Improved anti-oxMIF antibodies with reduced aggregation potential and reduced hydrophobicity

Abstract

The present invention relates to anti-oxMIF antibodies and their use in treating oxMIF-related disorders, said anti-oxMIF antibodies having improved properties, such as reduced aggregation potential and reduced hydrophobicity due to selected amino acid substitutions in the light and heavy chain variable domains, and optionally enhanced effector function due to further substitutions in the heavy chain constant region.

Description

Technical Field

[0001] The present invention relates to anti-oxMIF antibodies and their use in treating oxMIF-related disorders, said anti-oxMIF antibodies having improved properties, such as reduced aggregation potential and reduced hydrophobicity due to selected amino acid substitutions in the light and heavy chain variable domains, and optionally enhanced effector function due to further substitutions in the heavy chain constant region. Background Art

[0002] The cytokine macrophage migration inhibitory factor (MIF) was described as early as 1966 (David, JR, 1966; Bloom BR and Bennet, B., 1966). However, MIF is significantly different from other cytokines and chemokines in that it is constitutively expressed, stored in the cytoplasm, and present in the circulation of healthy subjects. Due to the ubiquitous nature of this protein, MIF can be considered an inappropriate target for therapeutic intervention. However, MIF appears in two immunologically distinct conformational isomers, referred to as reduced MIF (redMIF) and oxidized MIF (oxMIF) (Thiele M. et al., 2015). It was found that redMIF is an abundantly expressed MIF isomer that can be found in the cytoplasm and circulation of any subject. redMIF appears to represent a latent, inactive storage form (Schinagl. A. et al., 2018).

[0003] In contrast, oxMIF appears to be the physiologically relevant and disease-associated isoform, which can be detected in tumor tissues, particularly in patients with colorectal, pancreatic, ovarian and lung cancer, indicating the high tumor specificity of oxMIF (Schinagl.A. et al., 2016), and has also been detected in the circulation of patients with inflammatory diseases (Thiele et al., 2015).

[0004] The number of successful drug targets for treating cancer (such as the oxMIF-positive indications mentioned above) is limited. For example, more than 300 potential immuno-oncology targets have been described, but many clinical studies focus on anti-PD1 and anti-PDL1 antibodies (Tang J., et al. 2018). Therefore, the scientific and medical communities are eagerly awaiting potential drugs targeting tumor-specific antigens to increase treatment options for cancer patients with poor prognosis.

[0005] Monoclonal anti-MIF antibodies are described in WO 2009 / 086920 A1.

[0006] Antibodies targeting oxMIF have shown efficacy in in vitro and in vivo models of inflammation and cancer (Hussain F. et al., 2013; Schinagl. A. et al., 2016; Thiele et al., 2015). In phase 1 clinical trials, an oxMIF-specific antibody (Imalumab) demonstrated an acceptable safety profile, satisfactory tissue penetration, and signs of antitumor activity (Mahalingam D. et al., 2015; Mahalingam D. et al., 2020).

[0007] Bispecific anti-oxMIF / anti-CD3 antibodies are disclosed in WO 2019 / 234241A1.

[0008] Protein aggregation, especially antibody aggregation, is often observed during several stages of bioprocessing, including protein expression, purification, and storage. Antibody aggregation may affect the overall yield of the therapeutic protein manufacturing process and may affect the stability and immunogenicity of therapeutic antibodies.

[0009] Therefore, protein aggregation of antibodies has become an important issue in their development and remains a major concern in antibody production. Antibody aggregation may be triggered by partial unfolding of its domains, leading to monomer-monomer association, followed by nucleation and aggregate growth. Although the aggregation tendency of antibodies and antibody-based proteins may be affected by external experimental conditions, they are strongly dependent on intrinsic antibody properties as determined by their sequence and structure.

[0010] For example, aggregation resistance can be achieved by stabilizing the native state (i.e., resisting unfolding) or by reducing the aggregation tendency of proteins in an unfolded or partially folded state. The disadvantage of stabilizing the native state is that the protein will likely be exposed to an environment in which they will unfold. Typically, when a protein is denatured or unfolded, amino acid residues that are typically mediated by intramolecular contacts within the protein are exposed. Such exposure typically makes the protein prone to forming intermolecular contacts and aggregation. In contrast to proteins that resist unfolding, proteins whose aggregation tendency is reduced when unfolded will easily refold into a biologically active non-aggregated state after being exposed to such an environment.

[0011] The aggregation resistance or aggregation propensity of antibodies and proteins comprising their antigen binding domains is generally limited by the most aggregation-prone domain or domains contained therein and the strength of their interactions with surrounding domains, if any.

[0012] This is because once the domain unfolds, if it cannot refold, it may interact with other domains in the same protein or other proteins and form aggregates. The constant domains of antibodies generally do not aggregate and do not vary greatly. Therefore, in terms of aggregation potential and stability, the weakest domains of antibodies are generally considered to be the variable domains (e.g., the heavy chain variable domain (V H ) and / or light chain variable domain (V L ), Ewert S. et al., 2003). In this regard, the aggregation-prone V H or V LThe incorporation of domains into other stable recombinant antibody products usually confers these usually undesirable characteristics to new recombinant designs. Therefore, engineering variable domains into anti-aggregation properties is the most likely way to make the entire protein that may contain the variable domains anti-aggregation properties. Various strategies have been proposed to reduce variable domain aggregation, for example, the rational design of anti-aggregation proteins, complementary determining regions (CDR) transplantation, or the introduction of disulfide bonds in variable domains. The rational design of anti-aggregation proteins generally involves the use of computer simulation analysis to predict the effect of point mutations on protein aggregation tendency. However, this method has several difficulties. For example, it is not enough to identify mutations that may reduce the aggregation of unfolded proteins. On the contrary, mutations must not increase the aggregation of folded proteins or affect the function of folded proteins, especially binding properties, such as the affinity or specificity of antibodies. In addition, reasonable design requires detailed structural analysis of the specific protein to be improved, and therefore, it is difficult to use for proteins that have not been thoroughly characterized, and is not easily applicable to various different proteins. CDR transplantation involves transplanting CDRs from one variable domain to the framework region (FR) of another variable domain. This strategy has been shown to be useful in stabilizing anti-EGP-2 scFv (Willuda J. et al., 1999). The disadvantages of this approach include the possibility of reduced affinity after CDR transplantation. This loss of affinity can be overcome by introducing mutations into FR, however such mutations may produce immunogenic epitopes in the protein, making the protein no longer desirable from a therapeutic perspective. In addition, CDR transplantation generally requires analysis of the crystal structure or homology modeling of the donor and recipient variable domains to assess the suitability of the transplant. Such methods are laborious and require expertise. In addition, since each variable domain has a different structure, the method is not easily applicable to a variety of molecules. As for methods involving the introduction of disulfide bonds into variable domains, although disulfide bonds may contribute to the correct refolding of proteins, it also introduces rigidity into the variable domains. Such rigidity may reduce the affinity of antibodies to antigens. In addition, not all variable domains can support the introduction of cysteine ​​residues necessary for disulfide bond formation without loss of affinity or introduction of immunogenic epitopes. In addition, the formation of disulfide bonds at high protein concentrations may lead to protein aggregation, thereby offsetting any potential positive effects of disulfide bonds.

[0013] However, it has been shown that the reduction of aggregation tendency is accompanied by an increase in expression titer, showing that reducing protein aggregation is beneficial throughout the development process and can lead to a more efficient clinical research approach. For therapeutic proteins, aggregates are an important risk factor for harmful immune responses in patients and can be formed by a variety of mechanisms. Controlling aggregation can improve protein stability, manufacturability, loss rate, safety, formulation, titer, immunogenicity and solubility (Wei Li et al., 2016; Vander Kant R. et al., 2017).

[0014] Other intrinsic properties of proteins, such as hydrophobicity, also play an important role in antibody solubility. Low solubility of these therapeutic proteins due to surface hydrophobicity has been shown to make formulation development more difficult and may lead to poor biodistribution, undesirable pharmacokinetic behavior and in vivo immunogenicity. Therefore, reducing the overall surface hydrophobicity of a candidate monoclonal antibody may provide benefits and cost savings associated with purification and dosing regimens.

[0015] It is believed that the anticancer effect involves multiple mechanisms. Specifically, Fcγ receptor (FcγR) IIIA dependence and FcγRIIA dependence effector functions may be responsible for the two main key mechanisms of the clinical efficacy of therapeutic antibodies (Ab). It is known that FcγRIIIA is the main triggering receptor for antibody-dependent cell-mediated cytotoxicity (ADCC) in natural killer (NK) cells, and it is known that FcγRIIA is the main triggering receptor for antibody-dependent cellular phagocytosis (ADCP) in macrophages. Complement-dependent cytotoxicity (CDC), another effector function of antibodies, is also considered to be a possible anti-tumor mechanism. Most therapeutic antibodies that have been licensed and developed as medical agents are of human IgG1 isotype, which can induce ADCC and CDC. These effector functions are activated by Fc interacting with FcγR or complement, and the interaction is affected by the N-connected bi-branched complex-type oligosaccharides (N-linked biantennary complex-typeoligosaccharide) attached to the Fc region of the antibody (Natsume et al., 2009). Several in vitro and in vivo studies have demonstrated that antibody-dependent cell-mediated cytotoxicity (ADCC) is the main mode of action against cancer cells. It has been shown that the anti-tumor effects of these antibodies require direct interaction between Ab and the receptor (FcR) for the Fc region of the antibody. Therefore, the immune effector cells carrying FcR play an important role in mediating its effect (Ianello A. and Ahmad A., 2005). However, MIF antibodies known in the art neither induce complement-dependent cell lysis of MIF-responsive cells nor induce antibody-dependent cellular toxicity (Hussain et al., 2013).

[0016] US 2011 / 236375A1 describes Fc variants with enhanced effector function.

[0017] The combination of reduced aggregation potential and reduced hydrophobicity will result in very favorable antibody properties.There is a need in the art for anti-oxMIF antibodies with reduced hydrophobicity that resist aggregation, and also for anti-oxMIF antibodies with enhanced effector function, a need that has not been met by current antibody modifications. Summary of the invention

[0018] The object of the present invention is to provide improved antibodies or antigen-binding fragments thereof targeting oxMIF with reduced aggregation tendency and hydrophobicity.

[0019] This object is achieved by the subject matter of the present invention.

[0020] The present invention discloses a recombinant anti-oxMIF antibody or an antigen-binding fragment thereof with reduced aggregation potential and reduced hydrophobicity, which comprises the following variable domains:

[0021] (a1) a light chain variable domain comprising SEQ ID NO: 9 and at least one of the amino acid substitutions M30L, F49Y, A51G, P80S, W93F, or

[0022] (a2) comprising SEQ ID NO:9 having 1, 2, 3, 4 or 5 amino acid substitutions, further comprising a light chain variable domain comprising a conserved tyrosine at position 36 and at least one of the amino acid substitutions M30L, F49Y, A51G, P80S, W93F; and one of the following:

[0023] (b1) a heavy chain variable domain comprising SEQ ID NO: 6; or

[0024] (b2) a heavy chain variable domain comprising SEQ ID NO: 6 and amino acid substitutions L5Q and / or W97Y, or

[0025] (b3) a heavy chain variable domain comprising SEQ ID NO: 6 having 1, 2, 3, 4 or 5 amino acid substitutions and amino acid substitutions L5Q and / or W97Y; wherein the amino acid positions are numbered according to Kabat, wherein the aggregation potential and hydrophobicity are reduced compared to an antibody or antigen-binding fragment thereof comprising SEQ ID NO: 6 and SEQ ID NO: 9 lacking said amino acid substitutions.

[0026] Also disclosed herein is a recombinant anti-oxMIF antibody or antigen-binding fragment thereof with reduced aggregation potential and reduced hydrophobicity, comprising the following variable domains:

[0027] (a) a light chain variable domain comprising SEQ ID NO:9, or a light chain variable domain comprising a tyrosine at position 36 having at least 95% sequence identity to SEQ ID NO:9, and

[0028] (b) a heavy chain variable domain comprising SEQ ID NO:6 and amino acid substitutions W97Y and / or L5Q, or a heavy chain variable domain comprising SEQ ID NO:6 having 1, 2, 3, 4 or 5 amino acid substitutions and further comprising amino acid substitutions W97Y and / or L5Q, wherein the amino acid positions are numbered according to Kabat, wherein the aggregation potential and hydrophobicity are reduced compared to an antibody or antigen-binding fragment thereof comprising SEQ ID NO:6 and SEQ ID NO:9 lacking said amino acid substitutions.

[0029] Specifically, with reference to SEQ ID NO:9, the recombinant anti-oxMIF antibody comprises the amino acid substitution W93F.

[0030] Specifically, with reference to SEQ ID NO:6, the recombinant anti-oxMIF antibody comprises the amino acid substitution W97Y.

[0031] According to yet another specific embodiment, the recombinant anti-oxMIF antibody comprises the amino acid substitutions W93F and W97Y.

[0032] Surprisingly, it has been shown that even selected positions within the CDR domains can be substituted to reduce aggregation potential and / or hydrophobicity without any negative impact on oxMIF binding affinity.

[0033] Specifically, the light chain variable domain comprises 1, 2, 3, 4 or all of the amino acid substitutions M30L, F49Y, A51G, P80S, and W93F.

[0034] Specifically, the recombinant anti-oxMIF antibody comprises the constant light chain (CL) domain of SEQ ID NO:38.

[0035] According to a specific embodiment, the recombinant anti-oxMIF antibody described herein further has enhanced effector function and comprises a heavy chain constant region comprising:

[0036] (a) SEQ ID NO: 2 or a heavy chain constant region having at least 95% sequence identity to SEQ ID NO: 2 and comprising amino acids 239D and 332E; or

[0037] (b) SEQ ID NO:3 or a heavy chain constant region having at least 95% sequence identity to SEQ ID NO:3 and comprising amino acids 239D, 268F, 324T and 332E; or

[0038] (c) SEQ ID NO:4 or a heavy chain constant region having at least 95% sequence identity to SEQ ID NO:4 and comprising amino acids 239D and 332E; or

[0039] (d) SEQ ID NO:5 or a heavy chain constant region having at least 95% sequence identity to SEQ ID NO:5 and comprising amino acids 235V, 243L, 292P, 300L and 396L,

[0040] The amino acid positions are numbered according to the EU numbering index.

[0041] Specifically, the anti-oxMIF antibodies described herein comprise a VH or VL domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 8, 10, 11, 12 and 13.

[0042] According to an alternative embodiment, the anti-oxMIF antibody comprises SEQ ID NO:6, 11 and any one of SEQ ID NO:1, 2, 3, 4 or 5.

[0043] In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NO:7, 10 and any one of SEQ ID NO:1, 2, 3, 4 or 5.

[0044] In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NO:7, 11 and any one of SEQ ID NO:1, 2, 3, 4 or 5.

[0045] In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NO:8, 10 and any one of SEQ ID NO:1, 2, 3, 4 or 5.

[0046] In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NO:8, 11 and any one of SEQ ID NO:1, 2, 3, 4 or 5, or SEQ ID NO:39, or SEQ ID NO:40.

[0047] In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NO:8, 13 and any one of SEQ ID NO:1, 2, 3, 4 or 5, or SEQ ID NO:41, or SEQ ID NO:42.

[0048] Further provided herein is an anti-oxMIF antibody according to any one of claims 1 to 3, comprising SEQ ID NO: 39, 40, 41 or 42, or the anti-oxMIF antibody comprises SEQ ID NO: 39, 44, 41 or 45.

[0049] More specifically, the recombinant anti-oxMIF antibody or antigen-binding fragment thereof is a bispecific antibody.

[0050] In a specific embodiment, the anti-oxMIF antibody described herein is selected from monospecific and bispecific antibodies, scFv, (scFv)2, scFv-Fc, Fab, Fab', and F(ab')2, Fab'-SH, Fab-scFv fusion, Fab-(scFv)2-fusion, Fab-scFv-Fc, Fab-(scFv)2-Fc, fusion proteins of two single chain variable fragments (scFv) of different antibodies (e.g., BiTE), minibodies, DutaMab TM , DART, and

[0051] In particular, the antibodies described herein are used for the preparation of a medicament.

[0052] Specifically, provided herein is a pharmaceutical composition comprising the antibody of the present invention, optionally comprising a pharmaceutical carrier or adjuvant.

[0053] In particular, the composition comprises about 10 to 250 mg / ml of an antibody as described herein, especially >50 mg / ml.

[0054] In particular, the pharmaceutical composition is formulated for subcutaneous administration.

[0055] More specifically, the pharmaceutical composition can be used to treat oxMIF-related disorders.

[0056] In particular, the composition is intended for administration as a sole substance or in combination with another pharmaceutical composition comprising one or more active substances, preferably selected from antiviral, anti-inflammatory, anticancer, antiangiogenic and antibiotic substances.

[0057] In yet another embodiment, the pharmaceutical composition is used to treat patients suffering from inflammatory diseases, hyperproliferative disorders, infectious diseases or cancer, in particular for the treatment of asthma, vasculitis, arthritis, sepsis, septic shock, endotoxic shock, toxic shock syndrome, acquired respiratory distress syndrome, glomerulonephritis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, peritonitis, nephritis and psoriasis, colorectal cancer, ovarian cancer, breast cancer, prostate cancer, pancreatic cancer and lung cancer.

[0058] Further provided herein is an isolated nucleic acid encoding an antibody of the present invention.

[0059] Also provided herein is an expression vector comprising one or more nucleic acid molecules described herein.

[0060] In yet another embodiment, a host cell is provided, which contains the nucleic acid or expression vector described herein.

[0061] Also provided is a method of producing an antibody of the invention, comprising culturing a host cell and recovering the antibody from the cell culture.

[0062] Further provided herein is a method for producing an antibody of the present invention, comprising expressing a nucleic acid encoding the antibody in a host cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 : Amino acid sequence

[0064] Figure 2: Improved SEC (A, B) and HIC (C) profiles of the newly designed anti-oxMIF antibodies.

[0065] Figure 3 : Binding of the newly designed anti-oxMIF antibody to immobilized MIF (KD determination).

[0066] Figure 4 : Differential binding of newly designed anti-oxMIF antibodies to oxMIF and redMIF.

[0067] Figure 5 : Enhanced effector function of newly designed anti-oxMIF antibodies in a modified ADCC reporter bioassay.

[0068] Figure 6 : Tumor penetration of the newly designed anti-oxMIF antibody C0083. A: Biodistribution of C0008 (5 mg / kg / d); B: Biodistribution of C0083 (5 mg / kg / d); C: Untreated control mice.

[0069] Figure 7 : Improved production of the newly designed anti-oxMIF antibody C0083.

[0070] Figure 8 : Improved production of newly designed anti-oxMIF antibodies with enhanced effector function and reduced hydrophobicity and aggregation tendency compared to counterparts with enhanced effector function but containing the VH and VL domains of C0008 (comparison of C0097 with C0064, C0098 with C0065, and C0100 with C0067).

[0071] Fig. 9: Chromatographic profiles demonstrating reduced aggregation and hydrophobicity of the newly designed anti-oxMIF antibodies. (A) Comparison of elution profiles of C0008 (control antibody, grey area) and newly designed antibodies C0108 and C0109 with enhanced effector function on an Enrich 650 gel filtration column using 1x PBS as mobile phase; (B) Comparison of elution profiles of C0008 (control antibody, grey area) and newly designed antibodies C0083, C0090, C0108 and C0109 on a HiTrap Butyl HP HIC column.

[0072] Fig.10 :The newly designed anti-oxMIF antibody was confirmed by FACS in A2780MIF - / - Nonspecific binding to cells was reduced. - / - Cells were stained with the newly designed anti-oxMIF antibodies C0090, C0109 (A); C0083, C0108 (B) or control antibody C0008 and isotype IgG as a negative control. The geometric mean (GeoMean) of live cells (mean fluorescence intensity of AF488) was plotted against antibody concentration.

[0073] Fig.11 : Binding curves of the newly designed anti-oxMIF antibodies with enhanced effector functions to immobilized oxMIF (K D evaluation); anti-oxMIF antibody C0008 was used as a reference antibody.

[0074] Fig.12 : Differential binding of the newly designed antibodies to oxMIF and redMIF. C0008 was used as reference antibody and isotype IgG was used as negative control.

[0075] Fig.13 : Pharmacokinetics of the newly designed anti-oxMIF antibodies C0083 and C0090 compared with the control anti-oxMIF antibody C0008 in BALB / c nude mice. Plasma concentrations of the antibodies were determined by quantitative anti-oxMIF ELISA 4, 10, 24, 48 and 96 h after a single intravenous injection of 10 mg / kg of anti-oxMIF antibodies, and half-lives were calculated by a single-phase decay model in GraphPad Prism.

[0076] Fig.14: Enhanced effector function of the newly designed antibodies C0108 and C0109 as determined by reporter assays. (AB) ADCC reporter bioassays were performed using engineered Jurkat effector cells stably expressing FcyRIIIa (V high responder allotype, F low responder allotype) and HCT116-pMIF (A) or A2780-pMIF (B) target cells with newly designed antibodies C0108 and C0109 with enhanced effector function compared to anti-oxMIF control antibody C0008 with wt Fc; (C) ADCP reporter bioassays were performed using engineered Jurkat effector cells stably expressing FcyRIIa and HCT116-pMIF target cells with newly designed antibodies C0108 and C0109 with enhanced effector function compared to anti-oxMIF control antibody C0008 with wt Fc. Mean values ​​and SEM (n=2-4) are shown.

[0077] Fig.15 : ADCC activity enhancement of the newly designed anti-oxMIF antibodies C0108 and C0109 containing mutations that enhance ADCC / ADCP in the Fc portion as determined by PBMC-mediated cytotoxicity assay. ADCC bioassays using newly designed anti-oxMIF antibodies C0108 and C0109 and PBMC as effector cells (A: 22% NK cells, B: 7% NK cells) and HCT116-pMIF as target cells were performed compared to the anti-oxMIF control antibody C0008 with wt Fc. Mean values ​​and SEMs of two or three replicates using PBMCs from two healthy donors are shown.

[0078] Fig.16 :Newly designed anti-oxMIF antibody shows that the non-specific cytokine released from human PBMC is significantly reduced.Newly designed anti-oxMIF antibody C0108 and C0109 (70nM) and control anti-oxMIF antibody C0008 (70nM) containing the mutation enhancing ADCC / ADCP in the Fc part were incubated overnight with human PBMC, and the people IL-6 (A) and the people MCP-1 (B) of supernatant were analyzed in LegendPlex cell counting beads assay (BioLegend). Mean values ​​+ / -SEM of the cytokine concentration from three different PMBC donors are shown. DETAILED DESCRIPTION

[0079] Unless otherwise indicated or defined, all terms used herein have their usual meaning in the art, which will be clear to the skilled person. For example, reference is made to standard manuals, such as Molecular Cloning: A Laboratory Manual (Sambrook et al., "Molecular Cloning: A Laboratory Manual" (4th Ed.), Vols. 1-3, Cold Spring Harbor Laboratory Press (2012)); Lewin Genes XI (Krebs et al., "Lewin's Genes XI", Jones & Bartlett Learning, (2017)), and Janeway Immunobiology (Murphy & Weaver, "Janeway's Immunobiology" (9th Ed., or more recent editions), Taylor & Francis Inc, 2017).

[0080] The subject matter of the claims specifically relates to artificial products, which may be variants of natural (wild-type) products, or methods of using or producing such artificial products. Although there may be a certain degree of sequence identity with natural structures, it is understood that the materials, methods and uses of the present invention (e.g., specifically isolated nucleic acid sequences, amino acid sequences, fusion constructs, expression constructs, transformed host cells and modified proteins) are "artificial" or synthetic and therefore should not be considered to be the result of "the laws of nature".

[0081] The terms "include", "comprising", "having" and "including" as used herein may be used as synonyms and should be understood as open definitions, allowing for the presence of other components or parts or elements. "Consisting of" is considered to be the most closed definition, with no other elements other than the defining features. Therefore, "comprising" is broader and includes the definition of "consisting of".

[0082] The term "about" as used herein refers to the same value or a value that differs by + / - 5% from a given value.

[0083] As used herein and in the claims, the singular forms such as "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0084] As used herein, amino acids refer to the 20 naturally occurring amino acids encoded by 61 triplet codons. These 20 amino acids can be divided into neutrally charged, positively charged, and negatively charged amino acids:

[0085] “ neutralThe amino acids and their corresponding three-letter and 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 (Ile, I; nonpolar, neutral), Leucine (Leu, L; nonpolar, neutral), Methionine (Met, M; polar, neutral), ; 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, positive (10%), neutral (90%)).

[0086] bring" just The amino acids with the highest charge are: arginine (Arg, R; polar, positive) and lysine (Lys, K; polar, positive).

[0087] bring" burden The charged amino acids are: aspartic acid (Asp, D; polar, negative) and glutamic acid (Glu, E; polar, negative).

[0088] The antibodies or antigen-binding fragments of the present invention comprise at least one binding site that specifically recognizes oxMIF and exhibit reduced aggregation tendency and reduced hydrophobicity compared to an unmodified antibody lacking said amino acid substitutions due to targeted amino acid substitutions in the variable heavy and light chain domains.

[0089] The reduction in aggregation potential is due to amino acid substitutions at selected positions in the variable domains of the antibodies described herein.

[0090] Antibody aggregation levels can be measured using a variety of known techniques, including mass spectrometry, size exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC), dynamic light scattering (DLS), light obscuration (LO), dynamic imaging particle analysis (DIPA) techniques, such as microfluidics imaging (MFI), and Coulter counter (CC), differential scanning fluorimetry (DSF).

[0091] As used herein, reduced hydrophobicity and reduced aggregation potential refer to reduced surface hydrophobicity and reduced aggregation potential of newly designed antibodies compared to reference antibodies such as antibody C0008 (which includes SEQ ID NOs as disclosed herein: 1, 6, 38 and 9). The sequence of C0008 contains the sequence of Imalumab published in the proposed INN list 111 (WHO drug information, Vol. 28, No. 2, 2014), but lacks C-terminal lysine. Can use a variety of known techniques to measure, including but not limited to hydrophobic interaction chromatography (HIC) or affinity capture self-interaction nanoparticle spectroscopy (AC-SINS, Estep P. et al., 2015).

[0092] In one embodiment, the oxMIF antibody of the invention with reduced aggregation potential and reduced hydrophobicity specifically comprises a light chain variable domain having at least one of the amino acid substitutions M30L, F49Y, A51G, P80S, W93F according to 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:9 comprising a native tyrosine at position 36 and further comprising at least one of the amino acid substitutions M30L, F49Y, A51G, P80S, W93F; and a heavy chain variable domain comprising SEQ ID NO:6; or a heavy chain variable domain comprising SEQ ID NO:6 having the amino acid substitutions L5Q and / or W97Y according to Kabat numbering; or a heavy chain variable domain comprising SEQ ID NO:6 having 1, 2, 3, 4 or 5 amino acid substitutions and further comprising at least one of the amino acid substitutions L5Q or W97Y.

[0093] In an alternative embodiment, the light chain variable domain comprising SEQ ID NO: 9 has 1, 2, 3, 4, 5 amino acid substitutions, provided that the native tyrosine at position 36 is retained and at least one amino acid is further substituted at positions M30, F49, A51, P80, W93.

[0094] In particular, amino acid substitutions at positions W93 and W97 are preferred.

[0095] Furthermore, amino acid substitution at position F49 is preferred.

[0096] Further described herein are anti-oxMIF antibodies with reduced aggregation potential and reduced hydrophobicity, which specifically comprise: a light chain variable domain comprising SEQ ID NO: 9, or a light chain variable domain comprising a tyrosine at position 36 having at least 95% sequence identity to SEQ ID NO: 9; and a heavy chain variable domain comprising SEQ ID NO: 6 and amino acid substitution W97Y or amino acid substitutions L5Q and W97Y, or a heavy chain variable domain comprising SEQ ID NO: 6 having 1, 2, 3, 4 or 5 amino acid substitutions and further comprising amino acid substitution W97Y (optionally comprising L5Q).

[0097] The native tyrosine at position 36 of the light chain remains unmodified to preserve the binding properties of the antibodies described herein. Any modification at the amino acid position may result in undesirably impaired binding properties.

[0098] Specifically, the variable light chain domain comprises SEQ ID NO:10, and the variable heavy chain domain comprises SEQ ID NO:7.

[0099] Specifically, the variable light chain domain comprises SEQ ID NO:10, and the variable heavy chain domain comprises SEQ ID NO:8.

[0100] Specifically, the variable light chain domain comprises SEQ ID NO:11, and the variable heavy chain domain has SEQ ID NO:6.

[0101] Specifically, the variable light chain domain comprises SEQ ID NO:11, and the variable heavy chain domain comprises SEQ ID NO:7.

[0102] Specifically, the variable light chain domain comprises SEQ ID NO:11, and the variable heavy chain domain comprises SEQ ID NO:8.

[0103] Specifically, the variable light chain domain comprises SEQ ID NO:12, and the variable heavy chain domain has SEQ ID NO:6.

[0104] Specifically, the variable light chain domain comprises SEQ ID NO:12, and the variable heavy chain domain comprises SEQ ID NO:8.

[0105] Specifically, the variable light chain domain comprises SEQ ID NO:13, and the variable heavy chain domain has SEQ ID NO:6.

[0106] Specifically, the variable light chain domain comprises SEQ ID NO:13, and the variable heavy chain domain comprises SEQ ID NO:8.

[0107] As used herein, "effector function" refers to the biochemical events resulting from the interaction of an antibody Fc region with an Fc receptor or ligand. Effector functions include, but are not limited to, ADCC, ADCP, and CDC.

[0108] As used herein, "effector cell" means an immune system cell that expresses one or more Fc receptors and mediates one or more effector functions. Effector cells include, but are not limited to, monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, and T cells, and may be from any organism, including, but not limited to, humans, mice, rats, rabbits, and monkeys. In a specific embodiment, the anti-oxMIF antibodies described herein also have enhanced effector functions due to amino acid substitutions at selected positions in the heavy chain constant region. The effector functions of these antibodies that are enhanced due to enhanced complement-mediated and FcR-mediated activity may include enhanced complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and / or antibody-dependent cellular phagocytosis (ADCP). Fc receptor-mediated ADCC is an important mechanism of action by which antibodies target diseased cells for removal. When the Fc effector portion of the target-bound antibody also binds to the FcγRIIIA receptor on the cell surface of the effector cell, multiple cross-linking of the two cell types occurs, leading to activation of the ADCC pathway.

[0109] Antibodies with enhanced complement activity can be determined by cell-based CDC assays and improved binding to CIq by, for example, SPR or ELISA.

[0110] Compared to a reference (i.e., an unmodified wild-type antibody, such as C0008), the improved or increased CDC activity is determined to be at least 1.5-fold, specifically at least 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, more specifically at least 5-fold, and more specifically at least 6-fold increase.

[0111] Compared to a reference antibody (i.e., an unmodified wild-type antibody, such as C0008), the increased ADCC activity is determined to be at least 1.5-fold, specifically at least 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, more specifically at least 5-fold, and more specifically at least 6-fold increased potency.

[0112] In addition to the above-mentioned light chain and heavy chain variable domains, the oxMIF antibody may further comprise: a heavy chain constant region comprising SEQ ID NO: 1 having at least amino acid substitutions S239D and I332E according to the EU numbering index, or a functional variant having at least 95%, specifically at least 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1 and comprising at least amino acid substitutions S239D, I332E and optionally further comprising amino acid substitutions H268F and / or S324T.

[0113] Specifically, the oxMIF-antibody contains a heavy chain constant region comprising SEQ ID NO: 2, or a heavy chain constant region having at least 95% sequence identity to SEQ ID NO: 2 and comprising amino acids 239D and 332E.

[0114] Alternatively, the oxMIF-antibody contains a heavy chain constant region comprising SEQ ID NO: 3, or a heavy chain constant region having at least 95% sequence identity to SEQ ID NO: 3 and comprising amino acids 239D, 268F, 324T and 332E.

[0115] Alternatively, the oxMIF antibody may further contain: a heavy chain constant region comprising SEQ ID NO: 4, or a heavy chain constant region having at least 95% sequence identity to SEQ ID NO: 4 and comprising amino acids 239D and 332E.

[0116] In yet another alternative embodiment, the anti-oxMIF antibody may further comprise: a heavy chain constant region of SEQ ID NO:5, or a heavy chain constant region having at least 95% sequence identity to SEQ ID NO:5 and comprising amino acids 235V, 243L, 292P, 300L and 396L.

[0117] In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 1, 7, 10, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 2, 7, 10, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 3, 7, 10, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 4, 7, 10, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 5, 7, 10, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 1, 8, 10, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 2, 8, 10, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 3, 8, 10, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 4, 8, 10, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 5, 8, 10, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 1, 6, 11, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 2, 6, 11, and 38.

[0118] In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 3, 6, 11, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 4, 6, 11, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 5, 6, 11, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 1, 7, 11, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 2, 7, 11, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 3, 7, 11, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 4, 7, 11, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 5, 7, 11, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 1, 8, 11, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 2, 8, 11, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 3, 8, 11, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 4, 8, 11, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 5, 8, 11, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 1, 6, 12, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 2, 6, 12, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 3, 6, 12, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 4, 6, 12, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 5, 6, 12, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 1, 7, 12, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 2, 7, 12, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 3, 7, 12, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 4, 7, 12, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 5, 7, 12, and 38.In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 1, 8, 12, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 2, 8, 12, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 3, 8, 12, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 4, 8, 12, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 5, 8, 12, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 1, 6, 13, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 2, 6, 13, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 3, 6, 13, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 4, 6, 13, and 150. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 5, 6, 13, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 1, 7, 13, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 2, 7, 13, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 3, 7, 13, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 4, 7, 13, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 5, 7, 13, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 1, 8, 13, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 2, 8, 13, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 3, 8, 13, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 4, 8, 13, and 38. In yet another embodiment, the anti-oxMIF antibody comprises SEQ ID NOs: 5, 8, 13, and 38.

[0119] The oxMIF binding sites of the antibodies described herein are specific for oxidized forms of MIF, i.e., for animal oxMIF, particularly for mammalian oxMIF, such as, but not limited to, mouse, rat, monkey and human, particularly for human oxMIF, but do not show significant cross-reactivity to reduced MIF. oxMIF is a disease-associated structural isomer of MIF that can be specifically and predominantly detected in the circulation of subjects with inflammatory diseases and in tumor tissues of cancer patients. In one embodiment, the humanized or human anti-oxMIF binding site comprises one or more (e.g., all three) light chain complementary determining regions of a humanized or human anti-oxMIF binding domain described herein, such as, for example, the CDRs contained in SEQ ID NO: 9, 10, 11, 12 or 13, and / or one or more (e.g., all three) heavy chain complementary determining regions of a humanized or human anti-oxMIF binding domain described herein, such as, for example, SEQ ID NO: 6, 7 or 8.

[0120] oxMIF binding specificity can be determined by any assay suitable for determining selective binding to oxMIF, such as any competition assay for binding to oxMIF against a control antibody (such as imalutumab), or various assays known in the art and as disclosed herein.

[0121] The term "antibody" is used in the broadest sense herein and encompasses polypeptides or proteins consisting of or comprising antibody domains, which are understood to be constant and / or variable domains of immunoglobulin heavy and / or light chains with or without linker sequences. The term encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen binding activity, i.e., binding to oxMIF. The term also encompasses fusion proteins, such as fusions with immunotoxins or antibody conjugates, such as antibody drug conjugates bound to oxMIF.

[0122] The antibody domain may be of native structure or modified by mutagenesis or derivatization, e.g. to improve antigen binding properties or any other property, such as stability or functional properties, such as binding to an Fc receptor, e.g. FcRn and / or Fc-γ receptor. A polypeptide sequence is considered to be an antibody domain if it comprises a β-barrel structure consisting of at least two β strands of the antibody domain structure connected by a loop sequence.

[0123] It should be understood that the term "antibody" includes antigen-binding derivatives and fragments thereof. A derivative is any combination and / or fusion protein of one or more antibody domains or antibodies of the invention in which any domain of an antibody of the invention can be fused at any position of one or more other proteins (e.g., other antibodies or antibody forms, e.g., binding structures comprising CDR loops, receptor polypeptides, but also ligands, scaffold proteins, enzymes, markers, toxins, etc.).

[0124] The term "antibody" shall particularly refer to a polypeptide or protein that exhibits binding properties to the target antigen oxMIF.

[0125] "Antibody fragment" refers to a molecule other than an intact antibody, which comprises an antigen-binding portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, single-chain antibody molecules (e.g., scFv), Fab-scFv fusions, Fab-(scFv)2, Fab-scFv-Fc, Fab-(scFv)2-Fc, F(ab')2, ScFv-Fc, diabodies, cross-Fab fragments; linear antibodies; and multispecific antibodies formed from antibody fragments. In addition, antibody fragments comprise single-chain polypeptides having the characteristics of a VH domain, i.e., being able to assemble with a VL domain, or having the characteristics of a VL domain, i.e., being able to assemble with a VH domain into a functional antigen-binding site, and thereby providing the antigen-binding properties of a full-length antibody. Antibody fragments as referred to herein also encompass Fc domains comprising one or more structural loop regions containing an antigen-binding region, such as Fcab TM or a full-length antibody format having an IgG structure in which the Fc region has been replaced by an Fcab containing a second, different antigen-binding site TM Alternative.

[0126] The term "functional variant" or "functionally active variant" also includes naturally occurring allelic variants and mutants or any other non-naturally occurring variants. As known in the art, allelic variants, or also referred to as homologs, are alternative forms of nucleic acids or peptides, characterized by substitutions, deletions or additions of one or more nucleotides or amino acids that do not substantially alter the biological function of the nucleic acid or polypeptide. Specifically, functional variants may comprise substitutions, deletions and / or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid residues, or a combination thereof, wherein the substitutions, deletions and / or additions are conservative modifications and do not alter the antigen binding properties. Specifically, the functional variants as described herein comprise no more than or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid substitutions, deletions and / or additions, which are conservative modifications and do not change the function of the antibody. Specifically, the functionally active variants as described herein comprise at most 15, preferably at most 10 or 5 amino acid substitutions, deletions and / or additions, which are conservative modifications and do not change the function of the antibody.

[0127] Functional variants can be obtained by sequence changes in polypeptide or nucleotide sequences, for example, by one or more point mutations, wherein the sequence changes retain or improve the function of the unchanged polypeptide or nucleotide sequence when used in the combination of the present invention. Such sequence changes may include, but are not limited to, (conservative) substitutions, additions, deletions, mutations and insertions. Conservative substitutions are substitutions that occur in a family of amino acids that are related in their side chains and chemical properties. Examples of such families are amino acids with basic side chains, acidic side chains, non-polar aliphatic side chains, non-polar aromatic side chains, uncharged polar side chains, small side chains, large side chains, etc.

[0128] Point mutations are specifically understood as engineering of a polynucleotide that results in the expression of an amino acid sequence that differs from the non-engineered amino acid sequence by one or more single (non-consecutive) or paired amino acid substitutions or exchanges, deletions or insertions of different amino acids.

[0129] As used herein, the antibody fragment referred to by "Fab fragment or Fab" comprises a light chain fragment containing the VL domain and the constant domain of the light chain (CL) and the VH domain and the first constant domain (CH1) of the heavy chain. The antibody of the present invention may comprise at least one Fab fragment in which the variable regions or constant regions of the heavy and light chains are exchanged. Due to the exchange of the variable regions or constant regions, the Fab fragment is also referred to as a "cross-Fab fragment" or a "crossover Fab fragment". The crossover Fab molecule may consist of two different chains and is included in the antibody of the present invention: the variable regions of the Fab heavy and light chains may be exchanged, i.e., the crossover Fab molecule comprises a peptide chain consisting of a light chain variable region (VL) and a heavy chain constant region (CH1), and a peptide chain consisting of a heavy chain variable region (VH) and a light chain constant region (CL). This crossover Fab molecule is also referred to as CrossFab (VLVH). When the constant regions of the Fab heavy chain and light chain are exchanged, the crossover Fab molecule can contain a peptide chain consisting of the heavy chain variable region (VH) and the light chain constant region (CL), and a peptide chain consisting of the light chain variable region (VL) and the heavy chain constant region (CH1). This crossover Fab molecule is also called CrossFab (CLCH1).

[0130] "(scFv)2" refers to an artificial monoclonal antibody that is a fusion protein consisting of two single-chain variable fragments (scFv) of different antibodies or the same antibody, or amino acid sequences from four or two different genes on a single peptide chain of about 50 kilodaltons.

[0131] "bs(scFv)2" refers to an artificial monoclonal antibody that is a fusion protein consisting of two single-chain variable fragments (scFv) of antibodies with different target antigens, or amino acid sequences from four different genes on a single peptide chain of about 50 kilodaltons.

[0132] "Single-chain Fab fragment" or "scFab" is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein the antibody domains and the linker may have the following order in the N-terminal to C-terminal direction: VH-CH1-linker-VL-CL, VL-CL-linker-VH-CH1, VH-CL-linker-VL-CH1 or VL-CH1-linker-VH-CL; wherein the linker is a polypeptide of at least 20 amino acids, at least 30 amino acids, in particular 32 to 50 amino acids. The single-chain Fab fragments VH-CH1-linker-VL-CL, VL-CL-linker-VH-CH1, VH-CL-linker-VL-CH1 and VL-CH1-linker-VH-CL may be stabilized by a natural disulfide bond between the CL domain and the CH1 domain. Additionally, these single-chain Fab molecules can be further stabilized by creating interchain disulfide bonds via the insertion of cysteine ​​residues.

[0133] The term "N-terminus" refers to the last amino acid at the N-terminus.

[0134] The term "C-terminus" refers to the last amino acid at the C-terminus.

[0135] "BiTE" for "bispecific T cell engager" refers to an artificial monoclonal antibody that is a fusion protein consisting of two single-chain variable fragments (scFv) of different antibodies, or amino acid sequences from four different genes on a single-chain peptide chain of about 50 kilodaltons. One of the scFvs can, for example but not limited to, bind to T cells (e.g., via CD3) and the other binds to tumor cells via oxMIF. Specifically, BiTE is about 50 kDa.

[0136] The term "mini-antibody" refers to an antibody consisting of a pair of single-chain Fv fragments (single-chain Fv-CH3) connected via a CH3 domain and Fvs with different specificities paired with the previous part through a heterodimerization process (Hu SZ et al., 1996, Cancer Research, 56, 3055-3061). In order to promote heterodimerization efficiency, single residue mutations can be introduced into each CH3 domain to achieve the "knobs-into-hole" method. Not only that, additional cysteine ​​residues can also be introduced into the CH3 domain to stabilize the bispecific mini-antibody structure. TriBi mini-antibody (a mini-antibody format) includes chains designed to recognize antigens via its two Fv fragments, while another chain with an Fv fragment is responsible for recruiting effector cells, such as T cytotoxic cells or NK cells. Due to the addition of this additional binding domain, the affinity of TriBi mini-antibody is higher than that of bispecific mini-antibody. Specifically, mini-antibody is about 75kDa.

[0137] the term Refers to dual affinity retargeting antibodies, which are the simplest form of bispecific antibodies (BsAb). DART molecules consist of two engineered Fv fragments, the VH of which is exchanged with the other. Fv1 consists of VH from antibody A and VL from antibody B, while Fv2 consists of VH from Ab-B and VL from Ab-A. This mutual exchange of Fv domains releases the variant fragments from conformational constraints through a short connecting peptide.

[0138] The term "DutaMab TM ” refers to a BsAb format that is formed by the ligation of two independent paratopes in a single immunoglobulin chain.

[0139] the term Or "tandem antibody" refers to an antibody with two pairs of VL / VH from two different Fvs in series, which also means that the tandem antibody does not carry an Fc domain. TandAb is smaller than a complete IgG or IgG-derived bispecific Ab, but larger than a single-domain bispecific Ab. The moderate size also gives TandAb increased tissue penetration and a longer serum half-life. In addition, the tetravalent property (which means that each antigen is divalent) can improve its binding efficiency and subsequent therapeutic effect. Specifically, TandAb is about 100kDa.

[0140] The term "diabody" refers to a non-covalent dimer of a single-chain Fv (scFv) fragment, which is composed of a heavy chain variable (VH) and a light chain variable (VL) region connected by a small peptide linker. Another form of a diabody is a single chain (Fv) 2, in which two scFv fragments are covalently linked to each other. In addition, by connecting the genes in each chain in series with an internal linker, four VH and VL domains can be expressed in series and folded into a single-chain diabody (scDb), which is also an effective strategy for producing bispecific antibodies. In addition, the valence of the final product can be doubled by fusing the recombinant variable domain to the Fc region or the CH3 domain (scDb-Fc and scDb-CH3, diabody-CH3). The increased size can also extend the half-life of the diabody in serum. Specifically, diabody-CH3 is about 125kDa.

[0141] the term (where Mab refers to monoclonal antibody) is a bispecific Ab format derived from an independent parent antibody. Heavy chain mispairing is avoided by applying the knob-in-hole (KIH) method. Light chain mispairing is avoided because bispecific antibodies are produced by antibody domain exchange, and the variable domain or constant domain (CL and CH1) of one Fab arm is exchanged between the light chain and the heavy chain. This "crossing" maintains antigen binding affinity and also retains two different arms, thereby avoiding light chain mispairing. Examples of CrossMab can be, but are not limited to, Fab, VH-VL, and CH1-CL exchanged in different regions. In CrossMAb Fab, the complete VH-CH1 and VL-CL regions are exchanged; in the CrossMAb VH-VL format, only the VH and VL regions are exchanged; in the CrossMAb CH1-CL1 format, the CH1 and CL regions of the bispecific antibody are exchanged. Specifically, CrossMab is about 150kDa.

[0142] The term "IgG-scFv" refers to a bispecific antibody that is engineered to be bispecific by fusing two scFvs to a monospecific IgG, respectively. The specificity of each scFv can be the same or different. In addition, a pair of antibody variable domains can be attached to the amino or C-terminus of each light or heavy chain, which leads to the generation of diverse types of IgG-scFv BsAbs: IgG(H)-scFv or scFv-(H)IgG: IgG(H)-scFv, two scFvs with the same specificity attached to the C-terminus of the full-length IgG HC; scFv-(H)IgG, similar to IgG(H)-scFv, except that the scFv is attached to the HC N-terminus. IgG(L)-scFv or scFv-(L)IgG: two identical scFvs attached to the C-terminus or N-terminus of the IgG light chain, forming IgG(L)-scFv or scFv-(L)IgG, respectively. 2scFv-IgG or IgG-2scFv: Produced by fusing two pairs of scFvs with different specificities to the N-terminus (2scFv-IgG) or C-terminus (IgG-2scFv). Specifically, IgG(H)-scFv is about 200 kDa.

[0143] According to a specific embodiment, the antibodies described herein may comprise one or more tags for purification and / or detection, such as but not limited to affinity tags, solubility enhancing tags and monitoring tags.

[0144] Specifically, the affinity tag is selected from a polyhistidine tag, a polyarginine tag, a peptide substrate of an antibody, a chitin binding domain, an RNAse S peptide, protein A, β-galactosidase, a FLAG tag, a Strep II tag, a streptavidin binding peptide (SBP) tag, a calmodulin binding peptide (CBP), glutathione S-transferase (GST), maltose binding protein (MBP), an S-tag, an HA tag, and a c-Myc tag, specifically, the tag is a His tag comprising one or more Hs, such as a hexahistidine tag.

[0145] By "fused" or "linked" is meant that the components (eg, a Fab molecule and an Fc domain subunit) are linked by peptide bonds directly or via one or more peptide linkers.

[0146] The term "linker" as used herein refers to a peptide linker, and preferably a peptide of an amino acid sequence of 2, 3, 4, 5, 6, 7 or more amino acids in length, preferably 2-10 in length, more preferably 3-5 amino acids in length.

[0147] The term "immunoglobulin" refers to a protein with the structure of a naturally occurring antibody. For example, the immunoglobulin of the IgG type is a heterotetrameric glycoprotein of about 150,000 daltons consisting of two light chains and two heavy chains bound by disulfide bonds. From N-terminal to C-terminal, each heavy chain has a variable region (VH), also referred to as a variable heavy chain domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3), also referred to as a heavy chain constant region. Similarly, from N-terminal to C-terminal, each light chain has a variable region (VL), also referred to as a variable light chain domain or a light chain variable domain, followed by a constant light (CL) domain, also referred to as a light chain constant region. The immunoglobulin of the IgG type is essentially composed of two Fab molecules and an Fc domain connected by an immunoglobulin hinge region. The heavy chains of immunoglobulins can be assigned to one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which can be further divided into subtypes, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chains of immunoglobulins can be assigned to one of two types, called kappa (κ) and lambda (λ).

[0148] The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain is derived from a specific source or species, while the remainder of the heavy chain and / or light chain is derived from a different source or species, typically prepared by recombinant DNA technology. Chimeric antibodies may comprise a rabbit or mouse variable region and a human constant region. Chimeric antibodies are products of immunoglobulin genes expressing DNA segments encoding immunoglobulin variable regions and DNA segments encoding immunoglobulin constant regions. Methods for producing chimeric antibodies involve conventional recombinant DNA, and gene transfection techniques are well known in the art (Morrison, SL, et al., 1984).

[0149] "Human antibody" has an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell or derived from an amino acid sequence of an antibody derived from a non-human source utilizing a human antibody library or other human antibody encoding sequences. This definition of human antibody explicitly excludes humanized antibodies comprising non-human antigen binding residues. Also as mentioned for chimeric and humanized antibodies, the term "human antibody" as used herein also includes such antibodies modified in the constant region, for example, by "type switching", i.e., changes or mutations (e.g., IgG1 to IgG4 and / or IgG1 / IgG4 mutations) of the Fc portion.

[0150] The term "recombinant human antibody" as used herein is intended to include all human antibodies prepared, expressed, produced or isolated by recombinant means, such as antibodies isolated from host cells (e.g., HEK cells, NS0 or CHO cells) or from animals (e.g., mice) transgenic for human immunoglobulin genes, or antibodies expressed using recombinant expression vectors transfected into host cells. The amino acid sequences of the VH and VL regions of the recombinant antibodies, although derived from and related to human germline sequences, may not naturally exist in the human antibody repertoire in vivo.

[0151] A "human consensus framework" is a framework that represents the most frequently occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subset of variable domain sequences. Typically, the subset of sequences is a subset as described in Kabat et al., 1991.

[0152] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human framework regions (FRs) that have undergone humanization. In certain embodiments, a humanized antibody will comprise substantially all of at least one and typically two variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to the HVRs of a non-human antibody, and all or substantially all of the FRs correspond to the FRs of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. Other forms of humanized antibodies encompassed by the present invention include those in which additional modifications or changes to the constant region have been made from that of the original antibody to produce new properties, such as in terms of C1q binding and / or Fc receptor (FcR) binding.

[0153] As used herein, the term "bispecific" shall refer to a binding reaction with at least the oxMIF antigen and another antigen (such as, but not limited to, the CD3 antigen). A bispecific antibody may specifically comprise at least two sites with specific binding properties, wherein the antibody recognizes two different target antigens, oxMIF and another antigen. An exemplary bispecific antibody format may comprise two binding sites, wherein each binding site is capable of specific binding to a different antigen, such as CD3 and oxMIF. Another exemplary bispecific format may comprise more than two binding sites, wherein one or more binding sites bind to oxMIF and one or more binding sites are capable of specific binding to one or more different antigens, such as CD3.

[0154] "Bispecific antibodies" according to the present invention are antibodies with two different binding specificities. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments, as described herein. Immunoglobulin Fc heterodimers can be engineered by modification of the CH3 domain interface, wherein there are different mutations on each domain, so that the engineered Fc fragment carrying the CH3 variant pair preferentially forms heterodimers rather than homodimers (Ha JH. et al., 2016). Examples of bispecific antibody formats can be, but are not limited to, bispecific IgG (BsIgG), IgG with additional antigen binding moieties attached, BsAb fragments, bispecific fusion proteins, BsAb conjugates, hybrid bsIgG, variable domain-only bispecific antibody molecules, CH1 / CL fusion proteins, Fab fusion proteins, modified Fc and CH3 fusion proteins, HC fusions with IgG attached, LC fusions with IgG attached, HC and LC fusions with IgG attached, Fc fusions, CH3 fusions, IgE / IgM CH2 fusions, F(ab')2 fusions, CH1 / CL, modified IgG, non-immunoglobulin fusion proteins, Fc-modified IgG, diabodies, etc., as described in Spiess C. et al., 2015 and Brinkmann U. and Kontermann RE, 2017.

[0155] The oxMIF antibodies of the present invention may further comprise at least one binding site that specifically recognizes another epitope, e.g., an epitope of CD3, particularly an epitope of human CD3, including the CD3γ (γ) chain, CD3δ (δ) chain, and two CD3ε (ε) chains present on the cell surface. Clustering of CD3 on T cells, e.g., by immobilized anti-CD3 antibodies, results in T cell activation, similar to engagement of T cell receptors, but independent of their clone-typical specificity. In certain embodiments, the CD3 binding domain of the antibodies described herein not only exhibits effective CD3 binding affinity to human CD3, but also exhibits excellent cross-reactivity with the corresponding cynomolgus monkey CD3 protein. In some cases, the CD3 binding domain of the antibody cross-reacts with CD3 from cynomolgus monkeys. In one embodiment, the anti-CD3 binding site comprises one or more (e.g., all three) light chain complementary determining regions of an anti-CD3 binding domain described herein and / or one or more (e.g., all three) heavy chain complementary determining regions of an anti-CD3 binding domain described herein, e.g., the anti-CD3 binding domain comprises one or more (e.g., all three) LC CDRs and one or more (e.g., all three) HC CDRs.

[0156] In particular, the anti-oxMIF antibody comprises a second or further binding specificity for CD3, as described in WO2019234241A1. Such a bispecific anti-oxMIF antibody may comprise a binding site that specifically recognizes CD3, comprising:

[0157] (a) a heavy chain variable region, the heavy chain variable region comprising:

[0158] A CDR1-H2 sequence comprising any one of RYTMH (SEQ ID NO: 14), GYGMH (SEQ ID NO: 15) and SFPMA (SEQ ID NO: 16) or having at least 70%, in particular 80%, 90%, 95% or 99% sequence identity thereto, and

[0159] CDR2-H2, which comprises any one of the sequences selected from YINPSRGYTNYNQKFKD (SEQ ID NO: 17), VIWYDGSKKYYVDSVKG (SEQ ID NO: 18) and TISTSGGRTYYRDSVKG (SEQ ID NO: 19) or has at least 70%, in particular 80%, 90%, 95% or 99% sequence identity thereto, and

[0160] CDR3-H2 having at least 70%, in particular 80%, 90%, 95% or 99% sequence identity with any one of the sequences selected from YYDDHYCLDY (SEQ ID NO: 20), QMGYWHFDL (SEQ ID NO: 21), FRQYSGGFDY (SEQ ID NO: 22) and YYDDHYSLDY (SEQ ID NO: 23), and

[0161] (b) a light chain variable region, the light chain variable region comprising:

[0162] CDR1-L2 comprises any one of the sequences selected from RASSSVSYMN (SEQ ID NO: 24), SASSVSYMN (SEQ ID NO: 25), RASQSVSSYLA (SEQ ID NO: 26) and TLSSGNIENNYVH (SEQ ID NO: 27) or has at least 70%, in particular 80%, 90%, 95% or 99% sequence identity thereto, and

[0163] CDR2-L2, which comprises any one of the sequences selected from DTSKVAS (SEQ ID NO: 28), DTSKLAS (SEQ ID NO: NO 29), DASNRAT (SEQ ID NO: 30) and DDDKRPD (SEQ ID NO: 31) or has at least 70%, in particular 80%, 90%, 95% or 99% sequence identity thereto, and

[0164] CDR3-L2, which comprises any one sequence selected from QQWSSNPLT (SEQ ID NO:32), QQWSSNPFT (SEQ ID NO:33), QQRSNWPPLT (SEQ ID NO:34), SQWLYGMDV (SEQ ID NO:35) and QQWSSNP (SEQ ID NO:36) or has at least 70%, in particular 80%, 90%, 95% or 99% sequence identity thereto.

[0165] More specifically, the bispecific anti-oxMIF / anti-CD3 antibodies comprise 0, 1 or 2 point mutations in each of the CDR sequences listed above.

[0166] Another specific embodiment relates to anti-oxMIF / anti-CD3bs (scFv) 2, wherein the corresponding variable heavy chain region (VH) and the corresponding variable light chain region (VL) are arranged from N-terminus to C-terminus in the form of VH (oxMIF) -V L The order of (oxMIF)-VH(CD3)-VL(CD3), VH(CD3)-VL(CD3)-VH(oxMIF)-VL(oxMIF) or VH(CD3)-VL(CD3)-VL(oxMIF)-VH(oxMIF).

[0167] In yet another embodiment, the antibody is a bispecific antibody, in particular selected from a bispecific IgG, an IgG with an additional CD3 binding site, a BsAb fragment, a bispecific fusion protein, a BsAb conjugate.

[0168] The term "antigen", as used interchangeably herein with the term "target" or "target antigen", shall refer to the entire target molecule or a fragment of such a molecule that is recognized by an antibody binding site. Specifically, a substructure of an antigen (e.g., a polypeptide or carbohydrate structure, often referred to as an "epitope", such as an immune-related B cell epitope or T cell epitope) can be recognized by such a binding site.

[0169] As used herein, the term "epitope" specifically refers to a molecular structure that may fully constitute a specific binding partner for a binding site of an antibody form of the invention or may be a part of a specific binding partner. An epitope may be composed of a carbohydrate; a peptide structure; a fatty acid; an organic, biochemical or inorganic substance or a derivative thereof and any combination thereof. If the epitope is contained in a peptide structure (e.g., a peptide, a polypeptide or a protein), it will generally contain at least 3 amino acids, specifically 5 to 40 amino acids, and specifically less than 10 amino acids, specifically 4 to 10 amino acids. An epitope may be a linear epitope or a conformational epitope. A linear epitope is composed of a single segment of the primary sequence of a polypeptide or a carbohydrate chain. A linear epitope may be continuous or overlapping. A conformational epitope is composed of amino acids or carbohydrates that form a tertiary structure together by folding the polypeptide, and the amino acids are not necessarily adjacent to each other in the linear sequence. Such an oxMIF epitope may be the sequence EPCALCS (SEQ ID NO: 43) located within the central region of oxMIF.

[0170] The term "antigen binding domain" or "binding domain" or "binding site" refers to a portion of an antigen binding moiety that includes a region that specifically binds to and is complementary to part or all of an antigen. When the antigen is large, an antigen binding molecule may only bind to a specific portion of the antigen, which is called an epitope. The antigen binding domain can be provided, for example, by 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).

[0171] As used herein about the term "binding site" of the antibody of the present invention, it refers to a molecular structure that can interact with antigen binding. Typically, the binding site is located in the complementary determining region (CDR) of the antibody, also referred to herein as "CDR binding site", which is a specific region with different structures that give various antigen binding functions. Different structures can be derived from natural antibody libraries, such as mouse or human libraries, or can be recombined or synthesized, such as by mutagenesis and specifically by randomization techniques. These include the CDR regions of mutagenesis, the loop regions of variable antibody domains, particularly the CDR loops of antibodies, such as any CDR1, CDR2 and CDR3 loops in VL and / or VH antibody domains. As used according to the present invention, the antibody form typically comprises one or more CDR binding sites, and each site has specificity to a kind of antigen.

[0172] As used herein, the term "specificity" shall refer to a binding reaction that determines a target cognate ligand in a heterogeneous population of molecules. Herein, the binding reaction occurs at least with the oxMIF antigen. Thus, under specified conditions (e.g., immunoassay conditions), an antibody that specifically binds to its specific target will not bind to other molecules present in the sample in significant amounts, specifically, it will not show significant cross-reactivity with reduced MIF.

[0173] A specific binding site generally does not cross-react with other targets. Furthermore, a specific binding site may specifically bind to one or more epitopes, isoforms or variants of a target, or cross-react with other related target antigens, such as homologs or analogs.

[0174] Specific binding means that the binding is selective with respect to a selected target characteristic; high, medium or low binding affinity or avidity. Selective binding is typically achieved if the binding constant or binding kinetics for an antigen other than the target antigen differs by at least 10-fold, preferably by at least 100-fold, more preferably by at least 1000-fold, compared to the binding constant or binding kinetics for the target antigen, e.g., oxMIF.

[0175] The term "valency" as used in this application indicates that there is a specific number of binding sites in an antibody molecule. Thus, the terms "divalent", "tetravalent" and "hexavalent" indicate that there are two binding sites, four binding sites and six binding sites in an antibody molecule, respectively.

[0176] The term "monovalent" as used herein with respect to antibody binding sites shall refer to molecules comprising only one binding site for a target antigen. Thus, the term "valent" is understood as the number of binding sites for the same target antigen, which specifically bind to the same or different epitopes of the antigen.

[0177] The antibodies of the present invention are understood to comprise monovalent, bivalent, tetravalent or multivalent binding sites that specifically bind to oxMIF.

[0178] According to yet another embodiment, the antibody may comprise one or more additional binding sites which specifically recognize one or more antigens expressed on effector T cells, NK cells or macrophages, in particular one or more of CD3, ADAM17, CD2, CD4, CD5, CD6, CD8, CD11a, CD11b, CD14, CD16, CD16b, CD25, CD28, CD30, CD32a, CD40, CD 40L, CD44, CD45, CD56, CD57, CD64, CD69, CD74, CD89, CD90, CD137, CD177, CEAECAM6, CEACAM8, HLA-Dra cahin, KIR, LSECtin or SLC44A2.

[0179] According to an alternative embodiment, the antibody of the present invention is a bispecific antibody further comprising one or more of the CD3 variable binding domains of mosunetuzumab, pasotuxizumab, cibisatamab, otelixizumab, teplizumab, visilizumab or foralumab.

[0180] According to a specific embodiment, the anti-CD3 binding site comprises a complementary determining region (CDR) selected from the group consisting of muromonab-CD3 (OKT3), otelezumab (TRX4), teligrumab (MGA031), Nuvion, solitomab, blinatumomab, pertuximab, cerbituximab, motuzumab, SP34, X35, VIT3, BMA030 (BW 264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1 and WT-31 and any humanized derivatives thereof, if applicable.

[0181] Specifically, any CDR sequence comprising muromonab, otelimumab, teliguzumab, visilizumab, solituzumab, blinatumomab, pertuximab, cerbituximab, motuzumab, or an anti-CD3 variable region or one or more CDRs in a CDR having at least 70%, specifically 80%, 90%, 95% or 99% sequence identity thereto can be used as a CD3 binding domain according to the present invention.

[0182] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is highly variable in sequence and / or forms a structurally defined loop ("hypervariable loop"). Typically, a natural four-chain antibody comprises six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs typically comprise amino acid residues from hypervariable loops and / or from "complementarity determining regions" (CDRs), which have the highest sequence variability and / or are involved in antigen recognition (Kabat et al., 1991). Hypervariable regions (HVRs) are also referred to as complementary determining regions (CDRs), and these terms are used interchangeably herein with respect to portions of variable regions that form antigen binding regions. The exact number of residues comprising a particular CDR will vary depending on the sequence and size of the CDR. Given the variable region amino acid sequence of an antibody, a person skilled in the art can routinely determine which residues comprise a particular CDR.

[0183] Kabat defines a numbering system applicable to the variable region sequence of any antibody. Those of ordinary skill in the art can assign this "Kabat numbering" system to any variable region sequence without any doubt, without relying on any experimental data other than the sequence itself. For a given antibody, the Kabat numbering of the residue can be determined by comparing the homologous region of the antibody sequence with the "standard" Kabat numbering sequence. As used herein, "Kabat numbering" refers to the numbering system proposed by Kabat et al. (target immune protein sequence, Kabat et al., 1983, US Pept. of Health and Human Services, "Sequence of Proteins of Immunological Interest"). Unless otherwise stated, the numbering of specific amino acid residue positions in the antibody variable region is referenced according to the Kabat numbering system. The numbering of the constant region is based on the EU numbering index.

[0184] The CDRs also contain "specificity determining residues" or "SDRs," which are residues that contact the antigen. The SDRs are contained in CDR regions known as abbreviated-CDRs or a-CDRs. Unless otherwise indicated, HVR residues and other residues (e.g., FR residues) in the variable domains are numbered herein according to Kabat et al., supra.

[0185] "Point mutation" is specifically understood as engineering of polynucleotides, which results in the expression of an amino acid sequence that is different from a non-engineered amino acid sequence, wherein the difference is that one or more single (non-continuous) or paired amino acid substitutions or exchanges, deletions or insertions are performed on different amino acids. Preferred point mutations refer to the exchange of amino acids of the same polarity and / or charge. In this regard, amino acids refer to the 20 naturally occurring amino acids encoded by 61 triplet codons. These 20 amino acids can be divided into neutrally charged, positively charged, and negatively charged amino acids.

[0186] "Percentage (%) of sequence identity" with respect to the polypeptide sequences identified herein is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the specified polypeptide sequence, after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percentage of sequence identity, and any conservative substitutions are not considered part of the sequence identity. One skilled in the art can determine appropriate parameters for determining alignment, including any algorithms required to achieve maximum alignment over the full length of the compared sequences.

[0187] According to the invention, the variable or constant region sequences have a sequence identity of at least 95%, 96%, 97%, 98%, 99%, 99.5% or 100% to the corresponding sequences described herein.

[0188] A "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cattle, 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.

[0189] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0190] "Isolated nucleic acid encoding anti-oxMIF" refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including one or more such nucleic acid molecules in a single vector or separate vectors and one or more such nucleic acid molecules present in one or more locations in a host cell.

[0191] "No significant cross-reactivity" means that a molecule (e.g., an antibody) does not recognize or specifically bind to an antigen different from the actual target antigen of the molecule (e.g., an antigen closely related to the target antigen), specifically reduced MIF, particularly when compared to the target antigen. For example, the antibody can be less than about 10% to less than about 5% bound to an antigen different from the actual target antigen, or can be bound to the antigen different from the actual target antigen in an amount consisting of less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2% or 0.1%, preferably less than about 2%, 1% or 0.5% and most preferably less than about 0.2% or 0.1% bound to an antigen different from the actual target antigen. Binding can be determined by any method known in the art, such as, but not limited to, ELISA or surface plasmon resonance.

[0192] Recombinant production of the antibodies of the invention preferably utilizes an expression system, such as an expression construct or vector, comprising a nucleotide sequence encoding the antibody form.

[0193] The term "expression system" refers to a nucleic acid molecule containing an operably linked desired coding sequence and control sequences, so that a host transformed or transfected with these sequences can produce the encoded protein. To achieve transformation, the expression system can be contained on a vector; however, the relevant DNA can also be then integrated into the host chromosome. Alternatively, the expression system can be used for in vitro transcription / translation.

[0194] "Expression vector " used herein is defined as the DNA sequence required for the transcription of the cloned recombinant nucleotide sequence (that is, recombinant gene) and the translation of its mRNA in a suitable host organism. Expression vector comprises an expression cassette, and in addition generally comprises a starting point or a genome integration site for autonomous replication in a host cell, one or more selective markers (for example, an amino acid synthesis gene or a gene conferring antibiotic resistance, such as bleomycin, kanamycin, G418 or hygromycin), a number of restriction enzyme cleavage sites, a suitable promoter sequence and a transcription terminator, and the components are operably connected together. As used herein, the terms "plasmid" and "vector" include a nucleotide sequence for autonomous replication and a nucleotide sequence for genome integration.

[0195] Specifically, the term refers to a vector by which a DNA or RNA sequence (e.g., a foreign gene) (e.g., a nucleotide sequence encoding an antibody form of the invention) 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 invention.

[0196] A vector typically contains DNA of a transmissible agent into which the foreign DNA is inserted. A common method of inserting one DNA segment into another involves the use of enzymes called restriction enzymes, which cut the DNA at specific sites (specific groups of nucleotides) called restriction sites.

[0197] "Box" refers to a DNA coding sequence or DNA segment encoding an expression product, which can be inserted into a restriction site defined in a vector. Box restriction sites are designed to ensure that the box is inserted into the correct reading frame. Typically, foreign DNA is inserted into one or more restriction sites of the vector DNA and then brought into the host cell by the vector together with the transfer vector DNA. DNA segments or sequences to which DNA has been inserted or added, such as expression vectors, can also be referred to as "DNA constructs". A common type of vector is a "plasmid", which is usually an independent double-stranded DNA molecule that can easily accept additional (foreign) DNA and can be easily introduced into a suitable host cell. The vector of the present invention generally contains coding DNA and expression control sequences, such as promoter DNA, and has one or more restriction sites suitable for inserting foreign DNA. Coding DNA is a DNA sequence encoding a specific amino acid sequence of a specific polypeptide or protein (e.g., an antibody form of the present invention). Promoter DNA is a DNA sequence that starts, regulates, or otherwise mediates or controls the expression of coding DNA. Promoter DNA and coding DNA can come from the same gene or different genes, and can come from the same or different organisms. The recombinant cloning vectors of the invention will generally contain one or more replication systems for cloning or expression, one or more markers for selection in a host (eg, antibiotic resistance), and one or more expression cassettes.

[0198] The procedures for connecting DNA sequences (e.g., providing or encoding factors of the invention and / or proteins of interest, promoters, terminators and other sequences, respectively) and inserting them into a suitable vector containing the information required for integration or host replication are well known to those skilled in the art, for example as described by Sambrook et al., 2012.

[0199] Host cells are understood in particular to be cells, recombinant cells or cell lines which have been transfected with an expression construct, such as a vector according to the invention.

[0200] As used herein, the term "host cell line" refers to a clone of an established specific cell type that has acquired the ability to proliferate over an extended period of time. The term host cell line refers to a cell line such as that used to express endogenous or recombinant genes to produce polypeptides (e.g., recombinant antibody forms of the present invention).

[0201] "Production host cell" or "production cell" is generally understood to be a cell line or cell culture that is ready for cultivation in a bioreactor to obtain the product of the production process (recombinant antibody form of the invention). The host cell type according to the invention can be any prokaryotic or eukaryotic cell.

[0202] As used herein, the term "recombinant" shall mean "produced by genetic engineering" or "the result of genetic engineering", such as specifically using a heterologous sequence incorporated into a recombinant vector or a recombinant host cell.

[0203] The antibodies of the present invention can be produced using any known and established expression system and recombinant cell culture technology, for example, by expression in a bacterial host (prokaryotic system) or a eukaryotic system (e.g., yeast, fungi, insect cells or mammalian cells). The antibody molecules of the present invention can be produced in transgenic organisms, such as goats, plants or transgenic mice, i.e., engineered mouse strains with large fragments of human immunoglobulin loci and defects in mouse antibody production. Antibodies can also be produced by chemical synthesis.

[0204] According to a specific embodiment, the host cell is a production cell line of a cell selected from CHO, PerC6, CAP, HEK, HeLa, NSO, SP2 / 0, hybridoma and Jurkat. More specifically, the host cell is obtained from a CHO cell.

[0205] The host cells of the invention are specifically cultured or maintained in serum-free culture, eg comprising other components, such as plasma proteins, hormones and growth factors, as a replacement for serum.

[0206] The host cells are most preferred when established, adapted and cultured entirely under serum-free conditions and optionally in a medium that does not contain any animal-derived proteins / peptides.

[0207] The anti-oxMIF antibodies of the invention can be recovered from the culture medium using standard protein purification methods.

[0208] The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of the active ingredient contained therein to be effective, and which contains no additional components which would be unacceptably toxic to a subject to which the formulation would be administered.

[0209] "Pharmaceutically acceptable carrier" refers to a component of a pharmaceutical formulation other than the active ingredient that is non-toxic to the subject. Some examples of pharmaceutically acceptable carriers are water, saline, phosphate buffered saline, amino acids such as glycine or histidine, dextrose, glycerol, ethanol, etc. and combinations thereof. In many cases, it will be preferred to include an isotonic agent such as a sugar, a polyol such as mannitol, sorbitol or sodium chloride in the composition. Another example of a pharmaceutically acceptable substance is a wetting agent or a small amount of an auxiliary substance, such as a wetting agent or an emulsifier, a preservative or a buffer, which enhances the shelf life or effectiveness of the antibody.

[0210] As used herein, "treatment," "treat," or "treating" refers to a clinical intervention that attempts to alter the natural course of the individual being treated, and may be performed for prevention or during clinical pathology. Desired therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, the antibodies of the invention are used to delay the development of a disease or slow the progression of a disease.

[0211] The anti-oxMIF antibodies of the present invention and pharmaceutical compositions comprising the same can be administered in combination with one or more other therapeutic agents, diagnostic agents or prophylactic agents. Depending on the disease to be treated, additional therapeutic agents include other anti-neoplastic agents, anti-tumor agents, anti-angiogenic agents, chemotherapeutic agents, steroids or checkpoint inhibitors.

[0212] The pharmaceutical compositions of the present invention can be in various forms, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable solutions and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Typical preferred compositions are in the form of injectable solutions or infusible solutions, such as those compositions similar to compositions for passive immunity of people. The preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In a preferred embodiment, the antibody is administered by intravenous infusion or injection. In another preferred embodiment, the antibody is administered by intramuscular or subcutaneous injection. As will be appreciated by those skilled in the art, the route and / or mode of administration will vary according to the desired result.

[0213] Due to the optimized and advantageous properties of the antibodies of the present invention, high doses of antibody compositions can be administered. Specifically, the composition comprises about 10 to 250 mg / ml, specifically 25 to 100 mg / ml, specifically ≥50 mg / ml of the antibody.

[0214] The anti-oxMIF antibody can be administered once, but more preferably multiple times. For example, the antibody can be administered from three times a day to once every six months or longer. Administration can be performed on a schedule, such as three times a day, twice a day, once a day, once every two days, once every three days, once a week, once every two weeks, once a month, once every two months, once every three months, and once every six months.

[0215] The present invention also relates to compositions comprising anti-oxMIF antibodies or antigen binding portions thereof for use in treating a subject in need of treatment for a MIF-related disorder, particularly an immune disease, such as inflammatory diseases and hyperproliferative disorders. In some embodiments, the subject in need of treatment is a human.

[0216] As used herein, the term "cancer" refers to proliferative diseases, particularly solid cancers, such as colorectal cancer, ovarian cancer, pancreatic cancer, lung cancer, melanoma, squamous cell carcinoma (SCC) (e.g., head and neck cancer, esophageal cancer and oral cancer), hepatocellular carcinoma, colorectal adenocarcinoma, renal cancer, medullary thyroid carcinoma, papillary thyroid carcinoma, astrocytoma, neuroblastoma, Ewing's sarcoma, cervical cancer, endometrial cancer, breast cancer, prostate cancer and malignant seminoma, including refractory forms of any of the above cancers or a combination of one or more of the above cancers.

[0217] Hyperproliferative disorders, such as cancerous diseases or cancers, that can be treated with the anti-oxMIF antibodies of the invention can involve any tissue or organ, including but not limited to brain cancer, lung cancer, squamous cell carcinoma, bladder cancer, stomach cancer, pancreatic cancer, breast cancer, head cancer, neck cancer, liver cancer, kidney cancer, ovarian cancer, prostate cancer, colorectal cancer, esophageal cancer, gynecological cancer, nasopharyngeal cancer, or thyroid cancer; melanoma, lymphoma, leukemia or multiple myeloma. In particular, the anti-oxMIF antibodies of the invention can be used to treat ovarian cancer, pancreatic cancer, colon cancer and lung cancer.

[0218] In a specific embodiment, the recombinant anti-oxMIF antibody or antigen-binding fragment thereof with reduced aggregation potential and reduced hydrophobicity is an antibody very suitable for treating cancerous diseases, especially solid tumors, and comprises the following variable domains:

[0219] (a1) a light chain variable domain comprising SEQ ID NO: 9 and at least one of the amino acid substitutions M30L, F49Y, A51G, P80S, W93F, or

[0220] (a2) comprising SEQ ID NO:9 having 1, 2, 3, 4 or 5 amino acid substitutions, further comprising a light chain variable domain of a conserved tyrosine at position 36 and at least one of the amino acid substitutions M30L, F49Y, A51G, P80S, W93F; and one of the following:

[0221] (b1) a heavy chain variable domain comprising SEQ ID NO: 6; or

[0222] (b2) a heavy chain variable domain comprising SEQ ID NO: 6 and amino acid substitutions L5Q and / or W97Y, or

[0223] (b3) a heavy chain variable domain comprising SEQ ID NO: 6 with 1, 2, 3, 4 or 5 amino acid substitutions and amino acid substitutions L5Q and / or W97Y, wherein the amino acid positions are numbered according to Kabat, wherein the aggregation potential and hydrophobicity are reduced compared to an antibody comprising SEQ ID NO: 6 and SEQ ID NO: 9 lacking said amino acid substitutions.

[0224] Alternatively, treatment of solid tumors may be performed with a recombinant anti-oxMIF antibody comprising: a light chain variable domain comprising SEQ ID NO:9, or a light chain variable domain comprising a tyrosine at position 36 having at least 95% sequence identity to SEQ ID NO:9; and a heavy chain variable domain comprising SEQ ID NO:6 and amino acid substitutions W97Y and / or L5Q, or a heavy chain variable domain comprising SEQ ID NO:6 having 1, 2, 3, 4 or 5 amino acid substitutions and further comprising amino acid substitutions W97Y and / or L5Q.

[0225] In yet another specific embodiment, the recombinant anti-oxMIF antibody with enhanced effector function described herein is an antibody very suitable for treating cancerous diseases, especially for treating solid tumors, and its heavy chain constant region comprises:

[0226] (a) SEQ ID NO: 2, or a functional variant having at least 95% sequence identity to SEQ ID NO: 2 and comprising amino acids 239D and 332E; or

[0227] (b) SEQ ID NO: 3, or a functional variant having at least 95% sequence identity to SEQ ID NO: 3 and comprising amino acids 239D, 268F, 324T and 332E; or

[0228] (c) SEQ ID NO:4, or a functional variant having at least 95% sequence identity to SEQ ID NO:4 and comprising amino acids 239D and 332E; or

[0229] (d) SEQ ID NO: 5, or a functional variant having at least 95% sequence identity to SEQ ID NO: 5 and comprising amino acids 235V, 243L, 292P, 300L and 396L.

[0230] The present invention also includes methods of treating an inflammatory disease in a subject, including a human, such as vasculitis, arthritis, sepsis, septic shock, endotoxic shock, toxic shock syndrome, acquired respiratory distress syndrome, glomerulonephritis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, peritonitis, nephritis, atopic dermatitis, asthma, conjunctivitis, fever, malaria, NASH (non-alcoholic steatohepatitis), multiple sclerosis, acute and chronic pancreatitis, type 1 diabetes, IgA nephropathy, interstitial cystitis, post-COVID syndrome, psoriasis, glomerulonephritis, inflammatory bowel disease, nephritis and peritonitis, systemic lupus erythematosus (SLE - including lupus nephritis), asthma, rheumatoid arthritis (RA) and inflammatory bowel disease (IBD), comprising the step of administering to the subject in need thereof a therapeutically effective amount of an anti-oxMIF antibody, or an antigen-binding portion thereof.

[0231] In a specific embodiment, the recombinant anti-oxMIF antibody or antigen-binding fragment thereof with reduced aggregation potential and reduced hydrophobicity is an antibody that is very suitable for treating inflammatory or infectious diseases, and comprises the following variable domains:

[0232] (a1) a light chain variable domain comprising SEQ ID NO: 9 and at least one of the amino acid substitutions M30L, F49Y, A51G, P80S, W93F, or

[0233] (a2) comprising SEQ ID NO:9 having 1, 2, 3, 4 or 5 amino acid substitutions, further comprising a light chain variable domain of a conserved tyrosine at position 36 and at least one of the amino acid substitutions M30L, F49Y, A51G, P80S, W93F; and one of the following:

[0234] (b1) a heavy chain variable domain comprising SEQ ID NO: 6; or

[0235] (b2) a heavy chain variable domain comprising SEQ ID NO: 6 and amino acid substitutions L5Q and / or W97Y, or

[0236] (b3) a heavy chain variable domain comprising SEQ ID NO: 6 having 1, 2, 3, 4 or 5 amino acid substitutions and amino acid substitutions L5Q and / or W97Y; wherein the amino acid positions are numbered according to Kabat, wherein the aggregation potential and hydrophobicity are reduced compared to an antibody comprising SEQ ID NO: 6 and SEQ ID NO: 9 lacking said amino acid substitutions.

[0237] Alternatively, treatment of inflammatory or infectious diseases can be performed with a recombinant anti-oxMIF antibody comprising: a light chain variable domain comprising SEQ ID NO:9, or a light chain variable domain comprising a tyrosine at position 36 having at least 95% sequence identity to SEQ ID NO:9; and a heavy chain variable domain comprising SEQ ID NO:6 and amino acid substitutions W97Y and / or L5Q, or a heavy chain variable domain comprising SEQ ID NO:6 having 1, 2, 3, 4 or 5 amino acid substitutions and further comprising amino acid substitutions W97Y and / or L5Q.

[0238] In yet another specific embodiment, the recombinant anti-oxMIF antibodies described herein that are well suited for treating inflammatory or infectious diseases have increased preferential binding to the inhibitory receptor FcγRIIB or have high α2,6-N-linked sialylation.

[0239] The present invention further includes the following items:

[0240] 1. A recombinant anti-oxMIF antibody or antigen-binding fragment thereof with reduced aggregation potential and reduced hydrophobicity, comprising

[0241] i) the following variable domains:

[0242] (a) a light chain variable domain comprising SEQ ID NO:9 and at least one of the amino acid substitutions M30L, F49Y, A51G, P80S, W93F; or a light chain variable domain comprising SEQ ID NO:9 having 1, 2, 3, 4 or 5 amino acid substitutions, further comprising a conserved tyrosine at position 36 and further comprising at least one of the amino acid substitutions M30L, F49Y, A51G, P80S, W93F; and one of the following:

[0243] (b) a heavy chain variable domain comprising SEQ ID NO: 6; or

[0244] (c) a heavy chain variable domain comprising SEQ ID NO: 6 and at least one of the amino acid substitutions L5Q or W97Y; or a heavy chain variable domain comprising SEQ ID NO: 6 having 1, 2, 3, 4 or 5 amino acid substitutions and further comprising at least one of the amino acid substitutions L5Q or W97Y;

[0245] or

[0246] ii) the following variable domains

[0247] (a) a light chain variable domain comprising SEQ ID NO:9; or a light chain variable domain comprising a tyrosine at position 36 having at least 95% sequence identity to SEQ ID NO:9, and

[0248] (b) a heavy chain variable domain comprising SEQ ID NO: 6 and the amino acid substitution W97Y; or a heavy chain variable domain comprising SEQ ID NO: 6 having 1, 2, 3, 4 or 5 amino acid substitutions and further comprising the amino acid substitution W97Y.

[0249] 2. The recombinant anti-oxMIF antibody according to item 1, which has enhanced effector function, wherein the recombinant anti-oxMIF antibody comprises a heavy chain constant region, wherein the heavy chain constant region comprises

[0250] (a) SEQ ID NO: 2, or a heavy chain constant region having at least 95% sequence identity to SEQ ID NO: 2 and comprising amino acids 239D and 332E; or

[0251] (b) SEQ ID NO:3, or a heavy chain constant region having at least 95% sequence identity to SEQ ID NO:3 and comprising amino acids 239D, 268F, 324T and 332E; or

[0252] (c) SEQ ID NO:4, or a heavy chain constant region having at least 95% sequence identity to SEQ ID NO:4 and comprising amino acids 239D and 332E; or

[0253] (d) SEQ ID NO:5, or a heavy chain constant region having at least 95% sequence identity to SEQ ID NO:5 and comprising amino acids 235V, 243L, 292P, 300L and 396L.

[0254] 3. The anti-oxMIF antibody according to item 1 or 2, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 10, 11, 12 and 13.

[0255] 4. The anti-oxMIF antibody according to item 1 or 2, comprising any one of SEQ ID NO: 6, 11 and SEQ ID NO: 1, 2, 3, 4 or 5.

[0256] 5. The anti-oxMIF antibody according to item 1 or 2, comprising SEQ ID NO: 7, 10 and any one of SEQ ID NO: 1, 2, 3, 4 or 5.

[0257] 6. The anti-oxMIF antibody according to item 1 or 2, comprising SEQ ID NO: 7, 11 and any one of SEQ ID NO: 1, 2, 3, 4 or 5.

[0258] 7. The anti-oxMIF antibody according to item 1 or 2, comprising SEQ ID NO: 8, 11 and any one of SEQ ID NO: 1, 2, 3, 4 or 5, or SEQ ID NO: 39, or SEQ ID NO: 40.

[0259] 8. The anti-oxMIF antibody according to item 1 or 2, comprising SEQ ID NO: 8, 13 and any one of SEQ ID NO: 1, 2, 3, 4 or 5 or SEQ ID NO: 41 or SEQ ID NO: 42.

[0260] 9. The anti-oxMIF antibody according to item 1 or 2, comprising any one of SEQ ID NO: 8, 9 and SEQ ID NO: 1, 2, 3, 4 or 5.

[0261] 10. The anti-oxMIF antibody according to item 1 or 2, comprising SEQ ID NO: 8, 10 and any one of SEQ ID NO: 1, 2, 3, 4 or 5.

[0262] 11. The anti-oxMIF antibody according to any one of items 1 to 10, which is selected from bispecific antibodies, scFv, (scFv)2, scFvFc, Fab, Fab', and F(ab')2, Fab'-SH, Fab-scFv fusion, Fab-(scFv)2-fusion, Fab-scFv-Fc, Fab-(scFv)2-Fc, a fusion protein of two single-chain variable fragments (scFv) of different antibodies (e.g., BiTE), a mini antibody, DutaMab TM , DART, and

[0263] 12. The antibody according to any one of items 1 to 11, for use in the preparation of a medicament.

[0264] 13. A pharmaceutical composition comprising the antibody according to any one of items 1 to 11, optionally further comprising a pharmaceutical carrier or adjuvant.

[0265] 14. A pharmaceutical composition according to item 13, wherein the composition comprises about 10 to 250 mg / ml, in particular >50 mg / ml of the antibody according to any one of items 1 to 11.

[0266] 15. The pharmaceutical composition according to item 13 or 14, wherein the pharmaceutical composition is formulated for subcutaneous administration.

[0267] 16. A pharmaceutical composition according to any one of items 13 to 15, wherein the composition is administered as a sole substance, or wherein the treatment is combined with another pharmaceutical composition comprising one or more active substances, preferably the one or more active substances are selected from antiviral, anti-inflammatory, anticancer, antiangiogenic and antibiotic substances.

[0268] 17. A pharmaceutical composition according to any one of items 13 to 16 for use in treating patients suffering from inflammatory diseases, hyperproliferative disorders, infectious diseases or cancer, in particular for the treatment of asthma, vasculitis, arthritis, sepsis, septic shock, endotoxic shock, toxic shock syndrome, acquired respiratory distress syndrome, glomerulonephritis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, peritonitis, nephritis and psoriasis, colorectal cancer, ovarian cancer, breast cancer, prostate cancer, pancreatic cancer and lung cancer.

[0269] 18. An isolated nucleic acid encoding the antibody according to any one of items 1 to 11.

[0270] 19. An expression vector comprising one or more nucleic acid molecules according to item 18.

[0271] 20. A host cell comprising the nucleic acid according to item 18 or the expression vector according to item 19.

[0272] 21. A method of producing an antibody according to any one of items 1 to 11, comprising culturing the host cell according to item 20 and recovering the antibody from the cell culture.

[0273] 22. A method for producing the antibody according to any one of items 1 to 11, comprising expressing a nucleic acid encoding the antibody in a host cell.

[0274] Example

[0275] The embodiments described herein are illustrative of the present invention and are not intended to limit the present invention. Without departing from the scope of the present invention, many modifications and changes may be made to the technology described herein and shown herein. Therefore, it should be understood that these embodiments are merely illustrative and do not limit the scope of the present invention.

[0276] Example 1: Newly designed antibodies with reduced aggregation tendency and reduced hydrophobicity.

[0277] Table 1: Variant ID and molecular sequence combinations of newly designed antibodies in the examples

[0278]

[0279]

[0280] To evaluate hydrophobicity and aggregation, the newly designed antibodies were analyzed by gel filtration (SEC) using two different SEC columns and running buffer conditions and by hydrophobic interaction chromatography (HIC) and compared with the anti-oxMIF antibody C0008.

[0281] For SEC, the sample was diluted to 0.1 mg / ml in 5x phosphate buffered saline (5x PBST) containing 0.02% Tween, and 50 μl of the sample was applied to a Superdex 200increase 10 / 300GL (GE Healthcare) gel filtration column at a flow rate of 0.75 ml / min. Separation and equilibrium were performed in 5x PBST at 22°C. The protein peak was monitored using absorbance at 214 nm, and the spectrum was analyzed using the Unicorn emulation software package (GE Healthcare). In addition, the sample was diluted to 0.2 mg / ml in 1x phosphate buffered saline (1x PBS), and 100 μl of the sample was applied to an Enrich 650 (Bio-Rad) gel filtration column at a flow rate of 1 ml / min. Separation and equilibrium were performed in 1x PBS at 22°C. The protein peak was monitored using absorbance at 280 nm, and the spectrum was analyzed using the ChromLab software package (Bio-Rad). Results are reported as retention volume (Vr, ml) of the main peak and the presence of aggregates was manually ranked.

[0282] For HIC analysis, all samples were diluted to a final concentration of 0.2 mg / ml using 50 mM phosphate and 0.75 M ammonium sulfate (pH 6.9). Highly purified antibody samples were loaded independently onto 1 ml HiTrap Butyl HP columns. 50 μl of sample was injected and the column flow rate was maintained at 1 ml / min at 22 ° C. Peak separation was performed on a gradient of 0-100% B in 20 column volumes (CV) (buffer B: 50 mM phosphate, 20% isopropanol; pH 7.0). Protein peaks were monitored using absorbance at 280 nm and spectra were analyzed using the Unicorn emulation software package (GE healthcare). For each peak, the results were reported as Vr (ml) at the peak maximum.

[0283] Results: The control antibody (C0008) demonstrated a retention volume close to the bed volume of the size exclusion column (approximately 24 ml Superdex 200, approximately 18 ml Enrich 650), which corresponds to a molecular weight much smaller than that expected for human IgG ( Figure 2A The unusually long retention is mainly due to hydrophobic interactions with the stationary phase surface. In addition, for C0008, in addition to the high retention volume, there are significant amounts of IgG dimers and aggregates. All of the newly designed antibodies showed reduced retention volumes (Vr), demonstrating reduced interactions with the column and thus reduced hydrophobicity of the molecules (Table 2, Figure 2A When compared with the molecular weight standards, C0083 and C0090 showed the lowest retention volumes (Table 2, Figure 2B ), and Vr corresponds to the molecular weight of monomeric human IgG. In addition, in the samples of the newly designed antibodies C0073, C0078, C0083 and C0090, antibody dimers and aggregation were significantly reduced ( Figure 2A and B). In particular, C0083 and C0090 showed a single monomer peak.

[0284] Using HIC column retention volume, antibodies were sorted from least to most hydrophobic (lowest to highest retention volume) IgG ( Figure 2C , Table 3), the results confirmed the data of SEC analysis. The newly designed antibody C0083 (retention volume 12.37) had the lowest hydrophobicity, while C0008 (retention volume 14.92 ml) showed the highest hydrophobicity.

[0285] Table 2: Size Exclusion Chromatography

[0286] sample Running buffer column Vr(ml) Aggregates C0008 5x PBS Superdex 200 24.20 ++ C0070 5x PBS Superdex 200 16.43 + C0073 5x PBS Superdex 200 14.00 (+) C0076 5x PBS Superdex 200 21.10 + C0077 5x PBS Superdex 200 15.77 + C0078 5x PBS Superdex 200 14.02 (+) C0081 5x PBS Superdex 200 16.03 + C0083 5x PBS Superdex 200 13.01 - C0008 1x PBS Enrich 650 18.06 + C0083 1x PBS Enrich 650 12.39 - C0090 1x PBS Enrich 650 12.38 -

[0287] Table 3: Hydrophobic Interaction Chromatography

[0288] sample Vr(ml) C0008 14.92 C0070 13.67 C0073 13.37 C0076 14.84 C0077 14.05 C0078 13.41 C0081 13.48 C0083 12.37

[0289] Figure 2 shows chromatographic profiles demonstrating reduced aggregation and hydrophobicity of the newly designed anti-oxMIF antibodies. (A) Comparison of elution profiles of C0008 (control antibody, gray area) and the newly designed antibody on a Superdex 200increase 10 / 300GL gel filtration column using 5x PBST as the mobile phase. (B) Comparison of elution profiles of C0008 (control antibody, gray area) and the newly designed antibody on an Enrich 650 gel filtration column using 1x PBS as the mobile phase; (C) Comparison of elution profiles of C0008 (control antibody, gray area) and the newly designed antibody on a HiTrap Butyl HP HIC column.

[0290] Example 2: Binding of the newly designed anti-oxMIF antibody to immobilized MIF (K D Sure)

[0291] Recombinant human MIF diluted at 1 μg / ml in PBS was fixed in ELISA plates overnight at 4°C (MIF was converted to oxMIF according to Thiele et al., 2015). After blocking, serial dilutions of anti-oxMIF antibodies were added to the plates. Finally, bound antibodies were detected using protein L-HRP conjugate and tetramethylbenzidine (TMB) as substrate. The color reaction was stopped with 3M H2SO4 and the OD was measured at 450nm. The data from different experiments were normalized to the maximum OD (=100%) of the anti-oxMIF antibody C0008 of the corresponding experiment, and the EC was determined by 4-parameter fitting using GraphPad Prism. 50 Values ​​(mean values ​​of two experiments are shown).

[0292] Results: The binding of the newly designed antibody to immobilized MIF (oxMIF) was measured over a wide concentration range, and the resulting binding curves were shown in Figure 2. Figure 3 As shown. Anti-oxMIF antibody (C0008) was used as a reference for oxMIF binding. Binding curves and calculated EC 50 Value (represents K D ) clearly showed that the newly designed antibodies C0073, C0076, C0078, C0083 and C0090 retained their affinity for oxMIF (EC50 range 0.5-1.6 nM, within the assay variability) compared to C0008 (EC50 0.7 nM) ( Figure 3 A). Surprisingly, C0077 showed a 5-fold reduction in affinity for oxMIF compared to C0076 with only one additional VL point mutation (Y36F) in VL ( Figure 3 A), although the hydrophobicity of C0077 was significantly reduced ( Figure 2A ).

[0293] In another set of experiments, newly designed antibodies containing mutations in the Fc portion that enhance effector function showed EC values ​​in the range of 0.4-0.8 nM. 50 , which again fully matches the apparent affinity of the control antibody C0008 ( Figure 3 B).

[0294] Figure 3 The binding of the newly designed anti-oxMIF antibody to immobilized MIF is shown (K D(A) Binding curve of the newly designed antibody with a wild-type Fc part. (B) Binding curve of the newly designed antibody with a mutation in the Fc part that enhances effector function. C0008 was used as a reference antibody.

[0295] Table 4: K of anti-oxMIF antibodies D value

[0296] sample <![CDATA[K D (nM)]]> C0008 0.7 C0073 1.0 C0076 1.1 C0077 5.2 C0078 1.6 C0083 0.5 C0090 0.5 C0097 0.8 C0098 0.7 C0099 0.4 C0100 0.5

[0297] Example 3: Differential binding of newly designed anti-oxMIF antibodies to oxMIF and redMIF.

[0298] Anti-oxMIF antibodies were fixed in microplates at a concentration of 15 nM overnight at 4 ° C. After blocking, the wells were incubated with 50 ng / ml of redMIF or oxMIF surrogate NTB-MIF (Schinagl et al., 2018). The captured oxMIF was detected with biotinylated polyclonal rabbit anti-MIF antibody and streptavidin-HRP conjugate. The plates were stained with tetramethylbenzidine (TMB) and the color reaction was terminated by adding 30% H2SO4. OD was measured at 450nm. The data from different experiments were normalized to the maximum OD (= 100%) of the anti-oxMIF antibody C0008 of the corresponding experiment, and the average of two to three experiments is shown.

[0299] Results: Binding of the newly designed antibodies to soluble oxMIF and redMIF is depicted in Figure 4 The results clearly showed that the newly designed antibodies C0073, C0076, C0078, C0083 and C0090 exhibited strong binding to oxMIF but not to redMIF, thus the antibodies with reduced hydrophobicity and aggregation retained their ability to distinguish oxMIF from redMIF. Compared with the control antibody C0008, the newly designed antibodies showed similar OD ( Figure 4 A), the control antibody C0008 has been described as being able to distinguish oxMIF from redMIF (Thiele et al., 2015). Surprisingly, C0077 showed significantly reduced binding to oxMIF compared to C0076 by only one additional point mutation (Y36F) in VL ( Figure 4 A). C0077 showed significantly reduced hydrophobicity ( Figure 2A ), but failed to maintain the binding properties.

[0300] In another set of experiments, the newly designed antibodies (C0097-C0100) containing mutations in the Fc portion that enhance effector function also retained their ability to distinguish oxMIF from redMIF and showed similar OD values ​​compared to antibody C0008 ( Figure 4B).

[0301] Figure 4 Differential binding of the newly designed antibodies to oxMIF and redMIF is shown. (A) Differential binding of the newly designed antibodies with wild-type Fc parts. (B) Differential binding of the newly designed antibodies containing mutations in the Fc part that enhance effector function. C0008 was used as a reference antibody.

[0302] Example 4: Enhanced effector function of the newly designed anti-oxMIF antibodies with Fc mutations.

[0303] As mentioned above, the ADCC potential of antibody can be attempted to be increased by mutation in the Fc part.Fc receptor-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) is an important mechanism of action, and antibody targets diseased cells to remove by the mechanism.When the Fc effector part of the antibody of target binding also binds to the FcγRIIIA receptor on the cell surface of effector cells, multiple crosslinking occurs in two cell types, resulting in ADCC pathway activation.Use the engineered Jurkat cells of the NFAT response element of stably expressing FcγRIIIA receptor, V158 (high affinity) variant and driving firefly luciferase expression as effector cells to determine the ADCC activity of antibody, and the activity is quantified by the luciferase produced as a result of NFAT pathway activation.

[0304] ADCC reporter assays were performed essentially as recommended by the manufacturer (Promega # G7010) (with some modifications). In brief, recombinant human MIF diluted in PBS at 1 μg / ml was fixed in ELISA plates overnight at 4°C (according to Thiele et al., 2015, MIF was converted to oxMIF). After blocking, serial dilutions of the newly designed antibodies (C0097-C0100) described herein were added together with Jurkat effector cells, and the plates were incubated in a humidified incubator at 37°C / 5% CO2 for 6h. Finally, Bio-Glo luciferase reagent was added, and luminescence (RLU) (0.5s integration time) was measured on a Tecan multi-plate reader. C0008 was used as a reference antibody.

[0305] Results: All newly designed variants containing Fc mutations (C0097-C0100) showed a significant dose response with increasing concentrations of the newly designed antibodies, whereas antibody C0008 did not induce FcγRIIIA-mediated activation of Jurkat reporter cells ( Figure 5 ).

[0306] Figure 5The enhanced effector function of the newly designed anti-oxMIF antibodies in a modified ADCC reporter bioassay (Promega #G7010) is shown. In contrast to C0008, all newly designed anti-oxMIF antibodies induced FcγRIIIA-mediated activation of Jurkat reporter cells (n=2).

[0307] Example 5: Biodistribution of the newly designed anti-oxMIF antibodies

[0308] The biodistribution of the newly designed anti-oxMIF antibody C0083 compared to C0008 was studied in female Balb / c mice bearing subcutaneous tumors of the syngeneic colon cancer model CT26. Female Balb / c mice received 3x10 5 CT26 cells were injected subcutaneously unilaterally (100 μl / animal). When the tumor volume reached 150-300 mm 3 At 14:00, mice were assigned to treatment groups and received a single intravenous dose of 5 mg / kg / d IRDye 800CW labeled C0008 or IRDye 800CW labeled C0083. Two mice served as untreated "no signal" controls.

[0309] C0008 and C0083 were labeled with IRDye 800CW using the IRDye 800CW protein labeling kit (high MW) from LI-COR Biosciences according to the manufacturer's instructions. After the labeling treatment and before the labeled antibodies were injected into mice, the protein concentration and labeling efficiency of the IRDye 800CW labeled antibodies were determined using Nanodrop technology, and mice were dosed based on the protein concentration after labeling. After administration of the labeled antibodies at the following time points: 1h, 6-8h, 24h, 48h, 72h, 96h, 168h after administration, the labeled antibodies were injected into mice at LI-COR In vivo imaging was performed in a Trilogy imaging device. Image analysis was performed to quantify the relative fluorescence units (RFU) of the antibody in the tumor (RFU = RFU tumor area - RFU background).

[0310] Results: The intravenously administered 800CW labeled C0008 ( Figure 6 A) and C0083( Figure 6 (B) with a significant intratumoral distribution, where the peak occurs at 24 h and is retained in the tumor for up to 7 days. This clearly demonstrates that the tumor penetration properties of C0083 are retained or even improved compared to the control anti-oxMIF antibody C0008. The two antibodies were tested in separate experiments and the scaling of the images was optimized for the respective experiments. However, the same scaling was used for both experiments, and C0083 ( Figure 6The improvement in tumor penetration and retention of C0083 becomes much more obvious if the pixel intensity of the tumor area is evaluated numerically ( Figure 6 E). No signal was detected in untreated control mice ( Figure 6 C).

[0311] Figure 6 Tumor penetration of the newly designed anti-oxMIF antibody C0083 is shown by infrared in vivo imaging of mice bearing subcutaneous CT26 tumors. Photos were taken 1h, 6h, 24h, 48h, 72h, 96h and 168h after injection of IRDye 800CW labeled antibodies. A: Mice receiving IRDye 800CW labeled C0008 (5mg / kg); B: Mice receiving IRDye 800CW labeled C0083 (5mg / kg / d); C: Untreated control mice; D: Mice receiving IRDye 800CW labeled C0083 (5mg / kg / D), and images are displayed using the same scale as in A; E: Tumor penetration and retention of C0083 and C0008 were quantified by digital image analysis.

[0312] Example 6: Improved production of the newly designed antibody C0083.

[0313] Compared with C0008, the newly designed anti-oxMIF antibody C0083 was produced by transient expression in ExpiCHO cells (Thermo Fisher). In brief, 50-200ml exponentially growing ExpiCHO cells were transiently transfected using the ExpiFectamine CHO kit (Thermo Scientific), and cultivated for 8 days at 37°C in a humidified incubator according to the manufacturer's instructions "standard protocol" (ThermoScientific). Cells were removed by centrifugation, and the supernatant was diluted 1:1 with 40mM sodium phosphate, 300mM sodium chloride (pH 7.2). The diluted supernatant was filtered through 0.2 μm, and applied to the Mab Select Prism A column (Cytiva) using NGC QUEST 10 chromatography systems (Bio-Rad) at room temperature. Post is washed with 20mM sodium phosphate, 150mM sodium chloride (pH 7.2) of 10 column volumes, and with 100mM glycine (pH 3.5) elution antibody of 10 column volumes.Antibody is neutralized to pH 5 immediately, and is prepared in 1x PBS pH 7.2 by Bio-Scale P-6 desalting cartridge, and is concentrated to about 1mg / ml using Amicon Ultra-15 centrifugal device (Merck Millipore).Protein concentration is determined using NanoDrop technology and its corresponding extinction coefficient.The amount (final yield of corresponding antibody) of purified antibody after Mab Select Prism A and neutralization is divided by cell culture volume, and the values ​​obtained are referred to as antibody titer (mg / L) in this article.

[0314] Results: The three individual productions of C0083 clearly showed that the production of the newly designed antibody C0083 was significantly (unpaired t-test, p=0.03) better compared to C0008. A higher antibody titer (117 mg / L) was obtained compared to C0008 (93 mg / L) ( Figure 7 ).

[0315] Figure 7 Improved production of the newly designed anti-oxMIF antibody C0083 is shown.

[0316] Example 7: Improved production of newly designed anti-oxMIF antibodies with enhanced ADCC activity.

[0317] Anti-oxMIF antibodies (C0097, C0098 and C0100) were produced by transient expression in ExpiCHO cells (Thermo Fisher), which contained mutations that reduced hydrophobicity and aggregation tendency and also contained Fc mutations that improved ADCC activity. In brief, 50 ml of exponentially growing ExpiCHO cells were transiently transfected using the ExpiFectamine CHO kit (Thermo Scientific), and were cultivated for 8 days at 37 ° C in a humidified incubator according to the manufacturer's instructions "standard protocol" (Thermo Scientific). Cells were removed by centrifugation, and 1 / 10 volume of 200 mM sodium phosphate, 1500 mM sodium chloride (pH 7.2) were added to the supernatant. The diluted supernatant was filtered through 0.2 μm, and applied to the HiTrap Fibro PrismA column (Cytiva) using the NGC QUEST10 chromatography system (Bio-Rad) at room temperature. The post is washed with 20mM sodium phosphate, 150mM sodium chloride (pH 7.2) of 10 column volumes, and the antibody is eluted with 100mM glycine (pH 3.5) of 10 column volumes. Antibody is immediately neutralized to pH 5 with glycine, and final concentration is 250mM glycine. Protein concentration is determined using NanoDrop technology and its corresponding extinction coefficient. The amount (final yield of corresponding antibody) of purified antibody after HiTrap Fibro Prism A and neutralization is divided by cell culture volume, and the resulting value is referred to as antibody titer (mg / L) in this article. For comparison, anti-oxMIF antibodies (C0064, C0065 and C0067) are produced by the same procedure, which are not optimized through VH / VL but contain the same Fc mutations that improve ADCC activity in terms of hydrophobicity / aggregation.

[0318] Results: Generation of anti-oxMIF antibodies with improved hydrophobicity / aggregation propensity (C0097, C0098, and C0100) resulted in significantly higher titers (P < 0.05) compared to their respective counterparts (C0064, C0065, and C0067) containing the VH and VL domains of C0008. Figure 8 ).

[0319] Figure 8 Improved production of newly designed anti-oxMIF antibodies with enhanced effector function and reduced hydrophobicity and aggregation propensity compared to counterparts with enhanced effector function but comprising the VH and VL domains of C0008 (comparison of C0097 with C0064, C0098 with C0065, and C0100 with C0067) is shown.

[0320] Example 8: Reduced aggregation tendency and reduced hydrophobicity of newly designed anti-oxMIF antibodies with enhanced effector function

[0321] To evaluate hydrophobicity and aggregation, the newly designed antibodies were analyzed by gel filtration (SEC) and by hydrophobic interaction chromatography (HIC) and compared with the anti-oxMIF antibody C0008.

[0322] For SEC, the sample was diluted to 1 mg / ml in 1x phosphate buffered saline (1x PBS), and 100 μl of the sample was applied to an Enrich 650 (Bio-Rad) gel filtration column at a flow rate of 1.25 ml / min. Separation and balance were performed at room temperature in 1x PBS. The protein peak was monitored using the absorbance at 280 nm, and the ChromLab software package (Bio-Rad) was used to analyze the spectrum. The results were reported with the retention volume (Vr, ml) of the main peak, and the aggregates present were manually sorted.

[0323] For HIC analysis, all samples were diluted to a final concentration of 1 mg / ml using 50 mM phosphate and 0.75 M ammonium sulfate (pH 6.8). Highly purified antibody samples (approximately 100 μg) were independently loaded onto 1 ml HiTrap Butyl HP columns. 100 μl of sample was injected and the column flow rate was maintained at 1 ml / min at room temperature. Peak separation was performed on a gradient of 0-100% B (buffer B: 50 mM phosphate, 20% isopropanol; pH 7.0) of 20 column volumes (CV). Protein peaks were monitored using the absorbance at 280 nm, and the spectrum was analyzed using the Unicorn emulation software package (GE healthcare). For each peak, the results were reported as Vr (ml) at the peak maximum.

[0324] result : The control antibody C0008 demonstrated a retention volume close to the void volume of the size exclusion column (approximately 16 mL), which corresponds to a molecular weight much smaller than that expected for human IgG ( Fig. 9 A). The unusually long retention is mainly due to hydrophobic interactions with the stationary phase surface. In addition, for C0008, in addition to the high retention volume, there are significant amounts of IgG dimers and aggregates. The newly designed antibodies C0108 and C0109 showed significantly reduced retention volumes (Vr), demonstrating reduced interactions with the column and, therefore, reduced hydrophobicity of the molecules ( Fig. 9 A, Table 5). When compared with the molecular weight standard, Vr corresponds to the molecular weight of monomeric human IgG. In addition, antibody dimers and aggregation were not present in the samples of the newly designed antibodies C0108 and C0109 ( Fig. 9 A).

[0325] Using HIC, Vr is proportional to the hydrophobicity of the antibody being analyzed, i.e., the higher the Vr, the more hydrophobic the molecule. HIC analysis of the newly designed antibodies (C0083, C0090, C0108, and C0109) confirmed the similarity to the control antibody C0008 ( Fig. 9 B, retention volume of 20.8 ml, Table 6), which are significantly less hydrophobic ( Fig. 9 B, retention volume 15.2-16.2 ml, Table 6).

[0326] in conclusion : It is evident from SEC and HIC analysis that the newly designed antibodies have improved biochemical properties, especially reduced hydrophobicity and aggregation tendency.

[0327] Table 5: Size Exclusion Chromatography

[0328]

[0329] Table 6: Hydrophobic Interaction Chromatography

[0330]

[0331] Fig. 9 Chromatographic profiles demonstrating reduced aggregation and hydrophobicity of the newly designed anti-oxMIF antibodies are shown. (A) Comparison of elution profiles of C0008 (control antibody, gray area) and newly designed antibodies C0108 and C0109 with enhanced effector function on an Enrich 650 gel filtration column using 1x PBS as the mobile phase; (B) Comparison of elution profiles of C0008 (control antibody, gray area) and newly designed antibodies C0083, C0090, C0108 and C0109 on a HiTrap ButylHP HIC column.

[0332] Example 9: Newly designed anti-oxMIF antibodies C0083 and C0090 and A2780MIF - / - Non-specific binding of cells is reduced.

[0333] Gene editing of the human MIF gene in the A2780 ovarian cancer cell line by CRISPR / Cas9 to generate A2780MIF - / - Cell lines. A2780MIF was confirmed by Sanger sequencing and by Western blotting using a polyclonal anti-human MIF antibody. - / - Endogenous human MIF protein is not present in the cell line.

[0334] A2780MIF - / -The cells were detached with a cell stripper (Corning, Cat#25-056-Cl), washed with staining buffer (PBS + 5% BSA), and stained at 2×10 5 Cells were plated in 96-well U-shaped bottom plates. Cells were stained with fixable viability dye eFluo780 (1:2000 dilution in PBS) at 4°C for 20 min and washed with staining buffer. Cells were resuspended in 50 μl staining buffer and 50 μl of newly designed anti-oxMIF antibodies C0083, C0090, C0108, C0109, control anti-oxMIF antibodies C0008 or serial dilutions of isotype IgG were added (final concentration 37 nM-9.4 nM). After incubation at 4°C for 40 min, cells were washed with staining buffer and resuspended in 100 μl secondary antibodies (goat anti-human IgG (H+L)-AlexaFluor 488, 1:100 dilution). After incubation at 4°C for 30 min, cells were washed with staining buffer, resuspended in PBS+2% BSA, and collected on a Cytoflex-S flow cytometer. Data were analyzed with FlowJo, and the geometric mean of viable cells (mean fluorescence intensity of AF488) was plotted against antibody concentration in GraphPad Prism.

[0335] Results and Conclusions :from Fig.10 It can be clearly seen that the newly designed anti-oxMIF antibodies C0083, C0090, C0108 and C0109 do not bind to A2780 MIF - / - cells, as their geometric means were very close to those of the isotype IgG negative controls. In contrast, the reference anti-oxMIF antibody C0008 showed similar - / - Therefore, the reduction of hydrophobicity led to the binding of the newly designed anti-oxMIF antibodies C0083, C0090, C0108, and C0109 to A2780 MIF. - / - The nonspecific binding of oxMIF was significantly reduced or eliminated, while the anti-oxMIF control antibody C0008 nonspecifically bound to the cell surface due to its hydrophobic nature.

[0336] Fig.10 The newly designed anti-oxMIF antibody was confirmed by FACS to be active in A2780MIF. - / - Nonspecific binding to cells was reduced. - / -Cells were stained with the newly designed anti-oxMIF antibodies C0090, C0109 (A); C0083, C0108 (B) or control antibody C0008 and isotype IgG as a negative control. The geometric mean (GeoMean) of live cells (mean fluorescence intensity of AF488) was plotted against antibody concentration.

[0337] Example 10: Binding of the newly designed anti-oxMIF antibody with enhanced effector function to immobilized MIF (K D Evaluate)

[0338] Recombinant human MIF diluted at 1 μg / ml in PBS was fixed in ELISA plates overnight at 4°C (MIF was converted to oxMIF according to Thiele et al., 2015). After blocking, serial dilutions of anti-oxMIF antibody were added to the plate. Finally, bound antibodies were detected with goat anti-human IgG (Fc)-HRP conjugate and tetramethylbenzidine (TMB) as substrate. The color reaction was stopped with 3M H2SO4 and the OD was measured at 450nm. The data from different experiments were normalized to the maximum OD (=100%) of the anti-oxMIF antibody C0008 of the corresponding experiment, and the EC was determined by 4-parameter fitting using GraphPad Prism. 50 Values ​​(mean + / - SEM of two experiments are shown).

[0339] Results and Conclusions : The binding of the newly designed antibodies with enhanced effector function to immobilized MIF (oxMIF) was measured over a wide concentration range. The resulting binding curves are shown in Figure 2. Fig.11 As shown. Anti-oxMIF antibody C0008 was used as a reference for oxMIF binding. Binding curves and calculated EC 50 Value (represents K D ) clearly showed that the newly designed antibodies C0108 and C0109 retained their low nanomolar affinity for oxMIF compared to C0008 ( Fig.11 , Table 7).

[0340] Fig.11 Figure 2 shows the binding curves (K) of the newly designed anti-oxMIF antibodies with enhanced effector function to immobilized oxMIF. D evaluation); anti-oxMIF antibody C0008 was used as a reference antibody.

[0341] Table 7: Affinity estimation of anti-oxMIF antibodies (ELISA)

[0342]

[0343] Example 11: Differential binding of newly designed anti-oxMIF antibodies with enhanced effector functions to oxMIF and redMIF.

[0344] Anti-oxMIF antibodies and irrelevant human IgG1 controls were fixed in microplates at a concentration of 15 nM overnight at 4 ° C. After blocking, the wells were incubated with 50 ng / ml of redMIF or oxMIF surrogate NTB-MIF (Schinagl et al., 2018). Captured oxMIF was detected with polyclonal rabbit anti-MIF antibody and goat anti-rabbit IgG HRP. The plates were stained with tetramethylbenzidine (TMB) and the color reaction was terminated by adding 30% H2SO4. OD was measured at 450nm. The data from two experiments were normalized to the maximum OD (= 100%) of the anti-oxMIF antibody C0008 of the corresponding experiment, and the average + / - SEM of two to three experiments is shown.

[0345] Results and Conclusions: Binding of newly designed antibodies with enhanced effector functions to soluble oxMIF and redMIF Fig.12 As shown. The results clearly show that the newly designed antibodies C0108 and C0109 exhibited strong binding to oxMIF, but no binding to redMIF was detected. The newly designed antibodies showed very comparable ODs compared to the reference antibody C0008, which has been described as being able to distinguish oxMIF from redMIF (Thiele et al., 2015). For the irrelevant IgG1 control, no specific binding was observed. Therefore, the newly designed antibodies with enhanced effector function and reduced hydrophobicity and aggregation retain their ability to distinguish oxMIF from redMIF.

[0346] Fig.12 Differential binding of the newly designed antibodies to oxMIF and redMIF is shown. C0008 was used as reference antibody and isotype IgG was used as negative control.

[0347] Example 12: Determination of the affinity of the newly designed antibodies to human and mouse oxMIF using SPR

[0348] The newly designed antibodies C0108 and C0109 and the control anti-oxMIF antibody C0008 were immobilized onto Biacore CM5 optical sensor chips (GE Healthcare) using standard amine coupling conditions to a density of approximately 2500, 1500 or 100 RU on flow cells FC2, FC3 and FC4, respectively. FC1 was used as a reference cell and reacted with buffer only. Mouse or human recombinant MIF was diluted to a concentration of 50, 75, 100 or 150 nM in HBS-EP buffer (GE Healthcare) in the presence of 0.2% Proclin300 (active component 5-chloro-2-methyl-4-isothiazolin-3-one; Sigma) to convert MIF to an oxMIF substitute within 3 h at room temperature (Thiele M et al., 2015). Using Biacore TM Proclin300 treated MIF was applied to the immobilized anti-oxMIF antibody at a flow rate of 30 μl / min using a 3000 instrument (GE Healthcare) for 3 min. The dissociation time was 300 s, and the chip was regenerated for 3 min using 10 mM HCl. The kinetics of the concentration series were analyzed by fitting the local simultaneous association / dissociation of each binding curve after subtracting the background from the reference cell (FC1) using an iterative Langmuir 1:1 interaction model with mass transfer compensation provided by BiaEvaluation 4.1 software (GE Healthcare). Data are presented as calculated K for flow cells 2-4 D Values ​​are mean + / - SD (standard deviation).

[0349] Results and Conclusions: The binding of the newly designed antibodies with enhanced effector function to soluble human and mouse oxMIF as determined by SPR is shown in Table 8. The results clearly show that the newly designed antibodies have a nanomolar affinity of about 3 nM, which is in the same range as the control anti-oxMIF antibody C0008. For all anti-oxMIF antibodies, the affinity for mouse oxMIF is about 5-8 times lower than for human oxMIF. Therefore, the newly designed antibodies with enhanced effector function and reduced hydrophobicity and aggregation retain their affinity for human and mouse oxMIF.

[0350] Table 8: Affinity of anti-oxMIF antibodies (SPR)

[0351]

[0352] Example 13: Pharmacokinetics of the newly designed anti-oxMIF antibodies C0083 and C0090 compared with the control anti-oxMIF antibody C0008 in BALB / c nude mice

[0353] Newly designed anti-oxMIF antibodies C0083 and C0090 and control anti-oxMIF antibody C0008 were injected intravenously into female BALB / c nude mice at a dose of 10 mg / kg. 20 μl of blood was collected by tail vein puncture using a K2-EDTA-coated capillary at 4, 10, 24, 48 and 96 h after injection and transferred to a K2-EDTA-coated vial containing 60 μl PBS. After centrifugation, the supernatant (= 1:4 diluted plasma) was analyzed by quantitative anti-oxMIF ELISA.

[0354] In brief, recombinant human MIF (OncoOne) diluted with 1 μg / ml in PBS was fixed to ELISA plates (MaxiSorp, Nunc) overnight at 4°C. After blocking with dilution buffer (DB, 2% fish gelatin / TBST), diluted plasma samples (final dilution 1:100-1:100,000) were added to the plate. Goat anti-human IgG (Fc)-HRP conjugate (1:40,000 in TBST, Thermo Scientific #32420) was used to detect the bound antibody. Tetramethylbenzidine (TMB, Thermo Scientific #34029) was added as substrate, and 50 μl 30% H2SO4 was used to stop the color development reaction, and OD was measured at 450nm. Using the hyperbolic regression analysis in GraphPad Prism, a standard curve was generated using the serial dilutions of C0008, C0083 and C0090, and the plasma concentration of the antibody was calculated. The mean antibody concentration at each time point was plotted against the collection time, and half-lives were calculated by a one-phase decay model in GraphPad Prism.

[0355] result: Fig.13 The pharmacokinetics of anti-oxMIF antibodies following a monoexponential decay function are shown. The calculated half-life of the control anti-oxMIF antibody C0008 is 11 hours, and the calculated half-lives of the newly designed anti-oxMIF antibodies C0083 and C0090 are 15 hours, respectively. In addition, the area under the curve (AUC) below the monoexponential decay function is calculated to estimate the total antibody exposure and bioavailability. The AUCs of C0008, C0083 and C0090 are calculated to be 1600, 3784 and 3266, respectively.

[0356] The pharmacokinetics of the newly designed anti-oxMIF antibodies C0083 and C0090 compared with the control anti-oxMIF antibody C0008 in BALB / c nude mice are shown. The plasma concentrations of the antibodies were determined by quantitative anti-oxMIF ELISA 4, 10, 24, 48 and 96 h after a single intravenous injection of 10 mg / kg of anti-oxMIF antibodies, and the half-lives were calculated by a single-phase decay model in GraphPad Prism.

[0357] in conclusion: Substitutions in the variable regions of the newly designed anti-oxMIF antibodies C0083 and C0090 resulted in a 1.4-fold longer half-life and more than 2-fold higher bioavailability (AUC) compared to the control anti-oxMIF antibody C0008.

[0358] Example 14: Enhanced effector function of the newly designed anti-oxMIF antibodies C0108 and C0109 as determined by reporter assay.

[0359] Using engineered Jurkat cells stably expressing the human FcγRIIIa receptor, V158 (high affinity allotype) or F158 (low affinity allotype) (for demonstrating ADCC) (or engineered Jurkat cells stably expressing the human FcγRIIa H131 allotype receptor (for demonstrating ADCP)) and a NFAT response element driving expression of firefly luciferase as effector cells, antibody bioactivity was quantified by luciferase produced as a result of NFAT pathway activation.

[0360] In order to generate highly responsive target cells, HCT116 and A2780 cells were transfected with huMIF-pDisplay plasmid (Invitrogen), screened with geneticin, and sorted by FACS to produce cell lines that stably express membrane-anchored monomeric human MIF (HCT116-pMIF or A2780-pMIF), i.e., MIF is displayed as a monomeric protein, wherein the oxMIF epitope is accessible to anti-oxMIF antibodies (Schinagl et al., Biochemistry 2018). These cell lines show that oxMIF is presented on the cell surface and is therefore a sensitive tool for in vitro analysis.

[0361] ADCC or ADCP reporter assays were performed essentially according to the manufacturer's recommendations (Promega #G7010 and #G9991). Briefly, 1×10 4 cells / well of HCT116-pMIF( Fig.14A and C) or A2780-pMIF( Fig.14 B) Cells were seeded into 96-well plates and incubated overnight in a humidified incubator at 37°C / 5% CO2 for attachment. Afterwards, serial dilutions of the newly designed anti-oxMIF antibodies C0108 and C0109 with enhanced effector function or the control antibody C0008 with wt Fc (final concentration 0.01-100 nM) and Jurkat effector cells ( Fig.14 AB, FcγRIIIa effector cells; Fig.14 C, FcγRIIa effector cells) were incubated in a humidified incubator at 37°C / 5% CO2 for 6 h at a ratio of effector to target cells of approximately 6:1. Finally, the assay plate was equilibrated to room temperature and Bio-Glo luciferase reagent was added. Luminescence (RLU) was measured after 10-20 min incubation using a Tecan multiplate reader (0.5 s integration time).

[0362] result :from Fig.14 It can be clearly seen in AC that when compared with HCT116-pMIF( Fig.14 A, C) or A2780-pMIF( Fig.14 B) When cells were co-incubated, the newly designed antibodies C0108 and C0109 containing mutations in the Fc portion that enhance ADCC / ADCP showed ADCC ( Fig.14 AB) or ADCP( Fig.14 C) Strong activation of reporter cells, while control antibody C0008 induced no or only a small amount of FcγRIIIa (ADCC, Fig.14 AB) or FcγRIIa (ADCP, Fig.14 C) mediated activation of Jurkat reporter cells.

[0363] in conclusion: The newly designed anti-oxMIF antibodies C0108 and C0109 containing ADCC / ADCP enhancing mutations in the Fc portion showed highly enhanced ADCC and ADCP activities in reporter bioassays compared to the control antibody C0008 carrying wt Fc.

[0364] Fig.14The effector function enhancement of the newly designed antibodies C0108 and C0109 determined by the reporter assay is shown. (AB) Compared with the anti-oxMIF control antibody C0008 with wt Fc, the newly designed antibodies C0108 and C0109 with enhanced effector function were used to perform ADCC reporter bioassays using engineered Jurkat effector cells stably expressing FcyRIIIa (V high responder allotype, F low responder allotype) and HCT116-pMIF (A) or A2780-pMIF (B) target cells; (C) Compared with the anti-oxMIF control antibody C0008 with wt Fc, the newly designed antibodies C0108 and C0109 with enhanced effector function were used to perform ADCP reporter bioassays using engineered Jurkat effector cells stably expressing FcyRIIa and HCT116-pMIF target cells. Mean values ​​and SEM (n=2-4) are shown.

[0365] Example 15: The enhanced ADCC function of the newly designed anti-oxMIF antibodies C0108 and C0109 was determined by PBMC cell lysis bioassay.

[0366] This assay was performed to determine cell lysis caused by antibody-dependent cellular cytotoxicity (ADCC) induced by the newly designed anti-oxMIF antibody. Cytotoxicity was measured by the HiBiT detection assay (Promega), which quantifies HiBiT-tagged proteins released from target cells using a non-lytic detection reagent containing LgBit (LargeBiT) and Furimazine (imidazopyrazinone-based luminescent substrate). HiBiT and LgBiT spontaneously assemble into functional enzyme and emits a quantifiable luminescent signal in the presence of a substrate.

[0367] In order to produce highly responsive reporter target cells, HCT116 cells were transfected with HaloTag-HiBiT plasmid (Promega # CS1956B17), screened with blasticidin and sorted by FACS as cell pools.Then the stable cell pools expressing HiBiT were transfected with huMIF-pDisplay plasmid (Invitrogen), screened with geneticin, and sorted by FACS to produce cell pools of monomeric human oxMIF (HCT116-HiBiT-pMIF) that stably express HiBiT and membrane anchored in cells, i.e. MIF is displayed as monomeric protein, wherein the oxMIF epitope is accessible to anti-oxMIF antibodies (Schinagl et al., Biochemistry 2018).These cell lines show that oxMIF is presented on the cell surface and increases, and are therefore sensitive tools for in vitro analysis.

[0368] Briefly, 1×10 4 HCT116-HiBiT-pMIF target cells were seeded into 96-well plates at 10 cells / well and incubated overnight in a humidified incubator at 37°C / 5% CO2 for attachment. Thereafter, 50 μl of serial dilutions of the newly designed anti-oxMIF antibodies C0108 and C0109 or the control anti-oxMIF antibody C0008 with wt Fc in culture medium (final concentration 0.01-100 nM) and 50 μl of human PBMC effector cells (4×10 5 Cells / well; effector to target cell ratio: 40:1) were added to the plate. PBMCs from two different donors were characterized by FACS analysis to evaluate their NK cell content, which is a relevant cell type for this assay. After adding antibodies and PBMCs, the plates were incubated overnight in a humidified incubator at 37°C and 5% CO2. The next day, 10 μl of Nano-Glo HiBiT extracellular detection reagent (Promega #N2421) was added and the luminescent signal (RLU, integration time 0.1 s) was measured on a Tecan plate reader. The assays performed included analysis samples and donors in duplicate or triplicate.

[0369] result :from Fig.15 As can be clearly seen in Figure 2, the newly designed antibodies C0108 and C0109 containing mutations in the Fc portion that enhance ADCC / ADCP showed a significant increase in ADCC activity with PBMCs from two donors compared to the control anti-oxMIF antibody C0008 ( Fig.15 A, 22% NK cells; Fig.15 B, 7% NK cells).

[0370] in conclusion: The newly designed anti-oxMIF antibodies C0108 and C0109 containing ADCC / ADCP-enhancing mutations in the Fc portion indeed showed highly increased ADCC activity in a PBMC-mediated cell killing assay compared to a control anti-oxMIF antibody carrying a wt Fc.

[0371] Fig.15The ADCC activity enhancement of the newly designed anti-oxMIF antibodies C0108 and C0109 containing mutations that enhance ADCC / ADCP in the Fc portion as determined by PBMC-mediated cytotoxicity bioassay is shown. ADCC bioassays using the newly designed anti-oxMIF antibodies C0108 and C0109 and PBMC as effector cells (A: 22% NK cells, B: 7% NK cells) and HCT116-pMIF as target cells were compared to the anti-oxMIF control antibody C0008 with wt Fc. The mean and SEM of two or three replicates using PBMCs from two healthy donors are shown.

[0372] Example 16: Newly designed anti-oxMIF antibodies showed significant reduction in non-specific cytokine release from human PBMCs.

[0373] Cytokine release syndrome (CRS) is a form of systemic inflammatory response syndrome (SIRS), which may be triggered by multiple factors such as infection. Known CRS is also an adverse reaction of some monoclonal antibody drugs. When a large number of leukocytes including B cells, T cells, natural killer cells, macrophages, dendritic cells and monocytes are activated and release inflammatory cytokines (such as IL-6, IFN-γ, IL-8, MCP-1, etc.), CRS occurs, and these cytokines activate more leukocytes in the positive feedback loop of pathogenic inflammation. When this process is dysregulated, due to systemic excessive inflammation, hypotensive shock and multiple organ failure, life may be endangered. Therefore, the potential of newly designed anti-oxMIF antibodies to release inflammatory cytokines from PMBC was evaluated in in vitro assays.

[0374] Briefly, the newly designed anti-oxMIF antibodies C0108 and C0109 (both 70 nM) and the anti-oxMIF control antibody C0008 (70 nM) were incubated with freshly thawed PBMCs (4-5x10 596-well plate (cells / well) were incubated. After overnight incubation at 37°C / 5%CO2 in a humidified incubator, the plate was centrifuged for 5min at 300x g to precipitate cells, and the supernatant was transferred to a 96-well V-shaped bottom plate. According to the manufacturer's scheme, the supernatant clarified was analyzed by BioLegend LegendPlex cytometric bead cell counting beads. Measured on the CytoFlex-S hemocytometer with a 96-well plate loader (Beckman-Colter). The microbead sorting channel is APC (laser 638nm excitation, bandpass filter 660 / 10nm), and the reporter channel is PE (yellow laser 561nm excitation, bandpass filter 585 / 42nm). LegendPlex analysis software (BioLegend) was used to analyze data, and a figure was generated in GraphPad Prism. Mean values ​​+ / - SEM from 3 different PMBC donors are shown.

[0375] Results and conclusions: From Fig.16 As can be clearly seen in the Figures, the newly designed antibodies C0108 and C0109 containing mutations in the Fc part that enhance ADCC / ADCP surprisingly show no or only negligible release of IL-6 and MCP-1 from PBMCs at concentrations up to 70 nM compared to C0008. It can be expected that C0108 and C0109 induce higher levels of cytokine release due to their high affinity for Fcy receptors compared to the reference anti-oxMIF antibody C0008 containing the wt Fc part. Nevertheless, C0008 induced significant release of IL-6 and MCP-1 at the same concentration (70 nM), which is apparently due to its higher aggregation tendency and non-specific binding caused by its hydrophobicity. It is known that aggregation of antibody therapeutics, even in small amounts, strongly enhances cytokine release from immune cells.

[0376] Fig.16 Shown, the non-specific cytokine of new design anti-oxMIF antibody display release from human PBMC significantly reduced.New design anti-oxMIF antibody C0108 and C0109 (70nM) and control anti-oxMIF antibody C0008 (70nM) containing the mutation enhancing ADCC / ADCP in Fc part were incubated overnight with human PBMC, and the people IL-6 (A) and the people MCP-1 (B) of supernatant were analyzed in LegendPlex cell counting beads assay (BioLegend).The mean value + / -SEM of the cytokine concentration from 3 different PMBC donors is shown.

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SEQUENCE LISTING <110> Onkoone Research & Development LLC <120> Improved anti-oxMIF antibodies with reduced aggregation potential and reduced hydrophobicity <130> P23JM1WN00205AT <150> EP20199829.1 <151> 2020-10-02 <160> 45 <170> PatentIn version 3.5 <210> 1 <211> 329 <212> PRT <213> Artificial Sequence <220> <223> Anti-oxMIF constant heavy chain (CH), CH1-CH3 <400> 1 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly 325 <210> 2 <211> 329 <212> PRT <213> Artificial Sequence <220> <223> Anti-oxMIF constant heavy chain (CH), CH1-CH3 <400> 2 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Asp Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly 325 <210> 3 <211> 329 <212> PRT <213> Artificial Sequence <220> <223> Anti-oxMIF constant heavy chain (CH), CH1-CH3 <400> 3 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Asp Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser Phe Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Thr Asn 195 200 205 Lys Ala Leu Pro Ala Pro Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly 325 <210> 4 <211> 329 <212> PRT <213> Artificial Sequence <220> <223> Anti-oxMIF constant heavy chain (CH), CH1-CH3 <400> 4 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Asp Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Glu Glu Lys Thr Ile Ser Lys Thr Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly 325 <210> 5 <211> 329 <212> PRT <213> Artificial Sequence <220> <223> Anti-oxMIF constant heavy chain (CH), CH1-CH3 <400> 5 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Val Gly Gly Pro Ser Val Phe Leu Leu Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Pro Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Leu Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Leu Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly 325 <210> 6 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> anti-oxMIF VH <400> 6 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Gly Ser Ser Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Ser Gln Trp Leu Tyr Gly Met Asp Val Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 7 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> anti-oxMIF VH <400> 7 Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Gly Ser Ser Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Ser Gln Trp Leu Tyr Gly Met Asp Val Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 8 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> anti-oxMIF VH <400> 8 Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Gly Ser Ser Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Ser Gln Tyr Leu Tyr Gly Met Asp Val Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 9 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Anti-oxMIF VL <400> 9 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Arg Ile Met Thr Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Phe Val Ala Ser His Ser Gln Ser Gly Val Pro Ser Arg Phe Arg Gly 50 55 60 Ser Gly Ser Glu Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu Gln Pro 65 70 75 80 Glu Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Ser Phe Trp Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 10 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Anti-oxMIF VL <400> 10 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Arg Ile Met Thr Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Val Gly Ser His Ser Gln Ser Gly Val Pro Ser Arg Phe Arg Gly 50 55 60 Ser Gly Ser Glu Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu Gln Pro 65 70 75 80 Glu Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Ser Phe Trp Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 11 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Anti-oxMIF VL <400> 11 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Arg Ile Met Thr Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Val Gly Ser His Ser Gln Ser Gly Val Pro Ser Arg Phe Arg Gly 50 55 60 Ser Gly Ser Glu Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu Gln Pro 65 70 75 80 Glu Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Ser Phe Phe Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 12 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Anti-oxMIF VL <400> 12 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Arg Ile Leu Thr Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Val Gly Ser His Ser Gln Ser Gly Val Pro Ser Arg Phe Arg Gly 50 55 60 Ser Gly Ser Glu Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu Gln Pro 65 70 75 80 Glu Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Ser Phe Phe Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 13 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Anti-oxMIF VL <400> 13 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Arg Ile Leu Thr Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Val Gly Ser His Ser Gln Ser Gly Val Pro Ser Arg Phe Arg Gly 50 55 60 Ser Gly Ser Glu Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu Gln Ser 65 70 75 80 Glu Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Ser Phe Phe Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 14 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Anti-oxMIF CDR1-H2 <400> 14 Arg Tyr Thr Met His 1 5 <210> 15 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Anti-oxMIF CDR1-H2 <400> 15 Gly Tyr Gly Met His 1 5 <210> 16 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Anti-oxMIF CDR1-H2 <400> 16 Ser Phe Pro Met Ala 1 5 <210> 17 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Anti-oxMIF CDR2-H2 <400> 17 Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys 1 5 10 15 Asp <210> 18 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Anti-oxMIF CDR2-H2 <400> 18 Val Ile Trp Tyr Asp Gly Ser Lys Lys Tyr Tyr Val Asp Ser Val Lys 1 5 10 15 Gly <210> 19 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Anti-oxMIF CDR2-H2 <400> 19 Thr Ile Ser Thr Ser Gly Gly Arg Thr Tyr Tyr Arg Asp Ser Val Lys 1 5 10 15 Gly <210> 20 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Anti-oxMIF CDR3-H2 <400> 20 Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr 1 5 10 <210> twenty one <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Anti-oxMIF CDR3-H2 <400> twenty one Gln Met Gly Tyr Trp His Phe Asp Leu 1 5 <210> twenty two <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Anti-oxMIF CDR3-H2 <400> twenty two Phe Arg Gln Tyr Ser Gly Gly Phe Asp Tyr 1 5 10 <210> twenty three <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Anti-oxMIF CDR3-H2 <400> twenty three Tyr Tyr Asp Asp His Tyr Ser Leu Asp Tyr 1 5 10 <210> twenty four <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Anti-oxMIF CDR1-L2 <400> twenty four Arg Ala Ser Ser Ser Val Ser Tyr Met Asn 1 5 10 <210> 25 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Anti-oxMIF CDR1-L2 <400> 25 Ser Ala Ser Ser Ser Val Ser Tyr Met Asn 1 5 10 <210> 26 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Anti-oxMIF CDR1-L2 <400> 26 Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 <210> 27 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Anti-oxMIF CDR1-L2 <400> 27 Thr Leu Ser Ser Gly Asn Ile Glu Asn Asn Tyr Val His 1 5 10 <210> 28 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Anti-oxMIF CDR2-L2 <400> 28 Asp Thr Ser Lys Val Ala Ser 1 5 <210> 29 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Anti-oxMIF CDR2-L2 <400> 29 Asp Thr Ser Lys Leu Ala Ser 1 5 <210> 30 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Anti-oxMIF CDR2-L2 <400> 30 Asp Ala Ser Asn Arg Ala Thr 1 5 <210> 31 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Anti-oxMIF CDR2-L2 <400> 31 Asp Asp Asp Lys Arg Pro Asp 1 5 <210> 32 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Anti-oxMIF CDR3-L2 <400> 32 Gln Gln Trp Ser Ser Asn Pro Leu Thr 1 5 <210> 33 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Anti-oxMIF CDR3-L2 <400> 33 Gln Gln Trp Ser Ser Asn Pro Phe Thr 1 5 <210> 34 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Anti-oxMIF CDR3-L2 <400> 34 Gln Gln Arg Ser Asn Trp Pro Pro Leu Thr 1 5 10 <210> 35 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Anti-oxMIF CDR3-L2 <400> 35 Ser Gln Trp Leu Tyr Gly Met Asp Val 1 5 <210> 36 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Anti-oxMIF CDR3-L2 <400> 36 Gln Gln Trp Ser Ser Asn Pro 1 5 <210> 37 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Anti-oxMIF VL <400> 37 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Arg Ile Met Thr Tyr 20 25 30 Leu Asn Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Val Gly Ser His Ser Gln Ser Gly Val Pro Ser Arg Phe Arg Gly 50 55 60 Ser Gly Ser Glu Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu Gln Pro 65 70 75 80 Glu Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Ser Phe Trp Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 38 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Anti-oxMIF CL <400> 38 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 39 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Anti-oxMIF C0083 light chain <400> 39 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Arg Ile Met Thr Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Val Gly Ser His Ser Gln Ser Gly Val Pro Ser Arg Phe Arg Gly 50 55 60 Ser Gly Ser Glu Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu Gln Pro 65 70 75 80 Glu Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Ser Phe Phe Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 40 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> Anti-oxMIF C0083 heavy chain <400> 40 Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Gly Ser Ser Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Ser Gln Tyr Leu Tyr Gly Met Asp Val Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 <210> 41 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Anti - oxMIF C0090 light chain <400> 41 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Arg Ile Leu Thr Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Val Gly Ser His Ser Gln Ser Gly Val Pro Ser Arg Phe Arg Gly 50 55 60 Ser Gly Ser Glu Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu Gln Ser 65 70 75 80 Glu Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Ser Phe Phe Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 42 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> Anti-oxMIF C0090 <400> 42 Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Gly Ser Ser Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Ser Gln Tyr Leu Tyr Gly Met Asp Val Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 <210> 43 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> oxMIF epitope <400> 43 Glu Pro Cys Ala Leu Cys Ser 1 5 <210> 44 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> C0108 heavy chain <400> 44 Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Gly Ser Ser Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Ser Gln Tyr Leu Tyr Gly Met Asp Val Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Asp 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val 290 295 300 Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Glu Glu Lys Thr 325 330 335 Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 <210> 45 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> CO109 heavy chain <400> 45 Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Gly Ser Ser Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Ser Gln Tyr Leu Tyr Gly Met Asp Val Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Asp 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val 290 295 300 Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Glu Glu Lys Thr 325 330 335 Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445

Claims

1. A recombinant anti-oxMIF antibody or antigen-binding fragment thereof with reduced aggregation potential and reduced hydrophobicity, comprising: The heavy chain variable domain of SEQ ID NO: 8, the light chain variable domain of SEQ ID NO: 11, the heavy chain constant region of SEQ ID NO: 4, and the light chain constant region of SEQ ID NO: 38; or The heavy chain variable domain of SEQ ID NO: 8, the light chain variable domain of SEQ ID NO: 13, the heavy chain constant region of SEQ ID NO: 4, and the light chain constant region of SEQ ID NO: 38; or The heavy chain variable domain of SEQ ID NO:8, the light chain variable domain of SEQ ID NO:9, the heavy chain constant region of SEQ ID NO:1, and the light chain constant region of SEQ ID NO:38; or The heavy chain variable domain of SEQ ID NO:6, the light chain variable domain of SEQ ID NO:11, the heavy chain constant region of SEQ ID NO:1, and the light chain constant region of SEQ ID NO:38; or The heavy chain variable domain of SEQ ID NO:7, the light chain variable domain of SEQ ID NO:10, the heavy chain constant region of SEQ ID NO:1, and the light chain constant region of SEQ ID NO:38; or The heavy chain variable domain of SEQ ID NO:7, the light chain variable domain of SEQ ID NO:11, the heavy chain constant region of SEQ ID NO:1, and the light chain constant region of SEQ ID NO:38; or The heavy chain variable domain of SEQ ID NO: 8, the light chain variable domain of SEQ ID NO: 10, the heavy chain constant region of SEQ ID NO: 1, and the light chain constant region of SEQ ID NO: 38; or The heavy chain variable domain of SEQ ID NO:8, the light chain variable domain of SEQ ID NO:11, the heavy chain constant region of SEQ ID NO:1, and the light chain constant region of SEQ ID NO:38; or The heavy chain variable domain of SEQ ID NO:8, the light chain variable domain of SEQ ID NO:13, the heavy chain constant region of SEQ ID NO:1, and the light chain constant region of SEQ ID NO:

38.

2. The anti-oxMIF antibody according to claim 1, for use in the preparation of a medicament.

3. A pharmaceutical composition comprising the antibody according to claim 1 or 2. The pharmaceutical composition according to claim 3 , further comprising a pharmaceutical carrier or adjuvant.

5. The pharmaceutical composition according to claim 3 or 4, wherein the composition comprises 10 to 250 mg / ml of the antibody according to claim 1 or 2.

6. The pharmaceutical composition of claim 5, wherein the composition comprises >50 mg / ml of the antibody of claim 1 or 2.

7. The pharmaceutical composition according to claim 3 or 4, wherein the pharmaceutical composition is formulated for subcutaneous administration.

8. The pharmaceutical composition according to claim 3 or 4, wherein the composition is intended for administration as a sole substance or for administration together with another pharmaceutical composition comprising one or more active substances.

9. The pharmaceutical composition according to claim 8, wherein the one or more active substances are selected from antiviral, anti-inflammatory, anticancer, anti-angiogenic and antibiotic substances.

10. An isolated nucleic acid encoding the antibody according to claim 1 or 2. An expression vector comprising one or more nucleic acids according to claim 10.

12. A host cell comprising the nucleic acid according to claim 10 or the expression vector according to claim 11.

13. A method of producing the antibody according to claim 1 or 2, the method comprising culturing the host cell according to claim 12 and recovering the antibody from the cell culture.

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