / 0240037A; with underlined CDRs are indicated and defined using the IMGT / Kabat combined antibody numbering system.

[0313] Table II. Anti-HSG light chain variable region (VL) sequences and their closest human germline sequences.

[0314]

[0315]

[0316] *US2009 / 0246131A1; **US2009 / 0240037A; with underlined CDRs are marked and defined using the IMGT / Kabat combined antibody numbering system.

[0317] Table III. Anti-oxMIF monoclonal antibody sequences, constant regions, and linkers.

[0318]

[0319]

[0320]

[0321] *WO / 2022 / 069712A1; FcyR silencing: mutations in the Fc region of the monoclonal antibody eliminate binding to FcyR and complement (bold italicized ); FcRn silencing: mutations in the Fc region of the monoclonal antibody reduce or eliminate binding to FcRn ( bold underlined ); KIH (knobs-into-holes): knobs and holes mutations (bold).

[0322]

[0323]

[0324]

[0325] C0181 Polypeptide 1 ((LC oxMIF) SEQ ID NO: 47 DIQMTQSPSSLSASVGDRVTITCRSSQRIMTYLNWYQQKPGKAPKLLIYVGSHSQSGVPSRFRGSGSETDFTLTISGLQPEDSATYYCQQSFFTPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Polypeptide 2 (HC oxMIF) SEQ ID NO: 48 EVQLQESGGGLVQPGGSLRLSCAASGFTFSIYSMNWVRQAPGKGLEWVSSIGSSGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGSQYLYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Polypeptide 3 ((HSG HC) SEQ ID NO: 49 EVQLVESGGDLVQPGGSLRLSCAASGFTFSIYTMSWVRQAPGKGLEWVATLSGDGDDIYYPDSVKGRFTISRDNAKNNLYLQMNSLRSADTALYYCARVRLGDWDFDVWGQGTTVTVSSGGSGGSGGSGGSGGSDVVMTQSPSSLAVSLGERVTINCKSSQSLFNSRTRKNYLGWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQSEDVAVYYCTQVYYLCTFGQGTKLEIKGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 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 EVQLVESGGDLVQPGGSLRLSCAASGFTFSIYTMSWVRQAPGKGLEWVATLSGDGDDIYYPDSVKGRFTISRDNAKNNLYLQMNSLRSADTALYYCARVRLGDWDFDVWGQGTTVTVSSGGSGGSGGSGGSGGSDIVMTQSPSSLAVSLGERATITCKSSQSLFNSRTRKNYLGWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQSEDLAVYYCTQVYYLCTFGQGTKLEIKGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 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 QVQLVESGGDLVKPGGSLRLSCAASGFTFSIYTMSWLRQTPEKRLEWVSTLSGDGDDIYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCARVRLGDWDFDVWGQGTLVTVSSGGSGGSGGSGGSGGSDVVMTQSPSSLAVSLGERVTINCKSSQSLFNSRTRKNYLGWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQSEDVAVYYCTQVYYLCTFGQGTKLEIKGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 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 EVQLVESGGGLVKPGGSLRLSCAASGFTFSIYTMSWLRQTPEKRLEWVSTLSGDGDDIYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVRLGDWDFDVWGQGTTVSVSSGGSGGSGGSGGSGGSDIVMTQSPSSLAVSLGERATITCKSSQSLFNSRTRKNYLGWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQSEDLAVYYCTQVYYLCTFGQGTKLEIKGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG oxMIF epitopes EPCALCS SEQ ID NO: 53.

[0326] C0239 Polypeptide 1 (LC oxMIF) SEQ ID NO: 47 Polypeptide 2 (HC oxMIF) SEQ ID NO: 54 EVQLQESGGGLVQPGGSLRLSCAASGFTFSIYSMNWVRQAPGKGLEWVSSIGSSGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGSQYLYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLAQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG Polypeptide 3 (HC HSG) SEQ ID NO: 55 EVQLVESGGDLVQPGGSLRLSCAASGFTFSIYTMSWVRQAPGKGLEWVATLSGDGDDIYYPDSVKGRFTISRDNAKNNLYLQMNSLRSADTALYYCARVRLGDWDFDVWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLAQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG Polypeptide 4 (LC HSG) SEQ ID NO: 56 DIVMTQSPSSLAVSLGERVTINCKSSQSLFNSRTRKNYLGWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQSEDVAVYYCTQVYYLCTFGQGTKLEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC 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 DIVMTQSPSSLAVSLGERATITCKSSQSLFNSRTRKNYLGWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQSEDLAVYYCTQVYYLCTFGQGTKLEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC Figure 1 Exemplary Fab-scFv-Fc, CrossMab, and IgG-central scFv (IgG-central scFv) formats of bispecific anti-tumor target X x anti-HSG monoclonal antibodies are shown. Table I and Table II show the anti-HSG heavy chain variable region (Table I) and anti-HSG light chain variable region (Table II) sequences and their respective closest human germline sequences, respectively. Table III shows the anti-oxMIF monoclonal antibody sequences, monoclonal antibody 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.

[0327] Example 2: Comparison of in silico immunogenicity (humanization score) of new humanized anti-HSG sequence VH and VL variants 1-5 to hz679.

[0328] To generate new humanized VH and VL sequences, the CDRs of the murine anti-HSG antibody mo679 (from US 20090246131 Al) were determined using the IMGT and Kabat joint numbering system, which allows for optimal preservation of CDR loop conformation. The IgBLAST search algorithm (NCBI) was used to search the human IgGl / Igk sequence database for sequences that compared to the murine VH and VL regions of mo679, and candidate human VH and VL domains were selected from the top 200 BLAST results. These candidate sequences were reduced to a few based on a combination of framework homology, maintenance of key framework residues, and canonical loop structures. The murine CDRs of mo679 were grafted into defined human frameworks as "recipient" frameworks, resulting in 5 new humanized VH (VH1-VH5, see Table I) and 8 new humanized VL variants (VL1, VL2, VL3, VL4, VL5, VL3.1, VL4.1, VL5.1, see Table II).

[0329] Determination of the degree of antibody humanization Analysis of the variable (V) regions of the new humanized variants (VH1-VH5 and VL1-VL5, VL3.1, VL4.1, VL5.1) and the previously humanized anti-HSG antibody hz679 (from US 2009 / 0240037 Al) was performed to determine: 1) their percent identity to the closest human germline; 2) their degree of humanization as determined by the IMGT® DomainGapAlign tool (https: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi) according to the World Health Organization (WHO) definition of a humanized antibody.

[0330] Results and conclusions: As is evident from Table VII and Table VIII, all 5 new humanized VH variants (VH1-5) and 4 new humanized VL variants (VL1, VL2, VL5 and VL5.1) show higher identity (%) to their respective closest human germline compared to the previously humanized anti-HSG antibody hz679 and its closest human germline (Table VII). Furthermore, according to the WHO definition of a humanized antibody, 2 new humanized VH variants VH1 and VH2, and 4 new humanized VL variants VL1, VL2, VL5 and VL5.1 are defined as fully humanized (Table VIII). The new humanized VH variants VH3 and VH4 are equally close to human and murine origin, VH5 is equally close to human, murine and simian origin, while the new humanized VL variants VL3, VL3.1, VL4 and VL4.1 are closest to simian origin (Table VIII). In contrast, the previously humanized anti-HSG antibody hz679 is still closer to murine origin according to the WHO definition of a humanized antibody (Table VIII). It is known in the art that the closer the sequence of an antibody to its closest human germline, the lower the immunogenicity of the antibody once infused into a human (Gao SH et al., 2013). Therefore, the improved humanization score of the new humanized anti-HSG VH and VL variants reduces the immunogenicity potential of antibodies comprising the new humanized variable region sequences.

[0331] Table VII. Percent identity (%) to the closest human germline.

[0332]

[0333] * (US2009 / 0240037 Al), ** (US2009 / 0246131 Al) Table VIII. Degree of humanization of anti-HSG VH and VL variant sequences according to the WHO definition of a humanized antibody.

[0334]

[0335] * (US2009 / 0240037 Al), ** (US2009 / 0246131 Al) Example 3: Production and preparation characteristics of anti-oxMIF x anti-HSG bispecific antibodies.

[0336] Materials and methodsTo generate novel anti-oxMIF x anti-HSG bispecific Fab-scFv-Fc monoclonal antibodies, novel humanized VH and VL sequences were linked to a scFv (VH-(GGS)5-VL) (SEQ ID NO: 88) and fused to an Fc gamma R silent human IgGl "pocket" type Fc via a linker containing a truncated hinge region (GGGGSDKTHTCPPCP, SEQ ID NO: 58) to form an anti-HSG scFv-Fc "pocket" type heavy chain. The VH and VL of the previously humanized anti-HSG antibody hz679 were used as a reference. Plasmids containing the synthetic anti-HSG scFv-Fc heavy chain (HC) were co-transfected with plasmids encoding the corresponding anti-oxMIF antibody chains (light and heavy chain, Fc gamma R silent human IgGl "knobs" type constant domain) in a 3:2:1 ratio (anti-oxMIF light chain: anti-oxMIF heavy chain: anti-HSG scFv-Fc) into ExpiCHO-S cells. Transient expression was performed using the MAX Titer protocol (Thermo Fisher Scientific) to generate anti-oxMIF x anti-HSG bispecific antibodies C0176-C0200 (containing novel humanized anti-HSG sequences) and C0132 (containing previously humanized anti-HSG sequences of hz679) (Table IV).

[0337] To generate anti-oxMIF x anti-HSG bispecific CrossMab antibodies, novel humanized anti-HSG VH sequences (VH1-VH3) were cloned in-frame to an Fc gamma R / FcRn silent human IgGl "pocket" heavy chain (HC) constant region (CLKcross CH2-CH3) to obtain anti-HSG heavy chains; novel humanized anti-HSG VL sequences (VL1-VL4) were cloned in-frame to the CH1 region of human IgGl (CH1cross) to obtain anti-HSG light chains. 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 co-transfected with plasmids encoding the corresponding anti-oxMIF antibody chains (light and heavy chain, Fc gamma R / FcRn silent human IgGl "knobs" constant region) in a 2:1:1:2 ratio (anti-oxMIF light chain: anti-oxMIF heavy chain: anti-HSG heavy chain: anti-HSG light chain). This resulted in anti-oxMIF x anti-HSG CrossMab antibodies C0238-C0252 (containing novel humanized anti-HSG sequences) and C0255 (containing previously humanized anti-HSG sequences from hz679) (Table V).

[0338] Heterodimerization of the two different heavy chains was achieved by the “knobs-into-holes” (KIH) technology (anti-oxMIF heavy chain “knob”: T366W and S354C mutations to stabilize disulfide bond; anti-HSG heavy chain “hole”: T366S / L368A / Y407V and Y349C mutations to stabilize disulfide bond). In addition, all heavy chains carried L234A / L235A (“LALA”) mutations to minimize binding to Fc gamma receptor-bearing immune cells and complement. The CrossMab format of the bispecific monoclonal antibody also comprised H310A / H435Q mutations to modulate binding to human FcRn, thereby optimizing the half-life of the monoclonal antibody for human pre-targeting. Mutation sites are numbered according to the EU numbering system.

[0339] ExpiCHO-S cultures were harvested 12 days post transfection and monoclonal antibodies were purified from cell culture supernatant by protein A affinity chromatography (MabSelect Prism A HiTrap column, Cytiva). Monoclonal antibodies were further purified by preparative cation exchange chromatography (CEX) using Poros XS columns and formulated in a solution of 10 mM sodium acetate / acetic acid, 200 mM sodium chloride, pH 5.8.

[0340] Monoclonal antibody expression titers were calculated as milligrams of protein A purified monoclonal antibody per liter of harvested cell culture supernatant (mg / L). The relative abundance of correctly assembled bispecific monoclonal antibodies (monomer purity) was assessed by analytical size exclusion chromatography (SEC using ENrich 650 columns, Bio-Rad) and analytical cation exchange chromatography (CEX using Poros XS columns, Cytiva) on protein A purified samples.

[0341] Monoclonal antibody purity and 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 Instant Blue® Coomassie blue staining (Abeam). Band intensities (BI) were quantified using ImageStudioLite 5.2 software (LI-COR) and the extent of cleavage was calculated by the following formula: (BI anti-HSG heavy chain / BI anti-oxMIF light chain) x 100.

[0342] Results and conclusionsOf the 25 Fab-scFv-Fc monoclonal antibodies expressed in 25 mL scale at the same time, monoclonal antibodies C0176, C0181, C0182, C0186 and C0192 (in which the new humanized anti-HSG VL1 and VL2 variants were paired with one of the new humanized VH1-VH4 variants), showed the best manufacturing properties in terms of the combination of monoclonal antibody expression titers, monomer purity and severity of anti-HSG scFv-Fc heavy chain cleavage (candidate antibodies with arrows pointing upwards in Table IX - -). Mass spectrometry analysis showed that the 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). Unexpectedly, antibodies containing VL sequences and variable regions as VL3-5 variants (C0178-0180, C0183-0185, C0188-0190, C0193-0195, C0198-0200) whose FR4 region (FGAGTKLELK, SEQ ID NO: 60) was identical to that of the previously humanized anti-HSG monoclonal antibody hz679 (C0132), showed significantly enhanced anti-HSG scFv heavy chain cleavage (see C0178-C0180, for example, Figure 2 ).

[0343] Therefore, the top five monoclonal antibodies C0176, C0181, C0182, C0186 and C0192, together with the reference monoclonal antibody C0132 (having the sequence of the previously humanized anti-HSG monoclonal antibody hz679), were re-expressed at large scale (200 mL) in order to directly compare the manufacturing properties. As shown in Table X, the expression titers and monomer purity of the new humanized bispecific monoclonal antibodies C0176, C0181, C0182, C0186 and C0192 were significantly improved compared to C0132 having the sequence of the previously humanized anti-HSG hz679. In summary, bispecific Fab-scFv-Fc antibodies comprising the new humanized anti-HSG variable regions VL1-2 in combination with VH1-4 showed more favorable properties compared to the previously humanized anti-HSG monoclonal antibody hz679.

[0344] Anti-oxMIF x anti-HSG bispecific CrossMab antibodies C0238-C0252 were expressed simultaneously at 100 mL scale with reference CrossMab C0255 (with previously humanized anti-HSG hz679 sequence) for direct comparison. CrossMab purification methods were as described previously. As shown in Table XI, CrossMabs pairing the new humanized VH variants (VH1-3) with the new humanized VL variants (VL1-2) (C0238, C0239, C0240, C0241, C0245, C0250) showed improved manufacturing characteristics compared to reference C0255. This improvement was reflected in increased expression titers and improved monomer purity as determined by SEC and CEX (candidate antibodies with arrows pointing upwards in Table XI - ^ / U -). In summary, again it was shown that bispecific CrossMabs comprising the new humanized anti-HSG variable regions VL1-2 in combination with VH1-3 showed more favorable characteristics compared to reference C0255 comprising the previously humanized anti-HSG monoclonal antibody hz679.

[0345] Table IX. Manufacturing characteristics of new humanized anti-oxMIF x anti-HSG bispecific Fab-scFv-Fc monoclonal antibodies C0176-C0200.

[0346]

[0347] 1 mg of protein A purified monoclonal antibody per liter of cell culture supernatant; 2 Relative abundance (%) of correctly assembled monoclonal antibody monomer was calculated from SEC chromatogram peak area divided by peak width (mL); 3 Band intensity (BI) of cleaved anti-HSG heavy chain normalized by band intensity (BI anti-oxMIF LC) of anti-oxMIF light chain (BI anti-HSG HC / BI anti-oxMIF LC x 100). For determination of best candidate antibodies, expression titers and purity values were normalized to range all values between 0-100% (highest / best value = 100%, lowest value = 0%): U > 75%, ^ > 50% to 75%, D < 50%. For cleavage: U < 12.5%, ^ > 12.5% to < 25%, D > 25%.

[0348] Figure 2Purity of monoclonal antibodies C0132 and C0176-C0180 and extent of cleavage of the anti-HSG scFv-Fc heavy chain were assessed by SDS-PAGE and Coomassie Blue staining. A total of 3 pg of protein A purified monoclonal antibodies were separated by NuPAGE™ 4-12% SDS-PAGE under reducing conditions (“red.”). Spectra MultiColor Wide Range Protein MW Standard was used as standard. Arrows indicate anti-oxMIF light chain (LC) and cleaved anti-HSG heavy chain.

[0349] Table X. Comparison of manufacturing properties of selected new humanized Fab-scFv-Fc monoclonal antibodies C0176, C0181, C0182, C0186, C0192 and reference monoclonal antibody C0132 comprising the hz679 sequence of the previously humanized anti-HSG.

[0350]

[0351] 1 mg of protein A purified monoclonal antibody per liter of cell culture supernatant; ^ > 300 mg / L, ^ > 250 mg / L to < 300 mg / L, ^ < 250 mg / L; 2 Relative abundance (%) of correctly assembled monoclonal antibody monomers calculated from SEC chromatogram integrated peak areas; ^ > 90%, ^ > 81% to < 90%, ^ < 80%.

[0352] Table XI. Comparison of manufacturing properties of new humanized anti-oxMIF x anti-HSG bispecific CrossMab antibodies C0238-C0252 and reference anti-oxMIF x anti-HSG CrossMab C0255 comprising the hz679 sequence of the previously humanized anti-HSG.

[0353]

[0354] 1 mg of protein A purified monoclonal antibody per liter of cell culture supernatant; 2The relative abundance (%) of correctly assembled monoclonal antibody monomers was calculated from the integration of peak areas of SEC chromatograms or CEX chromatograms. To determine the best candidate antibodies, the candidate antibodies were ranked as follows: titer, ^ 150 mg / L, ^ 130 mg / L < 150 mg / L, ^ 123 mg / L < 130 mg / L, < 123 mg / L; SEC purity: ^ 90%, ^ 80% < 90%, ^ 77% < 80%, < 77%; CEX purity: ^ 60%, ^ 50% < 60%, ^ 40% < 50%, < 40% Example 4: Assessment of cleavage of the anti-HSG scFv-Fc heavy chain in the anti-oxMIF x anti-HSG bispecific monoclonal antibody C0132 by mass spectrometry.

[0355] As described in Example 3, the anti-HSG scFv-Fc heavy chain of all bispecific Fab-scFv-Fc anti-oxMIF x anti-HSG monoclonal antibodies was cleaved to varying degrees. Unexpectedly, antibodies containing the VL sequence and having a VL3-5 variant in the variable region (C0178-0180, C0183-0185, C0188-0190, C0193-195, C0198-0200) with the same FR4 region (FGAGTKLELK; SEQ ID NO: 60) as the previously humanized anti-HSG monoclonal antibody hz679 (C0132) showed a significantly enhanced cleavage of the anti-HSG scFv heavy chain (see Table IX and Figure 2 ), whereas antibodies containing the new humanized VL1 and VL2 variants showed significantly less cleavage compared to C0132. In this example, the C0132 fractions purified by protein A and CEX containing a high amount of truncated anti-HSG heavy chains were subjected to mass spectrometry analysis to determine the cleavage site in more detail.

[0356] Materials and methods C0132 was diluted in 0.1% formic acid (FA) and separated on a reversed-phase column (MAbPac RP 4 pm 2.1 x 50 mm, Thermo Scientific) using an ultra-performance liquid chromatography (UPLC) system (Waters ACQUITY Premier). The eluents were 0.1% FA in water and 0.1% FA in acetonitrile. Mass spectrometry analysis was performed using a compact quadrupole time-of-flight (QTOF) mass spectrometer (Bruker Daltonik). For data analysis, the recorded liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) spectra were summed, deconvoluted, and smoothed using the DataAnalysis software (Bruker Daltonik).

[0357] Results and conclusions : Figure 3 The complete mass spectrum of monoclonal antibody C0132 is shown. There are two major peaks in the spectrum, at 126882.3 and 100701.9 Da, corresponding to the intact antibody and an antibody with a truncated heavy chain. The antibody with a molecular mass of 100701.9 Da can only be an anti-HSG scFv-Fc heavy chain truncated at amino acid 1-244, which means that the cleavage occurred between amino acid residues E105 and L106 in the "KL EL K" motif (Kabat numbering) in the FRW4 of the anti-HSG VL, which motif is present in the new humanized variants containing VL3-5 and the previously humanized hz679 monoclonal antibody. In contrast, the Fab-scFv-Fc monoclonal antibodies C0176, C0181, C0182, C0186 and C0192, which show only a slight cleavage of the anti-HSG scFv-Fc, all contain the new humanized VL variants VL1 or VL2 in FRW4 carrying the "KL EI K" motif (SEQ ID NO: 61).

[0358] Figure 3 The deconvoluted mass spectrum of monoclonal antibody C0132 is shown. The two major peaks correspond to the intact antibody (126882.3 Da) and an antibody with a truncated anti-HSG scFv-Fc heavy chain (100701.9 Da).

[0359] Example 5: Comparison of the stability of the new humanized anti-oxMIF x anti-HSG bispecific Fab-scFv-Fc monoclonal antibodies C0176, C0181, C0182, C0186 and C0192, with reference antibodies having the VH and VL sequences of the previously humanized anti-HSG antibody hz679, under different storage conditions.

[0360] Materials and methods : Comparison of the stability of the new humanized anti-oxMIF x anti-HSG bispecific Fab-scFv-Fc monoclonal antibodies C0176, C0181, C0182, C0186 and C0192, with reference antibodies having the VH and VL sequences of the previously humanized anti-HSG antibody hz679, under different storage conditions. Figure 4AAntibody stability was assessed after storage at 4°C or 4°C (Figure 4B) for 83 days. For SEC, samples were diluted to 1 mg / mL with running buffer (0.1 M phosphate buffer, 0.2 M arginine, pH 6.8), and 100 µL of sample was loaded onto an Enrich 650 (Bio-Rad) gel filter column at a flow rate of 1.25 mL / min. Protein peaks were monitored using 280 nm absorbance, and spectra were analyzed using the ChromLab software package (Bio-Rad). Peak areas were integrated using 280 nm absorbance, and the relative areas of different peaks were used to determine the relative abundance of aggregates, monomers, and fragments. To facilitate direct comparison of samples, the percentage of monomers (% monomer) for each antibody was normalized to day 0 (=100%).

[0361] Results and conclusions As shown in Figure 4, the bispecific antibody containing the novel humanized anti-HSG sequence exhibited improved stability (at -80℃). Figure 4A ): C0176, C0181, C0182, C0186, C0192; 4℃ (Figure 4B): C0186 and C0192) or comparable stability (4℃ (Figure 4B): C0181 and C0182), showing higher monomer content than C0132 (containing the previously humanized anti-HSG sequence) or similar to C0132 (containing the previously humanized anti-HSG sequence). In summary, this again demonstrates that the novel bispecific antibodies containing the new humanized VL variants VL1 and VL2 paired with the VH variants VH1-VH4 exhibit improved stability during long-term (83 days) storage compared to the reference C0132 containing the previously humanized anti-HSG hz679 sequence.

[0362] Figure 4 shows a stability comparison between the novel humanized anti-oxMIF×anti-HSG bispecific Fab-scFv-Fc monoclonal antibody and the C0132 monoclonal antibody containing a previously humanized anti-HSG sequence. Stability was assessed by size exclusion chromatography (SEC) before and after storage at -80°C (A) or 4°C (B) for 83 days (day 0). For ease of direct comparison of samples, the percentage of monomer (% monomer) for each antibody was normalized to day 0 (=100%).

[0363] Example 6: Binding of bispecific anti-oxMIF × anti-HSG monoclonal antibody to immobilized HSG.

[0364] Materials and methodsStreptavidin diluted to 1 µg / mL in PBS was immobilized in 96-well Maxisorb plates (Thermo Fisher Scientific) and incubated at room temperature for 4 hours. Blocking was performed overnight at 4°C with TBST containing 2% fish gelatin (FG). After washing with TBST, the plates were incubated with 100 μL per well of 0.1 µg / mL biotin-PEG4-dTyr-dLys(HSG)-dGlu-dLys(HSG)-NH2 (Eurogentec) at room temperature for 1.5 hours. After washing with TBST, 100 µL of serially diluted bispecific anti-oxMIF × anti-HSG monoclonal antibody or control monoclonal antibody was added, and the plates were incubated with the monoclonal antibody at room temperature for 1.5 hours. After washing with TBST, the bound antibody was detected using goat anti-human IgG (Fc specific)-HRP conjugate and tetramethylbenzidine (TMB) as substrates. The colorimetric reaction was stopped with 3M H₂SO₄, and the optical density (OD) was measured at 450 nm. EC was determined using a GraphPad Prism via 4-parameter logistic fitting. 50 value.

[0365] Results and conclusions The binding of bispecific anti-oxMIF × anti-HSG monoclonal antibody to immobilized HSG was measured over a wide concentration range, and the results are as follows: Figure 5 As shown. Combined with curve ( Figure 5 A) and the calculated representative appearance K D EC 50 The values ​​clearly indicate that all bispecific Fab-scFv-Fc monoclonal antibodies ( Figure 5 B, C0176, C0181, C0182, C0186 and C0192) and CrossMab ( Figure 5 C, C0238, C0239, C0240, C0241, C0245, C0250), containing novel humanized anti-HSG sequences, show improved or maintained affinity for HSG compared to reference monoclonal antibodies C0132 and C0255, which contain the previously humanized anti-HSG hz679 sequence (manifested as EC). 50 (Value decreased).

[0366] Figure 5The binding of bispecific anti-oxMIF×anti-HSG monoclonal antibodies to immobilized HSG is shown. Optical density (OD) values ​​at 450 nm (mean ± SEM, n=3) are plotted against monoclonal antibody concentrations, and curves are fitted using GraphPad Prism software with 4-parameter logistic fitting. (A) Binding curves of Fab-scFv-Fc bispecific monoclonal antibodies (BsMAb) C0132, C0176, C0181, C0182, C0186, and C0192 to HSG; (B) Half-maximal effective concentration (EC50) of A. 50 (C) CrossMab (C0255, C0238, C0239, C0240, C0241, C0245, C0250) combined with HSG EC 50 Values ​​(mean ± SEM, n=3). The dashed line represents the EC50 values ​​of the bispecific anti-oxMIF × anti-HSG antibody used for reference. 50 value.

[0367] Example 7: Conserved binding of anti-oxMIF×anti-HSG bispecific antibody with novel humanized anti-HSG sequence variants VH1-VH4 paired with VL1-VL2 to oxMIF. Materials and methods Recombinant human MIF was diluted to 1 μg / mL with PBS and coated overnight in ELISA plates at 4°C (MIF was converted to oxMIF according to Thiele et al. (2015)). After blocking, serially diluted anti-oxMIF×anti-HSG bispecific antibody was added to the plate. Finally, the bound antibody was detected using goat anti-human immunoglobulin G (IgG) (Fc specific)-HRP conjugate and tetramethylbenzidine (TMB) as substrates. The colorimetric reaction was terminated with 3M sulfuric acid (H2SO4), and the OD value was measured at 450 nm. Representative epigenetic K was determined using GraphPad Prism software via 4-parameter logistic fitting. D EC 50 The values ​​are displayed as mean (n=2 or 3) ± SEM.

[0368] Results and conclusions The binding of the anti-oxMIF×anti-HSG bispecific monoclonal antibody to immobilized MIF (oxMIF) was measured over a wide concentration range. Binding curves and calculated EC50 values ​​were obtained. 50 Values ​​such as Figure 6 As shown. Figure 6 Clearly showing two forms of anti-oxMIF × anti-HSG monoclonal antibody, namely the Fab-scFv-Fc form ( Figure 6 A, B) and CrossMab form ( Figure 6Both C) maintained low nanomolar affinity for oxMIF. As a control, the bivalent monospecific antibody C0008 (imalizumab, reference anti-oxMIF antibody) was used. Clearly, in this setting, affinity makes a significant contribution to oxMIF binding, as C0008 exhibits a stronger interaction with oxMIF compared to the monovalent anti-HSG / oxMIF bispecific antibody. In conclusion, neither the use of novel humanized VH / VL sequences as the anti-HSG arm of the bispecific monoclonal antibody, nor the choice of bispecific monoclonal antibody form (Fab-scFv-Fc and CrossMab) compromised the binding of monoclonal antibodies to oxMIF.

[0369] Figure 6 The conserved binding of the anti-oxMIF×anti-HSG bispecific monoclonal antibody to immobilized MIF (oxMIF) was demonstrated. The anti-oxMIF×anti-HSG Fab-scFv-Fc monoclonal antibodies (A, B) and anti-CrossMab (C) were bound to immobilized oxMIF, and the binding was detected by goat anti-human IgG (Fc-specific)-HRP conjugate. The binding curve (A) and EC50 of the Fab-scFv-Fc bispecific antibody are shown. 50 Values ​​(BC) (mean ± SEM, n = 2–3). C0008 (imacizumab) was used as the reference anti-oxMIF monoclonal antibody.

[0370] Example 8: Evaluation of off-target binding of anti-oxMIF×anti-HSG bispecific monoclonal antibody to A2780 MIF gene knockout cells Materials and methods In the A2780 ovarian cancer cell line (ECACC / Sigma # 93112519), the human MIF gene was edited using CRISPR / Cas9 to generate A2780 MIF. - / - Cell lines. In short, target gene sequences are analyzed, and target sites are located according to the general rules of designing targeting guide RNAs (gRNAs) based on the GenCRISPR™ system. A 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 cells containing Streptococcus pyogenes (…). S. pyogenes) Cas9 (SpCas9) nuclease in the PX459 (addgene) vector. A2780 cells were transiently transfected by electroporation and cells were seeded in 96-well plates by limiting dilution to generate homogenous monoclonal. Homogenous monoclonal were identified by Sanger sequencing screen, in which the endogenous MIF gene was effectively mutated, resulting in a corresponding reduction (or elimination) of MIF protein expression. The final clone showed a deletion of 10 base pairs at the +2 position after the start codon of the human MIF gene. A2780 MIF - / - Deletion of endogenous human MIF protein in cell lines was confirmed by Western blot using a polyclonal anti-human MIF antibody.

[0371] A2780 MIF - / - Cells were detached from culture flasks, washed with PBS and stained with the fixable live dye eFluor 780 (diluted 1 :2000 in PBS) for 15 min at 4°C. Afterwards, cells were washed with staining buffer (PBS + 5% FBS) and resuspended in staining buffer at a concentration of 4 x 10 6 Cells were resuspended in staining buffer at a concentration of 2 x 10 5 Cells were plated in 96-well U-bottom plates at a volume of 2 x 105cells / well / 50 μL / well. 50 μL of serial dilutions (2-fold stock) of the bispecific anti-oxMIF x anti-HSG monoclonal antibodies C0176, C0181, C0182, C0186, C0192 or the control monoclonal antibody rituximab (final concentrations of 75 nM, 37.5 nM and 18 nM) were added per well. After incubation for 40 min at 4°C, cells were washed with staining buffer and resuspended in 100 μL secondary antibody (goat anti-human IgG (H+L) - Alexa Fluor 488 (AF488), diluted 1 : 100). After another incubation for 40 min at 4°C, cells were washed with staining buffer, resuspended in staining buffer and measured on a Cytoflex-S flow cytometer (Beckman Coulter): 10,000 events were collected after gating on live cells (eFluor 780 negative) and data were analyzed using FlowJo software. The AF488 geometric mean fluorescence intensity (MFI) values of live cells (eFluor 780 negative) were plotted as a function of antibody concentration in GraphPad Prism software.

[0372] Results and conclusions : Figure 7The bispecific anti-oxMIF x anti-HSG monoclonal antibodies with the new humanized anti-HSG sequences VL1 and VL2 paired with VH1-VH4 (C0176, C0181, C0186, C0182, C0192) showed no relevant off-target binding higher than rituximab, which indicates a good safety profile, since rituximab has been used for many years as a therapeutic antibody. Rituximab was shown to have a certain surface hydrophobicity, which leads to its non-specific 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 that is not present on A2780 cells.

[0373] Figure 7 It was specifically shown that the anti-oxMIF x anti-HSG bispecific monoclonal antibodies (bsMAbs) were assessed for non-target binding to A2780 MIF knockout - / - cells. A2780 MIF knockout cells were stained with serially diluted anti-oxMIF x anti-HSG bispecific monoclonal antibodies C0176, C0181, C0182, C0186, C0192 and the control therapeutic monoclonal antibody rituximab. Binding was detected on live cells with AF488-coupled goat anti-human IgG (H+L) secondary antibody. Geometric mean fluorescence intensity (MFI) values of the AF488 channel were plotted against monoclonal antibody concentration in GraphPad Prism software. The dotted line indicates the MFI value of staining with secondary antibody only. - / - - / - cells. A2780 MIF knockout cells were stained with serially diluted anti-oxMIF x anti-HSG bispecific monoclonal antibodies C0176, C0181, C0182, C0186, C0192 and the control therapeutic monoclonal antibody rituximab. Binding was detected on live cells with AF488-coupled goat anti-human IgG (H+L) secondary antibody. Geometric mean fluorescence intensity (MFI) values of the AF488 channel were plotted against monoclonal antibody concentration in GraphPad Prism software. The dotted line indicates the MFI value of staining with secondary antibody only.

[0374] Example 9: Thermal stability of new humanized anti-oxMIF x anti-HSG CrossMabs The thermal stability of new humanized anti-oxMIF x anti-HSG CrossMabs comprising the new humanized VL variants VL1 and VL2 paired with VH variants VH1-VH3 (C0238, C0239, C0240, C0241, C0245 and C0250) was assessed in a direct comparison to the reference anti-oxMIF x anti-HSG CrossMab C0255 with the VH and VL sequences of the previously humanized anti-HSG antibody hz679.

[0375] Materials and methods : Assessment of antibody thermal stability using nano-differential scanning fluorimetry (nanoDSF) Figure 8The purified antibody was prepared into a 0.5 mg / mL solution in 50 mM HEPES and 50 mM NaCl (pH 7.2), and four aliquots were analyzed on a Prometheus NT.48 instrument (NanoTemper Technologies). 10 μL of antibody sample was loaded into a glass capillary tube, placed in the sample holder, and the temperature was increased from 20 °C to 95 °C at a rate of 1 °C / min. Simultaneously, the fluorescence intensity at 330 nm and 350 nm (excitation wavelength 280 nm) was monitored. Using the manufacturer's software, the emission intensity ratio (350 nm / 330 nm) was plotted as a function of temperature, its first derivative was calculated, and the inflection point temperature (T0) of the first unfolding transition was determined. IP (℃).

[0376] Results and conclusions :like Figure 8 As shown, compared to the C0255 CrossMab containing the previously humanized anti-HSG sequence, all CrossMabs containing the novel humanized anti-HSG sequences (C0238, C0240, C0241, C0245, C0250) exhibit superior stability against thermal defolding, as evidenced by the higher temperature at which the first defolding transition occurs, a measure of overall stability. In summary, compared to the reference C0255 containing the previously humanized anti-HSG hz679 sequence, CrossMabs containing novel humanized VL variants VL1 and VL2 paired with VH variants VH1-VH3 exhibit improved thermal stability.

[0377] Figure 8 The novel humanized anti-oxMIF×anti-HSG CrossMab showed the inflection point temperature (Ti) of the first unfolding transition compared to the C0255 CrossMab containing the previously humanized anti-HSG sequence. IP (℃). Data are expressed as averages (n=4±SD).

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Claims

1. A bispecific anti-tumor antigen / anti-HSG antibody comprising at least one binding site specifically recognizing a tumor antigen and a binding site specifically recognizing HSG, comprising: - a light chain variable (VL) domain comprising a sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 89, SEQ ID NO: 90 and SEQ ID NO: 91; or - a light chain variable (VL) domain comprising a sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 89, SEQ ID NO: 90 and SEQ ID NO: 91, which sequence has one or two additional amino acid substitutions and is, according to the Kabat numbering, glutamine at position 100 (Q100), glutamic acid at position 105 (E105) and isoleucine at position 106 (1106); and - a heavy chain variable (VH) domain comprising a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10; or - a heavy chain variable (VH) domain comprising a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10, which sequence has one or two additional amino acid substitutions and is, according to the Kabat numbering, arginine at position 19 (R19).

2. The antibody according to claim 1, comprising a single chain variable fragment (scFv) specifically recognizing HSG having 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 according to claim 1, comprising a VL and a VH domain specifically recognizing HSG, wherein the C-terminus of the VL domain is linked to SEQ ID NO: 30 and the C-terminus of the VH domain is linked to SEQ ID NO:

38.

4. The antibody according to any one of claims 1 to 3, comprising an Fc region having SEQ ID NO: 31, which Fc region comprises asymmetric mutations in each CH3 domain, resulting in heterodimerization of two CH domains from different antibodies, in particular, the "knobs" mutation T366W and S354C in one CH domain and the "cleft" mutations T366S, L368A, Y407V and Y349C in one CH domain, according to the EU numbering index.

5. The antibody according to any one of claims 1 to 4, comprising a variant Fc region with reduced or ablated effector function and / or FcRn binding, in particular, the variant Fc region has 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 according to EU numbering index of SEQ ID NO: 31 and optionally an aglycosylated Fc region.

6. The antibody according to 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 (FRa), in particular, the anti-tumor antigen is oxMIF.

7. The antibody according to claim 6, wherein the binding site specifically recognizing oxMIF comprises: - 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, wherein amino acid positions according to Kabat numbering.

8. The antibody according to any one of claims 1 to 7, 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-center scFv), Fab / Fab-crossFab-Fc, Fab / crossFab-Fc, IgG-scFv and IgG-(scFv)2.

9. The antibody according to any one of claims 1 to 8 for use in the treatment or detection of a malignant tumor, wherein, In a first step the antibody is administered to the subject and in a second step the HSG group is administered, wherein the HSG group binds to the antibody.

10. The antibody for use according to claim 9, wherein the HSG group is coupled or labeled with one or more diagnostic agent and / or therapeutic agent, in particular, the HSG group comprises one or more HSG hapten, one or more diagnostic agent and / or therapeutic agent and one chelator.

11. The antibody according to any one of claims 1 to 8, wherein the antibody is bound to a HSG group, which is coupled or labeled with one or more diagnostic agent and / or therapeutic agent, in particular, the HSG group comprises one or more HSG hapten, one or more diagnostic agent and / or therapeutic agent and one chelator, in particular, the therapeutic agent is a radionuclide or a cytotoxic drug; the diagnostic agent is a radionuclide.

12. The antibody of claim 11, wherein the chelator is bound to a radionuclide, in particular the chelator is selected from the group consisting of DOTA, DTPA, desferrioxamine B (DFO) and DFO*, in particular the radionuclide is selected from the group consisting of 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 Th.

13. Use of the antibody according to any one of claims 1 to 8 for the manufacture of a medicament.

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

15. The pharmaceutical composition of claim 14 for use in the treatment of a patient suffering from cancer, in particular, for use in the treatment of a solid tumor, more particularly, for use in the treatment of colorectal cancer, ovarian cancer, breast cancer, prostate cancer, pancreatic cancer, and lung cancer.

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