Anti-oxmif radioimmunoconjugates

Abstract

The present invention relates to the development of methods and tools effective in the treatment, prevention and diagnosis of cancer. In particular, the present invention relates to radioisotopes coupled to anti-oxMIF antibodies (anti-oxMIF radioimmunoconjugates) having improved properties, such as reduced aggregation potential and reduced hydrophobicity, due to the substitution of selected amino acids in the light and heavy chain variable domains, as well as to methods of treatment, prevention and diagnosis of cancer comprising the use of anti-oxMIF radioimmunoconjugates.

Description

Technical Field

[0001] This invention relates to the development of methods and tools for the effective treatment, prevention, and diagnosis of cancer. Specifically, the invention relates to radioisotopes conjugated with anti-oxMIF antibodies (anti-oxMIF radioimmunoconjugates), which possess improved properties, such as reduced aggregation potential and reduced hydrophobicity, due to the substitution of selected amino acids in variable domains of the light and heavy chains. The invention also relates to methods for the treatment, prevention, and diagnosis of cancer, including the use of anti-oxMIF radioimmunoconjugates. Background Technology

[0002] Radioimmunotherapy (RIT) for cancer treatment involves injecting patients with radioactive isotope "bullets" linked to specific cancer cell carriers (such as monoclonal antibodies) to selectively destroy targeted tumor cells. During radioactive decay, photons, electrons, and even heavier particles are emitted, damaging or killing cells along their trajectories. Radioimmunotherapy (RIT) is still a relatively new cancer treatment method, initially using radionuclides that emit beta rays. In the past decade, RIT medical devices using radioisotopes have seen increased use due to the addition of commercially available lutetium-177 (…). 177 Lutetium-177 is a medium-energy beta emitter (with a maximum beta of 0.13 MeV), with a range of 0.7 mm in tissue and a physical half-life of 6.7 days. 177 Lu demonstrated encouraging results in therapeutic clinical trials, particularly in the binding of somatostatin receptors to radiolabeled peptides. Due to its radionuclide chemistry, 177 Lu is a residual radioactive isotope that is primarily excreted via the hepatobiliary route (Phaeton R. et al., 2016). Recent experiments have also demonstrated that alpha rays can effectively destroy tumor cells. They are considered particularly attractive for treating hematopoietic cancers and micrometastases (cancer cells are usually present throughout the body). Another potential application area for alpha-immunotherapy is the treatment of a small number of residual cancer cells after high-dose chemotherapy or surgery. However, the application of radioimmunotherapy is mainly involved in hematologic malignancies. The therapeutic effect of radioimmunotherapy (RIT) in solid tumors is often limited by insufficient penetration of the radioimmunoconjugate (RIC), as insufficient tumor penetration is a key factor limiting the efficacy of RIT (Huang CY, et al., 2012).

[0003] Antibodies are used as cancer carriers for radiographic imaging of selected tumors. This involves injecting a patient with a radioactive isotope linked to a tumor-specific antibody, acting as a "diagnostic probe." Following injection, the patient undergoes non-invasive neuroimaging techniques such as SPECT or PET, which allow for tumor localization and detection of disease progression. 89The half-life of Zr, its physicochemical properties related to protein binding, and its availability. 89 Zr is considered the best nuclide for PET (Warram JMet al., 2014; Carter L.M. et al., 2018).

[0004] Radioimmunoassay (RIT) or radiographic imaging is performed on cancer patients by administering so-called radioimmunoassay conjugates, which are constructs containing a radionuclide with desired properties linked to an antibody. Typically, the linking of the radionuclide to the antibody is achieved through a chelating agent. In vivo, the antibody carries the radionuclide to diseased tissue expressing the corresponding antigen. RIT and radiographic imaging require tumor-specific targets that are specifically expressed in tumor tissue but not expressed or expressed sparingly in normal tissue.

[0005] The cytokine macrophage migration inhibitory factor (MIF) was described as early as 1966 (David, JR, 1966; Bloom, B. and Bennet, B., 1966). However, MIF differs significantly from other cytokines and chemokines because it is constitutively expressed, stored in the cytoplasm, and present in circulation in healthy subjects. Due to the ubiquitous nature of this protein, MIF could be considered an unsuitable target for therapeutic intervention. However, MIF exists in two immunologically distinct conformational isoforms, called reduced MIF (redMIF) and oxidized MIF (oxMIF) (Thiele M. et al., 2015). redMIF was found to be a highly expressed isoform of MIF, highly expressed in both healthy and diseased subjects, and may reflect the dormant proenzyme form of MIF (Schinagl. A. et al., 2018). Conversely, oxMIF appears to be a disease-associated isotype, detectable in tumor tissues (particularly in patients with colorectal, pancreatic, ovarian, and lung cancer), suggesting high tumor specificity (Schinagl.A. et al., 2016). While oxMIF appears to meet the requirements for a tumor-specific target, several factors prevent it from being a clear target. OxMIF has not been precisely characterized, the molecular mechanisms underlying disease-associated oxMIF production remain unclear, and oxMIF is not considered a cell surface receptor.

[0006] Antibodies targeting oxMIF have shown efficacy in in vitro and in vivo cancer models (Hussain F. et al., 2013; Schinagl. A. et al., 2016). An oxMIF-specific antibody (imalumab) demonstrated acceptable safety, satisfactory tissue penetration, and indications of antitumor activity in a phase 1 clinical trial. However, a phase IIa study investigating the combination of imalumab plus 5-fluorouracil / leucine or panitumab with standard treatment in patients with mCRC was terminated early based on the overall benefit-risk assessment by the data safety monitoring committee (Mahalingam D. et al., 2015; Mahalingam D. et al., 2020).

[0007] Thiele M. et al. (2015) reported the accumulation of radiolabeled anti-oxMIF antibodies in a mouse pancreatic tumor model.

[0008] WO2019 / 234241A1 discloses a bispecific antibody against oxMIF / antiCD3 that can be labeled with a radioisotope.

[0009] WO2009 / 086920A1 describes the anti-oxMIF antibody Bax69 (imarumab).

[0010] Protein aggregation, particularly antibody aggregation, is frequently observed at several stages of bioprocessing, including protein expression, purification, and storage. Antibody aggregation can affect the overall yield of therapeutic protein production processes and contribute to the stability and immunogenicity of therapeutic antibodies. Therefore, antibody protein aggregation remains an important issue in its development and a major area of ​​concern in antibody production. Antibody aggregation can be triggered by the partial unfolding of hydrophobic patches and their domains, leading to monomer-monomer binding, followed by nucleation and aggregation growth. Although the aggregation tendency of antibodies and antibody-based proteins can be influenced by external experimental conditions, they strongly depend on intrinsic antibody properties determined by their sequence and structure.

[0011] The aggregation resistance or tendency of antibodies and proteins containing their antigen-binding domains is generally limited by the most aggregate-prone domains they contain and the strength of their interactions with surrounding domains (if present). The constant domains of antibodies typically do not aggregate and do not change significantly. Therefore, the weakest domains of antibodies with respect to aggregation potential and stability are generally considered to be variable domains (e.g., heavy chain variable domains (V... H ) and / or light chain variable structural domains (V L (Ewert S. et al., 2003). At this point, V, which is prone to aggregation... Hor V L Integrating domains into other stable recombinant antibody products often introduces these undesirable features into novel recombinant designs. Therefore, modifying variable domains to be anti-aggregating can potentially make the entire protein containing that variable domain anti-aggregating. Various strategies have been proposed to reduce the aggregation of variable domains, such as the rational design of anti-aggregating proteins, complementarity-determining region (CDR) transplantation, the introduction of disulfide bonds into the variable domain, or the removal of exposed hydrophobic patches. However, these methods have drawbacks because they often alter binding properties, such as affinity or specificity; they are often laborious and not universally applicable due to the specialized knowledge they require; and they may introduce immunogenic epitopes.

[0012] Other intrinsic properties of proteins, such as hydrophobicity, also play an important role in antibody solubility. Due to their surface hydrophobicity, the low solubility of these therapeutic proteins can 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 candidate monoclonal antibodies can provide benefits related to purification and dosing regimens and save costs.

[0013] Despite increasing efforts in radioimmunotherapy (RIT) and radioimaging, and despite the growing number of monoclonal antibodies used in clinical trials, there is still a need to improve radioimmunotherapy and imaging to provide effective treatment and diagnosis in cancer, particularly involving antibodies that bind with high selectivity to tumor-associated antigens. Summary of the Invention

[0014] The purpose of this invention is to provide radioimmunoconjugates with highly selective tumor-binding properties.

[0015] This objective is addressed by the subject matter of this invention. Although oxMIF appears to meet the requirements as a tumor-specific target, several factors prevent it from being a clear target for radioimmunotherapy. oxMIF has not been precisely characterized, the molecular mechanisms underlying disease-related oxMIF production remain unclear, and oxMIF is not considered a cell surface receptor.

[0016] Nevertheless, it has been surprisingly shown that when used for radioimmunotherapy, radiolabeled anti-oxMIF antibodies or their antigen-binding fragments (anti-oxMIF radioimmunoconjugates) are highly selective due to their oxMIF specificity and highly cytotoxic due to the presence of radioisotopes.

[0017] When used for radioimaging, radioimmunoconjugates can be readily detected in tumor tissues due to their oxMIF specificity and the appropriate radionuclide.

[0018] When used in therapeutic diagnostic methods, radioimmunoconjugates exhibit high selectivity due to their oxMIF specificity and allow for both diagnosis and treatment due to their radionuclide content. The combination of radionuclide and anti-oxMIF antibody has surprisingly demonstrated that the radiolabeled anti-oxMIF antibody possesses relevant biodistribution, tumor uptake, and efficacy, as confirmed in mouse models. Therefore, the radioimmunoconjugates of this invention are highly effective for cancer treatment and also valuable for tumor detection and diagnosis.

[0019] This invention discloses a radioimmunoconjugate (RIC) comprising a recombinant anti-oxMIF antibody or its antigen-binding fragment.

[0020] Specifically, this invention provides a radioimmunoconjugate (RIC) comprising a recombinant anti-oxMIF antibody or its antigen-binding fragment, comprising:

[0021] (a1) A light chain variable structural domain, said light chain variable structural domain comprising SEQ ID NO:32 having at least one amino acid substitution of M30L, F49Y, A51G, P80S, W93F, or

[0022] (a2) A light chain variable domain, said light chain variable domain comprising SEQ ID NO:32 having 1, 2, 3, 4 or 5 amino acid substitutions, and further comprising

[0023] At position 36, the conserved tyrosine, and

[0024] Amino acid substitution for at least one of M30L, F49Y, A51G, P80S, and W93F; and

[0025] (b1) A heavy chain variable structural domain, wherein the heavy chain variable structural domain comprises SEQ ID NO:29; or

[0026] (b2) A heavy chain variable domain, wherein the heavy chain variable domain comprises SEQ ID NO:29 and amino acid substitutions L5Q and / or W97Y, or

[0027] (b3) A heavy chain variable domain comprising SEQ ID NO:29 having at least one amino acid substitution of L5Q or W97Y, and 1, 2, 3, 4 or 5 other amino acid substitutions.

[0028] The amino acid positions are determined according to Kabat numbering.

[0029] Compared to antibodies or antigen-binding fragments thereof containing SEQ ID NO:32 and SEQ ID NO:29 lacking amino acid substitutions, the antibodies or antigen-binding fragments thereof have reduced aggregation potential and reduced hydrophobicity.

[0030] The relevant germline VL sequence of the anti-oxMIF antibody exhibits high variability at positions D1, M4, S10, and L11 in framework 1.

[0031] According to one specific embodiment, the anti-oxMIF antibody may therefore further comprise a light chain variable domain, which comprises SEQ ID NO:32 having 1, 2, 3, 4 or 5 amino acid substitutions in addition to further substitutions at positions D1, M4, S10 and L11.

[0032] The related phylogenetic VH sequence is highly variable at the following positions: E1, S74, and T77.

[0033] According to a further specific implementation, the anti-oxMIF antibody may therefore further include a heavy chain variable domain, which includes SEQ ID NO:29 having 1, 2, 3, 4 or 5 amino acid substitutions in addition to further substitutions at positions E1, S74 and T77.

[0034] In an alternative embodiment of the invention, a radioimmunoconjugate (RIC) comprising a recombinant anti-oxMIF antibody or an antigen-binding fragment thereof is provided herein, comprising:

[0035] (a) An anti-oxMIF antibody light chain variable domain comprising a light chain variable domain having at least 95% sequence identity with SEQ ID NO:32, comprising a tyrosine residue at position 36, and further comprising an amino acid substitution of at least one of M30L, F49Y, A51G, P80S, and W93F.

[0036] (b) A heavy chain variable domain comprising SEQ ID NO:29 and amino acid substitution W97Y, or a heavy chain variable domain comprising at least 95% sequence identity with SEQ ID NO:29 and further comprising amino acid substitution W97Y.

[0037] According to another implementation, the radioimmunoconjugate comprises a light chain variable domain having an amino acid substitution W93F and a heavy chain variable domain having an amino acid substitution W97Y.

[0038] According to specific embodiments of the present invention, anti-oxMIF antibodies or their antigen-binding fragments are directly labeled or labeled using acyclic, monocyclic, or macrocyclic bifunctional chelating agents, particularly chelating agents selected from p-SCN-bn-DOTA, p-NH2-Bn-DOTA, DOTA-NHS, and p-SCN-Bn-deferroamine containing chelating groups and functional / reactive linkers.

[0039] According to one specific embodiment, the radioimmunoconjugate contains a chelating group, particularly a chelating group formed by linking an anti-oxMIF antibody or its antigen-binding fragment to a chelating agent, especially a chelating group selected from DOTA, hexadentate chelating agents (e.g., deferoxamine B, CAS No. 70-51-9), and octadentate chelating agents (e.g., DFO derivative DFO* or its oxygen-containing analogue, such as oxoDFO*).

[0040] Specifically, a DFO can have the following structure:

[0041]

[0042] Specifically, DFO* can have the following structure:

[0043]

[0044] According to the present invention, any alpha- or beta-radioactive isotope used for therapeutic applications can be linked to an anti-oxMIF antibody; specifically, the radioactive isotope can be... 11 C 13 N、 15 O, 18F, 32 P, 64 Cu、 67 Cu、 67 Ga、 89 Zr、 90 Y、 99 mTc, 103 Pd, 105 Rh、 109 Pd, 111 Ag、 111 In、 123 I, 124 I, 125 I, 131 I, 140 La、 149 Tb, 149 Pm, 153 Sm、 159 Gd, 165 Dy、 166 Dy、 166 Ho、 169 Yb、 175Yb、 177 Lu、 227 Th、 186 Re、 188 Re、 192 Ir、 193 mPt, 195 mPt, 198 Au、 199 Au、 211 At、 212 Pb, 212 Bi、 213 Bi、 225 Ac、 223 Ra、 227 Th, specifically, radioactive isotopes are 67 Ga、 89 Zr、 111 In、 124 I, 131 I, 177 Lu、 225 Ac.

[0045] In one specific embodiment, a radioimmunoconjugate as described herein is provided comprising an Fc variant domain having a constant domain of wild-type human IgG1 (SEQ ID NO:37), comprising at least one amino acid substitution at any of positions L234, L235, G236, G237, N297, L328, or P329, wherein the radioimmunoconjugate exhibits reduced binding to the Fcγ-receptor compared to a radioimmunoconjugate comprising the wild-type IgG1 Fc region.

[0046] In another specific embodiment, the radioimmunoconjugate comprises the Fc variant domain of the wild-type human IgG1 constant domain of SEQ ID NO:37, which contains one, two, or three amino acid substitutions at any of positions I253, H310, and H435, wherein the radioimmunoconjugate exhibits reduced affinity for human FcRn compared to radioimmunoconjugates containing the wild-type IgG1 Fc region.

[0047] Specifically, the radioimmunoconjugate comprises SEQ ID NO:38 and exhibits reduced affinity for human FcRn compared to radioimmunoconjugates containing the wild-type IgG1 Fc region.

[0048] In one specific embodiment, the radioimmunoconjugate comprises an amino acid sequence selected from the group consisting of SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36.

[0049] In another specific embodiment, the radioimmunoconjugate comprises any one of SEQ ID NO:29 and SEQ ID NO:34; SEQ ID NO:30 and SEQ ID NO:33, SEQ ID NO:30 and SEQ ID NO:34, SEQ ID NO:31 and SEQ ID NO:34, SEQ ID NO:31 and SEQ ID NO:36, or SEQ ID NO:31 and SEQ ID NO:33, and SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:40.

[0050] More specifically, the radioimmunoconjugates described herein may be, but are not limited to, IgG, IgG-fusion protein, Fv, scFv, scFv-fusion protein, biantibody, biantibody-fusion protein, Fab, Fab fusion protein, scFab, scFab fusion protein, Fab' and F(ab')2, Fab'-SH, Fcab, Fcab fusion protein, fusion protein of two or more single-chain antibodies, microantibodies, and small immune proteins (SIPs).

[0051] According to one implementation scheme, the radioimmunoconjugate described herein is used to prepare a drug.

[0052] In one specific embodiment, this document provides a pharmaceutical composition comprising the radioimmunoconjugate described herein, optionally including a drug carrier or adjuvant.

[0053] Specifically, the pharmaceutical composition is formulated for intravenous administration.

[0054] According to another embodiment, the pharmaceutical composition is used to treat a subject suffering from cancer, particularly for the treatment of tumors, especially hematologic malignancies or solid tumors, and more particularly for the treatment of colorectal cancer, ovarian cancer, breast cancer, brain tumors, prostate cancer, pancreatic cancer, and lung cancer.

[0055] One aspect of the invention relates to the use of an effective amount of the pharmaceutical composition of the invention in a method for treating any of the diseases or symptoms listed herein.

[0056] One embodiment of the present invention relates to the use of the radioimmunoconjugate of the present invention in combination with other therapies or in addition to other therapies.

[0057] In one embodiment of the invention, other therapies are selected from chemotherapy, monoclonal antibody therapy, surgery, radiotherapy and / or photodynamic therapy.

[0058] According to one implementation, the radioimmunoconjugate described herein is used to detect or measure oxMIF levels in cancer cells of a subject, specifically, the detection is performed ex vivo in a subject sample. Potential patients can then be subgrouped (layers, blocks) based on oxMIF levels through stratification, where each layer represents a specific subset of the patient population.

[0059] Specifically, radioimmunoconjugates are used for cancer diagnosis in subjects.

[0060] In one embodiment of this document, a method for in vitro diagnosis of cancer is provided, wherein a radioimmunoconjugate is used to detect tumor cells in a subject sample in vitro, or to detect tumor cells or tumors in a subject. The sample may be, for example, a blood sample, a tissue sample, such as a tissue sample from a biopsy.

[0061] This article also provides a method for diagnosing cancer, wherein the radioimmunoconjugate is used to detect tumor cells in a subject or in an in vitro subject sample.

[0062] Another implementation involves a diagnostic kit containing the radioimmunoconjugate described herein.

[0063] In another embodiment, a kit for producing a radioimmunoconjugate is provided, comprising two or more vials, one vial containing a conjugate comprising a chelating agent linked to an anti-ox-MIF antibody, and a second vial containing a radioisotope specifically selected from... 67 Ga、 89 Zr、 111 In、 124 I, 131 I, 177 Lu、 225 Ac. Specifically, the contents of one or more vials are in lyophilized or solution form. More specifically, the kit also includes a manual containing instructions for preparing the radioimmunoconjugates described herein.

[0064] This article also provides methods for treating cancer using the radioimmunoconjugates described herein or pharmaceutical compositions containing said radioimmunoconjugates.

[0065] Also provided are isolated nucleic acids of recombinant anti-oxMIF antibodies or antigen-binding fragments thereof encoding the radioimmunoconjugates described herein.

[0066] In another embodiment, the expression vector comprises a nucleic acid molecule encoding a recombinant anti-oxMIF antibody or an antigen-binding fragment thereof that encodes a radioimmunoconjugate.

[0067] In another embodiment, a host cell is provided containing a nucleic acid comprising the oxMIF antibody described herein or an expression vector comprising the nucleic acid molecule described herein.

[0068] In another embodiment, a method for producing a radioimmunoconjugate is provided, comprising expressing a nucleic acid encoding an oxMIF antibody or antigen-binding fragment in a host cell, and further labeling the antibody or antigen-binding fragment with a radioisotope. Attached Figure Description

[0069] Figure 1 A: DOTA-C0008 purity and integrity analysis; B: DOTA-C0008 SE-HPLC chromatogram (λ=280nm). C: Coomassie stained SDS PAGE: 1: Molecular weight standard; 2: C0008 non-reduced; 3: C0008 reduced; 4: Concentrated C0008 (labeled batch) non-reduced; 5: Concentrated C0008 (labeled batch) reduced; 6: DOTA-C0008 non-reduced; 7: DOTA-C0008 reduced.

[0070] Figure 2 The number of DOTA molecules grafted onto C0008 was determined by ITLC: DOTA-C0008 was separated by ITLC, and DOTA-C0008 was reacted with tracer amounts of radioactive indium ( 111 In) and stable indium ( 115 Incubate together with a mixture of (In) 111 / 115 In an overdose of 2-fold (lane 6), 4-fold (lane 7), 6-fold (lane 8), or 8-fold (lane 9) of DOTA conjugate antibody.

[0071] Figure 3 Analysis of radiolabeled C0008 by ITLC and autoradiography: A: ITLC of radiolabeled C0008 (lane 3: 177 The labeling reaction of Lu with DOTA-C0008; Lane 4: 177 B: Autoradiography of the ITLC film, lane 3 (after DTPA addition) 177 Lu-DOTA-C0008; Peak 1 reflects free... 177 Lu-DTPA, peak 2 reflects 177 Lu-DOTA-C0008).

[0072] Figure 4 : 177 Plasma stability (autoradiography) of Lu-DOTA-C0008: 177 Lu-DOTA-C0008 incubated in mouse plasma for 7 days showed autoradiography; 1: 177Lu-DOTA-C0008 derivatives and free 177 Lu; 2: 177 Lu-C0008.

[0073] Figure 5 : 177 Plasma stability of Lu-DOTA-C0008 (SDS-PAGE): mouse plasma 177 Reducing SDS-PAGE of Lu-DOTA-C0008 (Left: Coomassie staining; Right: Autoradiography). Lanes 1 and 10: Molecular weight standards; Lane 2: DOTA-C0008; Lane 3: In buffer solution. 177 Lu-DOTA-C0008(T0); Lane 5: 177 Lu-DOTA-C0008 (T0 hours in plasma); Lane 6: 177 Lu-DOTA-C0008 (1 day in plasma), Lane 7: 177 Lu-DOTA-C0008 (2 days in plasma); Lane 8: 177 Lu-DOTA-C0008 (3 days in plasma); Lane 9: 177 Lu-DOTA-C0008 (7 days in plasma).

[0074] Figure 6 Biodistribution of IRDye 800CW-labeled C0008 in female Balb / c mice carrying subcutaneous CT26 tumors: In vivo infrared imaging of mice carrying subcutaneous CT26 tumors. Infrared images of mice taken at 1, 6, 24, 48, 72, 96, and 168 hours after injection of A: IRDye 800CW-labeled C0008 (5 mg / kg) or B: untreated mice (scale bars applicable to A and B); C: tumor penetration and retention of IRDye 800CW-labeled C0008 quantified by digital image analysis.

[0075] Figure 7 Chromatographic results demonstrate reduced aggregation and hydrophobicity of the newly designed anti-oxMIF antibody. (A) Elution curves of C0008 (control antibody, gray area) and the newly designed antibody were compared on a Superdex 200increase 10 / 300GL gel filter column using 5xPBST as the mobile phase. (B) Elution curves of C0008 (control antibody, gray area) and the newly designed antibody were compared on an Enrich 650 gel filter column using 1xPBS as the mobile phase. (C) Elution curves of C0008 (control antibody, gray area) and the newly designed antibody were compared on a HiTrap butyl HP HIC column.

[0076] Figure 8 The binding curve of the newly designed anti-oxMIF antibody to immobilized MIF (K) D (Assay). C0008 is used as a reference antibody.

[0077] Figure 9 The newly designed antibody showed differential binding to oxMIF and redMIF. C0008 was used as a reference antibody.

[0078] Figure 10 The newly designed anti-oxMIF antibody C0083 showed increased yield compared to the control antibody C0008.

[0079] Figure 11 In vivo infrared imaging of mice carrying subcutaneous CT26 tumors compared the tumor penetration of newly designed anti-oxMIF antibodies C0083 and C0008. A: Infrared images of mice taken at 1, 6, 24, 48, 72, 96, and 168 hours after injection of 5 mg / kg of IRDye 800CW-labeled C0083; B: Tumor penetration and retention of IRDye 800CW-labeled C0083 quantified by digital image analysis; C: Tumor penetration and retention of IRDye 800CW-labeled C0083 compared to C0008 quantified by digital image analysis.

[0080] Figure 12 A: Purity and integrity analysis of DOTA-C0083; B: SE-HPLC chromatogram of DOTA-C0083 (λ=280nm). C: Coomassie staining SDS PAGE: 1: Molecular weight standard; 2: Non-reduced C0083; 3: Reduced C0083; 5: Non-reduced DOTA-C0083; 6: Reduced DOTA-C0083.

[0081] Figure 13 The number of DOTA molecules grafted onto C0083 was determined by ITLC: DOTA-C0083 was separated by ITLC, and the radioactivity of DOTA-C0083 was traced in 0.04N HCl. 177 Lu and stable lutetium ( 175 Incubate together with a mixture of Lu) 177 / 175 Lu is 2-fold (lane 1), 4-fold (lane 2), 6-fold (lane 3), or 8-fold (lane 4) overdosage of DOTA conjugate antibody.

[0082] Figure 14 Analysis of radiolabeled components using ITLC and autoradiography 177 Lu-DOTA-C0083: Autoradiography of ITLC thin films (after DTPA addition) 177Lu-DOTA-C0083; Peak 1 / 2 reflects free... 177 Lu-DTPA and 177 Lu-DOTA-C0083 derivative, peak 3 reflects 177 Lu-DOTA-C0083).

[0083] Figure 15 : 177 Plasma stability (autoradiography) of Lu-DOTA-C0083: 177 Autoradiography of Lu-DOTA-C0083 after 7 days of incubation in mouse plasma; 1: 177 Lu-DOTA-C0083 derivatives and free 177 Lu; 2: 177 Lu-DOTA-C0083.

[0084] Figure 16 : 177 Plasma stability of Lu-DOTA-C0083 (SDS-PAGE): Mouse plasma showed [symptoms related to...]. 177 Reducing SDS-PAGE of Lu-DOTA-C0083 (Left: Coomassie staining; Right: Autoradiography). Lane 1: Molecular weight standard; Lane 2: In buffer solution 177 Lu-DOTA-C0083(T0); Lane 3: 177 Lu-DOTA-C0083 (T0 hours in plasma); Lane 4: 177 Lu-DOTA-C0083 (1 day in plasma), Lane 5: 177 Lu-DOTA-C0083 (3 days in plasma); Lane 6: 177 Lu-DOTA-C0083 (7 days in plasma).

[0085] Figure 17 amino acid sequence

[0086] Figure 18 Chromatographic results demonstrate reduced aggregation and hydrophobicity of the newly designed Fc-silencing anti-oxMIF antibodies. (A) Elution curves of C0008 (control antibody, gray area) and the newly designed Fc-silencing antibodies C0115 and C0118 on an Enrich 650 gel filter column using 1xPBS as the mobile phase; (B) Elution curves of C0008 (control antibody, gray area) and the newly designed Fc-silencing antibodies C0115 and C0118, as well as their parental antibodies C0083 and C0090 (without Fc silencing), on a HiTrap butyl HP HIC column.

[0087] Figure 19The binding curve of the newly designed Fc-silencing anti-oxMIF antibody to immobilized oxMIF (K) D (Assay). Anti-oxMIF antibody was detected by anti-human IgG(Fc)-HRP conjugate, with C0008 used as a reference antibody.

[0088] Figure 20 The newly designed Fc silencing antibody was differentially bound to oxMIF and redMIF. C0008 was used as a reference antibody, and isotype IgG was used as a negative control.

[0089] Figure 21 FACS assays showed that the newly designed Fc-silenced anti-oxMIF antibody was effective against A2780 MIF. - / - Non-specific binding to cells was reduced. The newly designed anti-oxMIF antibody C0118 and its parental antibodies C0090(A) and C0115 and its parental antibody C0083(B), the control antibody C0008, and isotype IgG as a negative control were used to target A2780 MIF. - / - Cells were stained; the geometric mean of live cells (average fluorescence intensity of AF488) was plotted against antibody concentration.

[0090] Figure 22 Reporter gene analysis revealed significantly reduced ADCC effector function in the newly designed Fc silencing antibodies C0115 and C0118. ADCC reporter gene bioassays were performed using engineered Jurkat effector cells stably expressing FcyRIIIA and HCT116-pMIF target cells, comparing the newly designed Fc silencing antibodies C0115 and / or C0118 with their parental antibodies C0083 and C0090, which possess wild-type Fc. Mean values ​​and SEM (n=2) are shown.

[0091] Figure 23 In vivo infrared imaging of mice carrying subcutaneous HCT116 tumors to demonstrate tumor penetration and retention of the newly designed Fc-silenced anti-oxMIF antibody C0115 and reference antibody C0008. (A) Infrared images of mice taken at 1, 6, 24, 48, 72, 96, and 168 hours after injection of IRDye 800CW-labeled antibodies C0115 (top) and C0008 (bottom) at doses of 5 mg / kg; (B) Tumor penetration and retention of C0115 and C0008 quantified by digital image analysis.

[0092] Figure 24 In vivo PET / CT imaging of mice carrying subcutaneous CT26 or HCT116 tumors, coupled with DFO* 89Tumor penetration and preservation of newly designed Zr-labeled Fc-silencing anti-oxMIF antibodies C0115 and C0118. (Injection) 89 PET and CT images of mice taken 4, 7, and 10 days after administration of the Zr-DFO*-labeled antibody at a dose of approximately 10 MBq per mouse; representative digital vertical cross-sections of the mice are shown, with white arrows marking tumors. Detailed Implementation

[0093] Unless otherwise stated or defined, all terms used herein have their ordinary meaning in the art, which will be clear to those skilled in the art. References include, for example, standard manuals such as Sambrook et al., "Molecular Cloning: A Laboratory Manual" (4th Ed.), Vols. 1-3, Cold Spring Harbor Laboratory Press (2012); Krebs et al., "Lewin's Genes XI", Jones & Bartlett Learning, (2017); and Murphy & Weaver, "Janeway's Immunobiology" (9th Ed., or more recent editions), Taylor & Francis Inc, 2017.

[0094] The subject matter of the claims specifically relates to artificial products or methods of using or producing such artificial products, which may be variations of natural (wild-type) products. Although there may be a degree of sequence identity with natural structures, it is well understood that the materials, methods, and uses of the present invention, such as 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 construed as a result of “natural laws”.

[0095] The terms “comprise,” “contain,” “have,” and “include” used in this document can be used as synonyms and should be understood as open-ended definitions that allow for more members, parts, or elements. “Consisting” is considered the most closed definition, without any other elements characteristic of the constitutive definition. Therefore, “comprise” is broader and encompasses the definition of “consisting.”

[0096] The term “about” as used in this article refers to the same value or a value that differs from a given value by + / - 5%.

[0097] As used herein and in the claims, the singular forms, such as “a”, “an” and “the”, include the plural unless the context clearly specifies otherwise.

[0098] As used in this article, amino acids refer to the 20 naturally occurring amino acids encoded by 61 triplet codons. These 20 amino acids can be categorized into neutral, positive, and negatively charged amino acids:

[0099] The following are the "neutral" amino acids and their corresponding three-letter and one-letter codes and polarities: 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 (IIe, I; nonpolar, neutral), Leucine (Leu, L; nonpolar, neutral), Methionine (Met, M; nonpolar, neutral), phenylalanine (Phe, F; nonpolar, neutral), proline (Pro, P; nonpolar, neutral), serine (Ser, S; polar, neutral), threonine (Thr, T; polar, neutral), tryptophan (Trp, W; nonpolar, neutral), tyrosine (Tyr, Y; polar, neutral), valine (Val, V; nonpolar, neutral), and histidine (His, H; polar, positively charged (10%), neutral (90%)).

[0100] The amino acids with a "positive charge" are arginine (Arg, R; polar, positive charge) and lysine (Lys, K; polar, positive charge).

[0101] The amino acids with a "negative charge" are: aspartic acid (Asp, D; polar, negative charge) and glutamic acid (Glu, E; polar, negative charge).

[0102] The term "immunoconjugate" refers to a conjugate of an anti-oxMIF antibody or its antigen-binding fragment and second part, and the term "radioimmunoconjugate" refers to a conjugate of an anti-oxMIF antibody or its antigen-binding fragment and a radioactive isotope (radionucleoside).

[0103] Radioactive isotopes can be radioactive nuclides that emit beta rays, alpha rays, or positrons, including but not limited to... 11 C 13 N、 15 O, 18F, 32 P, 64 Cu、 67 Cu、 67 Ga、 89 Zr、 90 Y、 99 mTc, 103Pd, 105 Rh, 109 Pd, 111 Ag, 111 In, 123 I, 124 I, 125 I, 131 I, 140 La, 149 Tb, 149 Pm, 153 Sm, 159 Gd, 165 Dy, 166 Dy, 166 Ho, 169 Yb, 175 Yb, 177 Lu, 227 Th, 186 Re, 188 Re, 192 Ir, 193 mPt, 195 mPt, 198 Au, 199 Au, 211 At, 212 Pb, 212 Bi, 213 Bi, 225 Ac, 223 Ra, 227 Th, specifically, the radioisotope is 67 Ga, 89 Zr, 111 In, 124 I, 131 I, 177 Lu, 225 Ac.

[0104] Useful alpha-emitting radionuclides may include but are not limited to 149 Tb, 211 At, 212 Bi, 213 Bi and 225 Ac.

[0105] A particularly useful beta emitter is 177 Lu.

[0106] Positron emission or beta+ decay (β + decay) is a subtype of radioactive decay, called beta decay, in which a proton inside the radionuclide nucleus is converted into a neutron while releasing a positron and an electron neutrino (ν ePositron emission tomography (PET) uses radionuclides that emit electrons.

[0107] A particularly useful positron emitter is 11 C 13 N、 15 O、 18 F or 89 Zr, specifically 89 Zr.

[0108] Any type of connector with sufficient chelating ability and functional groups that allow direct or indirect coupling to proteins or peptides can be used. Examples of such connectors for attaching radioisotopes to antibodies are described in the literature (e.g., Brechbiel MW, 2008; Liu S., 2008; Zeglis & Lewis 2011). The radionuclide in this invention is preferably coupled directly to the anti-oxMIF antibody or using a bifunctional chelating agent. These chelating agents can be cyclic, linear, or branched. Particular reference can be made to polyaminopolyacid chelating agents comprising a linear, cyclic, or branched polyazine backbone with an acidic group (e.g., carboxylalkyl) attached to the nitrogen atom of the backbone.

[0109] Some useful, non-limiting examples are cyclic chelating agents or bifunctional chelating agents thereof, such as diethyltriaminepentaacetic acid (DTPA) and 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid, ca-DTPA, ibca-DTPA, 1B4M-DTPA, lys-DTPA, vinyl DTPA, glu-DTPA, p-SCN-bn-DOTA, DOTA-NHS-ester, deferoxamine B, or derivatives thereof; or linear chelating agents or bifunctional chelating agents thereof, such as p-SCN-Bn-DTPA, HOPO, and CH X-A″-DTPA, ethylenediaminetetraacetic acid (EDTA), DTPA, EDTMP, NOTA, TETA, DOTMP, N2S2, N3S, HEDP. Examples of other suitable chelating agents include DOTA derivatives, such as p-isothiocyanobenzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (p-SCN-Bz-DOTA), and DTPA derivatives, such as p-isothiocyanobenzyl-diethylenetriaminepentaacetic acid (p-SCN-Bz-DTPA), the former being a cyclic chelating agent and the latter a linear chelating agent.

[0110] Further examples of deferoxamine (DFO) and its derivatives include, but are not limited to, p-NCS-Bz-DFO, DFOSq, DFO*, oxoDFO*, DFO*Sq, DFO*-NCS, and DFO*pPhe-NCS.

[0111] Purification can then be performed to remove unconjugated chelating agents, and after the chelating agent antibody conjugate reacts with the radionuclide, purification can be performed to remove any unconjugated radionuclides. Alternatively, the chelating agent and radionuclide can be bound first, followed by conjugation with the antibody.

[0112] The radiolabeling process may be more convenient if the chelating agent is conjugated to the antibody prior to radiolabeling. The principle of preparing radiolabeled conjugates using chelating agents linked to antibodies is described more extensively in, for example, Liu S., 2008.

[0113] The antibody or antigen-binding fragment of the radioimmunoconjugate contains at least one binding site that specifically recognizes oxMIF. These antibodies are described in Kerschbaumer R. et al., 2012. Anti-oxMIF antibodies are specific for the β-sheet structure in MIF, which includes a highly conserved catalytic motif of a thiol protein oxidoreductase. 57 Cys-Ala-Leu-Cys 60 (SEQ ID NO:44), which is associated with the biological function of MIF (Kerschbaumer R. et al., 2012). This article describes a CDR specific to oxMIF.

[0114] Imarumab (C0008) CDR contains SEQ ID NO:7 to SEQ ID NO:12.

[0115] Alternatively, the CDR of other antibodies that specifically recognize oxMIF includes SEQ ID NO:1 to SEQ ID NO:6 (Bax8), or SEQ ID NO:13 to SEQ ID NO:18 (Bax74), or SEQ ID NO:19 to SEQ ID NO:24 (Bax94), or SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:21 (BaxA10), or any sequence having at least 80%, 90%, or 95% sequence identity with SEQ ID NO:7 to SEQ ID NO:12, SEQ ID NO:1 to SEQ ID NO:6, SEQ ID NO:13 to SEQ ID NO:18, or SEQ ID NO:19 to SEQ ID NO:24, or SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:21.

[0116] In the case of radioimmunoconjugates, reduced aggregation tendency and / or hydrophobicity are particularly important, as any aggregation and / or highly hydrophobic interface can lead to nonspecific binding of the radioimmunoconjugate, resulting in nonspecific binding of the radionuclide to non-tumor tissue.

[0117] In one specific implementation, due to the targeted amino acid substitutions in the variable heavy and light chain domains, the anti-oxMIF antibody or its antigen-binding fragment of the radioimmunoconjugate exhibits reduced aggregation tendency and reduced hydrophobicity compared to the unmodified antibody lacking said amino acid substitutions.

[0118] The reduced aggregation potential and hydrophobicity are due to amino acid substitutions at selected positions within the antibody variable domains described herein.

[0119] The level of antibody aggregation can be measured using a variety of known techniques, including mass spectrometry, volume exclusion chromatography (SEC), dynamic light scattering (DLS), light obscuration (LO), dynamic imaging particle analysis (DIPA) techniques such as microfluidic imaging (MFI) and Coulter counter (CC), and differential scanning fluorescence assay (DSF).

[0120] The reduced hydrophobicity and reduced aggregation potential used herein refer to the reduced surface hydrophobicity and reduced aggregation potential of the radioimmunoconjugate antibody of the present invention compared to the surface hydrophobicity and aggregation potential of imarumab comprising SEQ ID NO:29(VH), SEQ ID NO:32(VL), SEQ ID NO:37(CH1-CH3), and SEQ ID NO:41(CL). The sequence of C0008 is included in the sequence of imarumab published in the proposed INN list 111 (WHO Drug Information, Vol. 28, No. 2, 2014), but lacks the C-terminal lysine. Measurements can be performed using various known techniques, including but not limited to hydrophobic interaction chromatography (HIC) or affinity capture-from-interaction nanoparticle spectroscopy (AC-SINS, Estep P. et al., 2015).

[0121] In one embodiment, the oxMIF antibody, which has reduced aggregation potential and reduced hydrophobicity, specifically comprises:

[0122] The light chain variable domain, referring to SEQ ID NO:32 and according to the Kabat number, has 1, 2, 3, 4 or 5 amino acid substitutions from M30L, F49Y, A51G, P80S, and W93F, or

[0123] A light chain variable domain having at least 95%, particularly at least 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:32, wherein the light chain variable domain comprises a native tyrosine residue at position 36 and one, two, three, four, or five amino acid substitutions from M30L, F49Y, A51G, P80S, and / or W93F, or

[0124] In conjunction with a heavy chain variable structural domain, the heavy chain variable structural domain comprises SEQ ID NO:29, or

[0125] In conjunction with the heavy chain variable domain, referring to SEQ ID NO:29 and according to the Kabat number, the heavy chain variable domain comprises amino acid substitutions for L5Q and / or W97Y, or

[0126] The heavy chain variable domain contains at least 95%, particularly at least 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:29, and also contains at least one amino acid substitution of L5Q or W97Y.

[0127] In an alternative embodiment, the light chain variable domain comprising SEQ ID NO:32 has 1, 2, 3, 4, or 5 amino acid substitutions, provided that the native tyrosine at position 36 is retained, and 1, 2, 3, 4, or 5 amino acids at positions M30, F49, A51, P80, or W93 are additionally substituted.

[0128] In another embodiment, the radioimmunoconjugate anti-oxMIF antibody has reduced aggregation potential and reduced hydrophobicity, and specifically comprises:

[0129] Heavy chain variable structural domain, the heavy chain variable structural domain comprising SEQ ID NO:29 and amino acid substitution W97Y or amino acid substitution L5Q and W97Y, or

[0130] The heavy chain variable domain comprises SEQ ID NO:29 having 1, 2, 3, 4 or 5 amino acid substitutions, and further comprises amino acid substitution W97Y, optionally combined with L5Q.

[0131] The native tyrosine residue at position 36 of the light chain remains unmodified to preserve the binding properties of the anti-oxMIF antibody. Any modification at this amino acid position may result in undesirable or impaired binding properties.

[0132] The radioimmunoconjugates described herein specifically comprise a light chain variable domain having one of the following amino acid substitution combinations: M30L, F49Y, A51G, P80S, and W93F; F49Y, A51G, and W93F; F49Y and A51G, referring to the amino acid numbers of SEQ ID NO:32.

[0133] The radioimmunoconjugates of the present invention may comprise any of the following combinations of variable light chain variable domains and heavy chain variable domains:

[0134] SEQ ID NO:33 and SEQ ID NO:30,

[0135] SEQ ID NO:34 and SEQ ID NO:29,

[0136] SEQ ID NO:34 and SEQ ID NO:30,

[0137] SEQ ID NO:34 and SEQ ID NO:31,

[0138] SEQ ID NO:35 and SEQ ID NO:29,

[0139] SEQ ID NO:35 and SEQ ID NO:30, SEQ ID NO:31,

[0140] SEQ ID NO:36 and SEQ ID NO:29,

[0141] SEQ ID NO:36 and SEQ ID NO:30, SEQ ID NO:31.

[0142] In another embodiment, the anti-oxMIF antibody comprises any of the following sequence combinations:

[0143] SEQ ID NO:37, SEQ ID NO:30, SEQ ID NO:33 and SEQ ID NO:41,

[0144] SEQ ID NO:37, SEQ ID NO:31, SEQ ID NO:33 and SEQ ID NO:41,

[0145] SEQ ID NO:37, SEQ ID NO:29, SEQ ID NO:34 and SEQ ID NO:41,

[0146] SEQ ID NO:37, SEQ ID NO:30, SEQ ID NO:34 and SEQ ID NO:41,

[0147] SEQ ID NO:37, SEQ ID NO:31, SEQ ID NO:34 and SEQ ID NO:41,

[0148] SEQ ID NO:37, SEQ ID NO:29, SEQ ID NO:35 and SEQ ID NO:41,

[0149] SEQ ID NO:37, SEQ ID NO:30, SEQ ID NO:35 and SEQ ID NO:41,

[0150] SEQ ID NO:37, SEQ ID NO:31, SEQ ID NO:35 and SEQ ID NO:41,

[0151] SEQ ID NO:37, SEQ ID NO:29, SEQ ID NO:36 and SEQ ID NO:41,

[0152] SEQ ID NO:37, SEQ ID NO:30, SEQ ID NO:36 and SEQ ID NO:41, or

[0153] SEQ ID NO:37, SEQ ID NO:31, SEQ ID NO:36 and SEQ ID NO:41.

[0154] In one specific embodiment, due to amino acid substitutions at selected positions in the heavy chain constant region, particularly the CH2 region, the anti-oxMIF antibody of the radioimmunoconjugate of the present invention exhibits reduced binding to Fcγ receptor-bearing cells (reduced binding to FcγRI, FcγRII, and FcγRIII).

[0155] As used in this article, "effective function" refers to a biochemical event resulting from the interaction between the antibody's Fc region and the Fc receptor or ligand. Effector functions include, but are not limited to, ADCC, ADCP, and CDC.

[0156] As used herein, "effective cells" refers to immune system cells that express one or more Fc receptors and mediate 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 can be derived from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. According to the present invention, the anti-oxMIF antibodies described herein possess silenced effector functions due to amino acid substitutions at selected positions in the heavy chain constant region, particularly the Fc region. The reduction or silencing of effector functions of these antibodies due to decreased complement-mediated and FcγR-mediated activity may include a reduction or elimination of complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and / or antibody-dependent phagocytosis (ADCP).

[0157] As used herein, the term "Fc" or "Fc region" (or fragment crystallizable region) refers to a polypeptide containing the constant region of an antibody, excluding the first constant region, the immunoglobulin domain (CH1 domain), and in some cases, the hinge portion. The Fc region refers to the C-terminal region of the antibody. The Fc region consists of two identical protein fragments derived from the second and third constant domains of the antibody's two heavy chains (chain A and chain B), respectively. The second and third constant domains are referred to as the CH2 and CH3 domains, respectively. The CH2 domain contains the CH2 domain sequences of chain A and chain B. The CH3 domain contains the CH3 domain sequences of chain A and chain B. As used herein, the Fc region includes the hinge region or a portion thereof.

[0158] According to EU designations, the "CH2 domain" of the human IgG Fc region sequence typically extends from approximately amino acid 231 to approximately amino acid 340. The CH2 domain sequence is unique because it does not pair tightly with another domain sequence. Instead, two N-linked branched hydrocarbon chains are inserted between the two CH2 domain sequences of the intact natural IgG molecule.

[0159] The “CH3 domain” contains a segment of the Fc region sequence from the C-terminus of the residue to the CH2 domain sequence (i.e., approximately amino acid residue 341 to approximately amino acid residue 447 of IgG according to the EU number).

[0160] The “functional Fc region” possesses the “effective functions” of the natural Fc region. Exemplary “effective functions” include C1 q binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); etc. Such effector functions typically require the Fc region to bind to a binding domain (e.g., an antibody variable domain) and can be evaluated using various assays known in the art and disclosed herein.

[0161] The “natural Fc region” contains amino acid sequences identical to those found in naturally occurring Fc regions. Natural human Fc regions include the natural human lgG1 Fc region (non-A allotype and A allotype); the natural human lgG2 Fc region and the natural human lgG3 Fc region and their naturally occurring variants.

[0162] The “variant Fc region” comprises an amino acid sequence that differs from the native Fc region sequence due to “one or more amino acid substitutions”. Compared to the native Fc region sequence or the Fc region sequence of the parent polypeptide, the variant Fc region sequence has at least one amino acid substitution, for example, about 1 to about 20 amino acid substitutions, preferably about 1 to about 17 amino acid substitutions, in the native Fc region sequence or the Fc region sequence of the parent polypeptide. In some embodiments, the variant Fc region sequence herein has at least about 80% identity with the native Fc region sequence and / or with the Fc region sequence of the parent polypeptide, most preferably at least about 90% identity, and more preferably at least about 95% identity.

[0163] In one specific implementation, according to the EU designation, referring to IgG1, the amino acid substitution is located at any of the following positions: E233, L234, L235, G236, G237, P238, D265, S267, H268, N297, S298, T299, E318, L328, P329, A330, and P331. However, according to the EU designation, these silent mutations can also be introduced at the corresponding positions in the Fc of wild-type IgG2, IgG3, or IgG4.

[0164] Modifications to the Fc region that lead to Fc reduction or silencing are known in the art and have been described in Saunders K., 2019 and Liu R. et al., 2020.

[0165] In an alternative embodiment, the amino acid replaces any or all of the following positions in the CH2 domain: L234F, H268Q, K274Q, Y296F, A327G, A330S, P331S, and in the CH3 domain: R355Q, K409R, Q419E, P445L.

[0166] Specifically, the Fc-silencing anti-oxMIF antibody described herein comprises one or more of the following amino acid substitutions or deletions:

[0167] i) L235, G237 and E318, especially L235A, G237A and E318A;

[0168] ii) L234, L235, especially L234A and L235A;

[0169] iii) S228, L235, especially S228P and L235E;

[0170] iv) G236, L328, especially G236R, L328R;

[0171] v)S298, T299, especially S298G, T299A;

[0172] vi) L234, L235, P331, especially L234F, L235E, P331S;

[0173] vii) H268, V309, A330, P331, especially H268Q, V309L, A330S, P331S;

[0174] viii) E233, L234, L235, G236, S267, especially E233P, L234V, L235A, G236del, S267K;

[0175] ix)L234, L235, P329, especially L234A, L235A, P329G;

[0176] x)V234, G237, P238, H268, A330, P331, especially V234A, G237A, P238S, H268A, A330S, P331S;

[0177] xi)L234, L235, D265, especially L234F, L235E, D265A;

[0178] xii)D265, specifically D265A;

[0179] xiii) G237, especially G237A;

[0180] xiv)E318, specifically E318A;

[0181] xv)E233, especially E233P;

[0182] xvi)G236, L328, especially G236R, L328R;

[0183] xvii)L235, especially L235E;

[0184] xviii)L234, L235, P331, especially L234Q, L235F, P331S;

[0185] xix)L234, L235, G237, P238, H268, A330, P331, especially L234A, L235A, G237A, P238S, H268A, A330S, P331S.

[0186] xx)N297, especially N297A, N297Q or N297G, produces glycosylated antibodies.

[0187] Glycosylation, namely O-glycosylation and N-glycosylation, is a post-translational modification of antibodies that can be regulated by a series of B cell stimuli, including environmental factors (e.g., stress or disease), cytokine activity, and innate immune signaling receptors (e.g., Toll-like receptors). The glycosylation pattern of parental antibodies can be modified using methods known in the art. Specifically, O-linked glycosylation sites are located at CH2 and the hinge region.

[0188] The term "unglycosylated" means that the Fc region is not glycosylated. It is known that all constant regions of IgG isotypes are glycosylated at the asparagine residue at position 297, which forms part of the N-glycosylation motif asparagine 297-X 298-serine 299 or threonine 299, where X is any amino acid residue other than proline. The glycan has a heptasaccharide core and variable extensions, such as fucose, galactose, and / or sialic acid. Therefore, the antibody of the present invention can be deglycosylated by replacing asparagine 297 in this constant region with another amino acid that cannot be glycosylated or deglycosylated by enzymatic methods. Any other amino acid residue can be used, but alanine is preferred. Alternatively, glycosylation at position 297 of the asparagine motif can be prevented by altering another residue of the motif, for example by replacing residue 298 with proline, or by replacing residue 299 with any amino acid other than serine or threonine. The techniques for performing such site-directed mutagenesis are known to those skilled in the art, and can be performed, for example, using commercially available site-directed mutagenesis kits.

[0189] The term "silent Fc" refers to the Fc region of an antibody whose effector function is reduced or eliminated due to modifications such as amino acid substitutions or glycosylation patterns. This results in the modified glycans reducing or eliminating the antibody's binding to any FcγR and to complement factor C1 q proteins, such as FcγRIIaH, FcγRIIaR, FcyRIIb, FcyRIIIaF, FcyRIIIaV, and FcyRIa receptors. This reduction or elimination of binding leads to a decrease or elimination of effector function normally mediated by the wild-type IgG Fc region.

[0190] The term "Fc nullification" may be used herein if FcγR binds to any of the receptors, such as FcγRIIaH, FcγRIIaR, FcyRIIb, FcyRIIIaF, FcyRIIIaV, and FcyRIa, and if its binding to complement factor C1 q protein is completely eliminated.

[0191] Significant Fc silencing can be achieved through combinatorial mutations. L234 and L235, residues located near the hinge region, reduce FcyR binding when substituted with alanine. For example, the combination of L234A and L235A with P329G can result in almost complete suppression of FcyR interactions in all isoforms.

[0192] Compared to the parent peptide or a peptide containing a natural Fc region sequence, Fc-silencing antioxMIF antibodies or their antigen-binding fragments exhibit significantly reduced, silenced, negligible, or eliminated FcyR binding affinity and C1q binding affinity, and thus reduced FcyR and C1q binding activity. In some embodiments, Fc-silencing antioxMIF antibodies or their antigen-binding fragments exhibiting significantly reduced, silenced, negligible, or eliminated FcyR binding affinity and C1q binding affinity, compared to the parent peptide or a peptide containing a natural Fc region sequence, also exhibit significantly reduced, silenced, negligible, or eliminated ADCC, ADCP, and CDC activities. Fc-silencing antioxMIF antibodies or their antigen-binding fragments (exhibiting reduced or undetectable binding to FcyR) bind all FcyRs with lower binding strength compared to the parent peptide. Such variants exhibiting reduced FcyR binding may have little or no significant FcyR binding ability. In one specific embodiment, the variant exhibits binding to 0-20% of FcyR compared to the native IgG Fc region, as measured, for example, by a change in the equilibrium constant. In one embodiment, the variant exhibits binding to 0-10% of FcyR compared to the native IgG Fc region. In one embodiment, the variant exhibits binding to 0-5% of FcyR compared to the native IgG Fc region. In one embodiment, the variant exhibits binding to 0-1% of FcyR compared to the native IgG Fc region.

[0193] The antibodies described in this article that have complement-silencing activity can be identified by cell-based CDC assays and by, for example, SPR or ELISA, by reducing or eliminating binding to C1q.

[0194] Compared with a reference antibody (i.e., an unmodified wild-type antibody, such as C0008), the CDC activity was determined to be reduced or silenced by at least 1.5-fold, specifically at least 2-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, and more specifically at least 10-fold.

[0195] Compared to a reference antibody (i.e., an unmodified wild-type antibody, such as C0008), the determined reduced ADCC or ADCP activity was at least 2, 5, 6, 7, 8, or 9 times, and more specifically at least 10 times.

[0196] Compared to radioimmunoconjugates containing the Fc region of wild-type IgG1, the Fc domain of the radioimmunoconjugate antibody portion exhibiting reduced FcγR binding can specifically include the Fc variant domain of the wild-type human IgG1 constant domain (SEQ ID NO:37), which contains 1, 2, 3, 4, 5, 6 or 7 or more amino acid substitutions, more specifically 1, 2 or 3 amino acid substitutions at any of positions L234, L235, G236, G237, N297, L328 or P329, more specifically including substitutions at positions L234 and L235.

[0197] More specifically, the anti-oxMIF antibody portion contains an Fc variant domain of the wild-type human IgG1 constant region (SEQ ID NO:37), which contains amino acid substitutions at positions L234 and L235, specifically L234A and L235A.

[0198] On the other hand, the reduction or downregulation of FcγR binding by antioxMIF antibodies containing Fc variants is a decrease of 0, 2.5%, 5%, 10%, 20%, 50%, or 75% of the value observed by antioxMIF antibodies containing wild-type Fc regions.

[0199] In vitro assays can be performed to confirm the reduction / elimination of FcγR binding. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcγR binding. Such assays may include, but are not limited to, SPR binding studies or FACS on FcγR-specifically expressing cells.

[0200] In another embodiment, the anti-oxMIF antibody comprises any of the following sequence combinations:

[0201] SEQ ID NO:38, SEQ ID NO:30, SEQ ID NO:33 and SEQ ID NO:41,

[0202] SEQ ID NO:38, SEQ ID NO:31, SEQ ID NO:33 and SEQ ID NO:41,

[0203] SEQ ID NO:38, SEQ ID NO:29, SEQ ID NO:34 and SEQ ID NO:41,

[0204] SEQ ID NO:38, SEQ ID NO:30, SEQ ID NO:34 and SEQ ID NO:41,

[0205] SEQ ID NO:38, SEQ ID NO:31, SEQ ID NO:34 and SEQ ID NO:41,

[0206] SEQ ID NO:38, SEQ ID NO:29, SEQ ID NO:35 and SEQ ID NO:41,

[0207] SEQ ID NO:38, SEQ ID NO:30, SEQ ID NO:35 and SEQ ID NO:41,

[0208] SEQ ID NO:38, SEQ ID NO:31, SEQ ID NO:35 and SEQ ID NO:41,

[0209] SEQ ID NO:38, SEQ ID NO:29, SEQ ID NO:36 and SEQ ID NO:41,

[0210] SEQ ID NO:38, SEQ ID NO:30, SEQ ID NO:36 and SEQ ID NO:41, or

[0211] SEQ ID NO:38, SEQ ID NO:31, SEQ ID NO:36 and SEQ ID NO:41.

[0212] In one specific embodiment, due to amino acid substitution at selected positions in the heavy chain constant region, the antioxMIF antibody of the radioimmunoconjugate of the present invention exhibits reduced binding to the neonatal receptor (FcRn).

[0213] FcRn is expressed by endothelial cells, which internalize serum components, including soluble IgG from the bloodstream, through endocytosis. IgG binding to FcRn is pH-dependent; the acidic pH (pH 6.0) within the endosome compartment allows IgG to bind to FcRn. Upon recirculation back to the cell surface, IgG dissociates from FcRn at physiological pH (approximately pH 7.2), releasing it back into the bloodstream and preventing lysosomal degradation, thus prolonging the IgG half-life. Decreased binding of radioimmunoconjugates to FcRn leads to a reduced circulating half-life and accelerated clearance from the body.

[0214] FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, for example, Petkova, SB, et al., 2006).

[0215] Compared to radioimmunoconjugates containing the Fc region of wild-type IgG1, the Fc domain of the radioimmunoconjugate antibody moiety exhibiting reduced FcRn binding may preferably include an Fc variant domain exhibiting reduced FcγR binding (SEQ ID NO: 38), and the Fc moiety of the radioimmunoconjugate may further include one, two, or three amino acid substitutions at any of positions I253, H310, and H435.

[0216] In a further embodiment, the anti-oxMIF antibody comprises any of the following sequence combinations:

[0217] SEQ ID NO:39, SEQ ID NO:30, SEQ ID NO:33 and SEQ ID NO:41,

[0218] SEQ ID NO:39, SEQ ID NO:31, SEQ ID NO:33 and SEQ ID NO:41,

[0219] SEQ ID NO:39, SEQ ID NO:29, SEQ ID NO:34 and SEQ ID NO:41,

[0220] SEQ ID NO:39, SEQ ID NO:30, SEQ ID NO:34 and SEQ ID NO:41,

[0221] SEQ ID NO:39, SEQ ID NO:31, SEQ ID NO:34 and SEQ ID NO:41,

[0222] SEQ ID NO:39, SEQ ID NO:29, SEQ ID NO:35 and SEQ ID NO:41,

[0223] SEQ ID NO:39, SEQ ID NO:30, SEQ ID NO:35 and SEQ ID NO:41,

[0224] SEQ ID NO:39, SEQ ID NO:31, SEQ ID NO:35 and SEQ ID NO:41,

[0225] SEQ ID NO:39, SEQ ID NO:29, SEQ ID NO:36 and SEQ ID NO:41,

[0226] SEQ ID NO:39, SEQ ID NO:30, SEQ ID NO:36 and SEQ ID NO:41,

[0227] SEQ ID NO:39, SEQ ID NO:31, SEQ ID NO:36 and SEQ ID NO:41.

[0228] In another embodiment, the anti-oxMIF antibody comprises any of the following sequence combinations.

[0229] SEQ ID NO:40, SEQ ID NO:30, SEQ ID NO:33 and SEQ ID NO:41,

[0230] SEQ ID NO:40, SEQ ID NO:31, SEQ ID NO:33 and SEQ ID NO:41,

[0231] SEQ ID NO:40, SEQ ID NO:29, SEQ ID NO:34 and SEQ ID NO:41,

[0232] SEQ ID NO:40, SEQ ID NO:30, SEQ ID NO:34 and SEQ ID NO:41,

[0233] SEQ ID NO:40, SEQ ID NO:31, SEQ ID NO:34 and SEQ ID NO:41,

[0234] SEQ ID NO:40, SEQ ID NO:29, SEQ ID NO:35 and SEQ ID NO:41,

[0235] SEQ ID NO:40, SEQ ID NO:30, SEQ ID NO:35 and SEQ ID NO:41,

[0236] SEQ ID NO:40, SEQ ID NO:31, SEQ ID NO:35 and SEQ ID NO:41,

[0237] SEQ ID NO:40, SEQ ID NO:29, SEQ ID NO:36 and SEQ ID NO:41,

[0238] SEQ ID NO:40, SEQ ID NO:30, SEQ ID NO:36 and SEQ ID NO:41, or

[0239] SEQ ID NO:40, SEQ ID NO:31, SEQ ID NO:36 and SEQ ID NO:41.

[0240] The oxMIF binding site of the antibody described herein is specific to the oxidized form of MIF, i.e., specific to animal, specifically mammalian oxMIF (e.g., but not limited to mouse, rat, monkey, and human oxMIF, specifically human oxMIF), but does not show significant cross-reactivity to reduced MIF. oxMIF is a disease-associated structural isotype of MIF, which can be specifically and significantly detected 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 complementarity-determining regions of the humanized or human anti-oxMIF binding domain described herein, such as CDRs included in SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, or SEQ ID NO:36, and one or more (e.g., all three) heavy chain complementarity-determining regions of the humanized or human anti-oxMIF binding domain described herein, such as SEQ ID NO:29, SEQ ID NO:30, or SEQ ID NO:31.

[0241] The oxMIF binding specificity of radioimmunoconjugates can be determined by any assay suitable for determining selective binding to oxMIF, such as any competitive assay for binding to oxMIF of unlabeled and nonspecific control antibodies (e.g., unlabeled imarumab).

[0242] The term "antibody" is used in the broadest sense herein and encompasses polypeptides or proteins that consist of or contain 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 covers a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies), and antibody fragments, provided they exhibit the desired antigen-binding activity, i.e., binding to oxMIF. The term also covers fusion proteins, such as fusions with immunotoxins or antibody-drug conjugates, such as antibody-drug conjugates binding to oxMIF. Antibody also encompasses full-length antibody forms with an IgG structure, wherein the Fc region is bounded by an Fcab region containing a second, different antigen-binding site. TM replace.

[0243] Antibody domains can be native structures or modified through mutagenesis or derivatization, for example, modifying antigen-binding properties or any other properties, such as stability or functional properties, such as binding to Fc receptors, binding to, for example, FcRn and / or Fc-γ receptors, preferably with modifications reducing binding to Fc-γ receptors and / or FcRn. A polypeptide sequence is considered an antibody domain if it contains a β-barrel structure consisting of at least two β chains of an antibody domain structure linked by a loop sequence.

[0244] It should be understood that the term "antibody" includes antigen-binding derivatives and fragments thereof. A derivative is any combination of one or more antibody domains of the present invention or of antibodies and / or fusion proteins, wherein any domain of the antibody of the present invention may be fused at any site to one or more other proteins (e.g., other antibodies or antibody forms, such as binding structures containing CDR loops, receptor peptides and ligands, scaffold proteins, enzymes, labels, toxins, etc.).

[0245] The term "antibody" should specifically refer to a polypeptide or protein that exhibits the property of binding to the target antigen oxMIF.

[0246] "Antibody fragment" refers to a molecule other than a complete antibody that contains a portion of the complete antibody that binds to the antigen to which the complete antibody is bound. 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, (scFv)2, F(ab')2, biantibodies, cross-Fab fragments; and linear antibodies. In one specific embodiment, the antibody fragment of the radioimmunoconjugate is fused to a silenced Fc-part or a silenced Fc-domain via a hinge region.

[0247] In addition, the antibody fragment contains V H Single-chain polypeptides with domain characteristics, that is, those capable of binding with VL The structural domains are assembled together, or have V L Single-chain polypeptides with domain characteristics, that is, those capable of binding with V H The structural domains assemble together to form functional antigen-binding sites, thereby providing antigen-binding properties for full-length antibodies.

[0248] The antibody fragments referred to in this article may also include silenced Fc domains, which contain one or more loop regions containing antigen-binding regions, such as Fcab. TM Alternatively, it may be a full-length antibody with an IgG structure, wherein the silenced Fc region has been replaced by an Fcab containing a second, different antigen-binding site. TM What it replaced.

[0249] The term "functional variant" or "functionally active variant" also includes naturally occurring allelic variants, as well as mutants or any other non-naturally occurring variants. As known in the art, an allelic variant, or also called a homolog, is an alternative form of a nucleic acid or peptide, characterized by having substitutions, deletions, or additions of one or more nucleotides or amino acids that substantially do not alter the biological function of the nucleic acid or peptide. Specifically, a functional variant may contain 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 combinations thereof, which are conserved modifications and do not alter antigen-binding properties. Specifically, the functional variants described herein contain 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 alter the antibody's function. Specifically, the functionally active variants described herein contain up to 15, preferably up to 10 or 5, amino acid substitutions, deletions, and / or additions, which are conservative modifications and do not alter the antibody's function.

[0250] Functional variants can be obtained through sequence alterations in a polypeptide or nucleotide sequence, such as through one or more point mutations, wherein the sequence alteration, when used in combination with the present invention, preserves or improves the function of the unchanged polypeptide or nucleotide sequence. Such sequence alterations may include, but are not limited to, (conserved) substitutions, additions, deletions, mutations, and insertions.

[0251] Conservative substitutions are substitutions that occur within amino acid families, and these substitutions are related in terms of their side chains and chemical properties. Examples of these families are amino acids with basic side chains, acidic side chains, nonpolar aliphatic side chains, nonpolar aromatic side chains, uncharged polar side chains, small side chains, large side chains, etc. These conserved substitutions can be, for example, isoleucine replacing leucine, aspartic acid replacing glutamic acid, cysteine ​​replacing serine, etc.

[0252] Non-conservative substitutions are substitutions that occur in amino acids whose side chains and chemical properties are unrelated, such as, but not limited to, alanine replacing proline or valine, or cysteine ​​replacing phenylalanine, or phenylalanine replacing serine or valine.

[0253] Point mutation should be specifically understood as the engineering of polynucleotides that results in the expression of an amino acid sequence different from that of a non-engineered amino acid sequence, wherein the difference lies in the substitution, exchange, deletion, or insertion of one or more single (discontinuous) or paired amino acids. Preferred point mutations refer to the exchange of amino acids with 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 classified as neutrally charged, positively charged, and negatively charged amino acids.

[0254] As used herein, an antibody fragment referred to as a "Fab fragment" comprises a light chain fragment containing a VL domain and a constant domain (CL) of the light chain, and a VH domain and a first constant domain (CH1) of the heavy chain. The antibodies of the present invention may comprise at least one Fab fragment in which the variable or constant regions of the heavy and light chains are interchanged. Due to the interchange of the variable or constant regions, the Fab fragment is also referred to as a "cross-Fab fragment" or "crossover-Fab fragment." A cross-Fab molecule may have two different chain compositions and is included in the antibodies of the present invention: the variable regions of the Fab heavy and light chains can be interchanged, i.e., the cross-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). Such a cross-Fab molecule is also referred to as a cross-antibody (VLVH).

[0255] A "single-chain Fab fragment" or "scFab" is a polypeptide composed 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. The antibody domains and the linker may have the following sequence 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-VL. The linker is a polypeptide of at least 20 amino acids, at least 30 amino acids, and particularly 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 can be stabilized by native disulfide bonds between the CL and CH1 domains. Furthermore, these single-chain Fab molecules can be further stabilized by inserting cysteine ​​residues to generate interchain disulfide bonds.

[0256] Fv fragments are the smallest units of immunoglobulin molecules that have antigen-binding activity. "Single-chain variable fragments" or "scFv" are the heavy chains (V) of immunoglobulins. H ) and light chains (V L Fusion proteins of the variable region of V are linked via short linker peptides or disulfide bonds. Linkers are typically glycine-rich for flexibility, serine or threonine-rich for solubility, and can transport V... H The N-terminus and V L The C-terminus is linked, or vice versa. Single-stranded variable fragments lack a constant Fc region. Despite the removal of the constant region and the introduction of a linker, scFv retains the specificity of the original immunoglobulin.

[0257] The term "N-terminus" refers to the last amino acid at the N-terminus.

[0258] The term "C-terminus" refers to the last amino acid at the C-terminus.

[0259] The term "microantibody" refers to an antibody consisting of a pair of single-chain Fv fragments linked by a CH3 domain (single-chain Fv-CH3). Specifically, a microantibody is approximately 75 kDa.

[0260] The term "biantibody" refers to a non-covalent dimer of a single-chain Fv (scFv) fragment, consisting of a heavy chain variable region (V) linked by a small peptide linker. H ) and light chain variable region (V LThe composition is as follows: Another form of biantibody is single-chain (Fv)2, in which two scFv fragments are covalently linked to each other. Furthermore, by tandemly linking the genes in each chain with internal linkers, the four VH and VL domains can be expressed in tandem and folded into single-chain biantibodies (scDb), which is also an efficient strategy for generating bispecific antibodies. In addition, fusing recombinant variable domains to the Fc or CH3 domains (scDb-Fc and scDb-CH3, biantibody-CH3) can double the titer of the final product. Increased size can also prolong the half-life of the biantibody in serum. Specifically, biantibody-CH3 is approximately 125 kDa.

[0261] According to one specific implementation, the antibody described herein may contain one or more tags for purification and / or detection, such as, but not limited to, affinity tags, solubility enhancement tags, and monitoring tags.

[0262] Specifically, affinity tags are selected from the group consisting of polyhistidine tags, polyarginine tags, antibody peptide substrates, chitin-binding domains, RNase S peptides, protein A, β-galactosidase, FLAG tags, Strep II tags, streptavidin-binding peptide (SBP) tags, calmodulin-binding peptide (CBP) tags, glutathione S-transferase (GST), maltose-binding protein (MBP), S-tags, HA tags, and c-Myc tags. Specifically, the tag is a His tag containing four or more H atoms, such as a hexahistidine tag.

[0263] "Fusion" or "linkage" refers to the direct connection of components (such as Fab molecules and Fc domain subunits) via peptide bonds or via one or more peptide linkers.

[0264] As used herein, the term "linker" refers to a peptide linker, preferably a peptide having an amino acid sequence of 2, 3, 4, 5, 6, 7 or more amino acids in length, more preferably 2-10 amino acids in length, and more preferably 3-5 amino acids in length.

[0265] The term "immunoglobulin" refers to a protein with a structure resembling natural antibodies. For example, IgG immunoglobulins are heterotetraglycoproteins of approximately 150,000 Daltons, composed of two light chains and two heavy chains linked by disulfide bonds. Each heavy chain has a variable region (VH), also called a variable heavy chain domain or heavy chain variable domain, from the N-terminus to the C-terminus, followed by three constant domains (CH1, CH2, and CH3), also called heavy chain constant regions. Similarly, each light chain has a variable region (VL), also called a variable light chain domain or light chain variable domain, from the N-terminus to the C-terminus, followed by a constant light chain (CL) domain, also called a light chain constant region. IgG immunoglobulins essentially consist of two Fab molecules and an Fc domain, linked by immunoglobulin hinge regions. The heavy chains of immunoglobulins can be classified into five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which can be further subdivided into subtypes, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chains of immunoglobulins can be classified into two types, called kappa (κ) and lambda (λ).

[0266] The term "chimeric antibody" refers to an antibody in which a portion of the heavy and / or light chain originates from a specific source or species, while the remainder of the heavy and / or light chain originates from a different source or species, typically prepared using recombinant DNA technology. Chimeric antibodies may contain rabbit or mouse variable regions and human constant regions. A chimeric antibody is the product of an immunoglobulin-expressing gene containing a DNA fragment encoding the variable region of an immunoglobulin and a DNA fragment encoding the constant region of an immunoglobulin. Methods for producing chimeric antibodies include conventional recombinant DNA and gene transfection techniques, which are known in the art (Morrison, SL, et al., 1984).

[0267] A "human antibody" has an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell, or to an amino acid sequence derived from a non-human antibody source using a human antibody library or other human antibody encoding sequences. This definition of a human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues. The term "human antibody" as used herein also includes antibodies whose constant regions have been modified, for example, through "type switching," i.e., alterations or mutations in the Fc portion (e.g., from IgG1 to IgG4 and / or IgG1 / IgG4 mutations). Other forms of humanized antibodies included in this invention are antibodies in which the constant region has been further modified or altered from the constant region of the original antibody to produce novel properties, such as the property of binding to C1q and / or binding to the Fc receptor (FcR).

[0268] As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, produced, or isolated through recombinant means, such as antibodies isolated from host cells (e.g., HEK cells, NSO, or CHO cells), antibodies isolated from transgenic animals (e.g., mice) targeting 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 recombinant antibodies, while derived from and related to human germline sequences, may not naturally exist in the antibody library within the human body.

[0269] The "human common framework" is a framework representing the most frequently occurring amino acid residues in the selection of the VL or VH framework sequence of human immunoglobulins. Typically, the selection of the VL or VH sequence of human immunoglobulins comes from a subgroup of variable domain sequences. Typically, the subgroup of the sequence is as described in Kabat et al., 1991.

[0270] In this article, the term "antigen," which can be used interchangeably with "target" or "target antigen," refers to the complete target molecule or a fragment of such a molecule recognized by an antibody binding site. Specifically, the substructure of an antigen, such as a polypeptide or hydrocarbon structure, is often referred to as an "epitope," such as an immune-associated B-cell epitope or T-cell epitope, which can be recognized by such a binding site.

[0271] As used herein, the term "epitope" specifically refers to a molecular structure that can completely constitute a specific binding partner for the antibody form of the present invention, or is part of a specific binding partner. Epitopes can consist of hydrocarbons, peptide structures, fatty acids, organic, biochemical, or inorganic substances or derivatives thereof, and any combination thereof. If an epitope is contained in a peptide structure (e.g., peptide, polypeptide, or protein), it typically comprises at least 3 amino acids, specifically 5 to 40 amino acids, specifically less than 10 amino acids, specifically 4 to 10 amino acids. Epitopes can be linear or conformational. Linear epitopes consist of a single fragment of the primary sequence of a polypeptide or hydrocarbon chain. Linear epitopes can be continuous or overlapping. Conformational epitopes comprise amino acids or hydrocarbons that are folded together by the polypeptide to form a tertiary structure, and the amino acids are not necessarily adjacent to each other in the linear sequence. Such an oxMIF epitope can be a sequence EPCALCS (SEQ ID NO:45) located within the central region of an oxMIF.

[0272] The terms "antigen-binding domain," "binding domain," or "binding site" refer to a portion of an antigen-binding moiety that contains a region specifically binding to and complementary to the antigen, either partially or entirely. When the antigen is large, the antigen-binding molecule may bind only to a specific portion of the antigen, called an epitope. Antigen-binding domains can be provided, for example, by one or more antibody variable domains (also called antibody variable regions). Preferably, the antigen-binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0273] The term "binding site" used herein to refer to the molecular structure capable of binding and interacting with an antigen. Typically, the binding site is located within the complementarity-determining region (CDR) of the antibody, also referred to herein as the "CDR binding site," which is a specific region with different structures that confers various antigen-binding functions. These different structures can be derived from natural antibody libraries, such as mouse or human antibody libraries, or can be recombined or synthesized, for example, through mutagenesis, particularly through randomization techniques. These include mutagenized CDR regions, loop regions of variable antibody domains, and particularly CDR loops of antibodies, such as the CDR1, CDR2, and CDR3 loops of any VL and / or VH antibody domain. The antibody forms used according to the invention typically contain one or more CDR binding sites, each site being specific for one antigen.

[0274] As used herein, the term "specificity" refers to the binding reaction that determines the target homologous ligand within a heterogeneous population of molecules. In this context, the binding reaction occurs at least with the oxMIF antigen. Therefore, under specified conditions, such as immunoassay conditions, an antibody that specifically binds to its particular target will not bind to other molecules present in the sample in significant amounts; specifically, it will not exhibit significant cross-reactivity with reduced MIF.

[0275] Specific binding sites typically do not cross-react with other targets. In addition, specific binding sites can specifically bind to one or more epitopes, isotypes, or variants of a target, or cross-react with other related target antigens, such as homologs or analogs.

[0276] Specific binding refers to binding that is selective, depending on the selected target identity, high binding affinity, medium binding affinity, or low binding affinity. Selective binding is generally achieved if the binding constant or binding kinetics with a target antigen (e.g., oxMIF) differs by at least 10-fold, preferably at least 100-fold, and more preferably at least 1000-fold, compared to the binding constant or binding kinetics with a non-target antigen.

[0277] The term "valence" as used in this application refers to the presence of a specific number of binding sites in an antibody molecule. Antibody titer refers to the number of antigenic determinants that a single antibody molecule can bind to. Therefore, the terms "bivalent," "tetravalent," and "hexavalent" respectively indicate the presence of two, four, and six binding sites in an antibody molecule.

[0278] The higher the antibody titer (the number of antigen-binding sites), the more antigens the antibody can bind to.

[0279] In this article, the term "monovalent" for antibody binding sites refers to a molecule that contains only one binding site against the target antigen.

[0280] The antibodies of the present invention are understood to contain monovalent, bivalent, tetravalent or multivalent binding sites that specifically bind to oxMIF.

[0281] As used herein, the term "hypervariant region" or "HVR" refers to each region of an antibody variable domain that is sequence-hypervariant and / or forms a structurally defined loop ("hypervariant loop"). Typically, a natural tetrachain antibody contains six HVRs; three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). HVRs typically contain amino acid residues from the hypervariant loop and / or the "complementarity-determining region" (CDR), which has the highest sequence variability and / or is involved in antigen recognition (Kabat et al., 1991). The hypervariant region (HVR) is also called the complementarity-determining region (CDR), and these terms are used interchangeably herein to refer to the portion of the variable region that forms the antigen-binding region. The exact number of residues containing a particular CDR will vary depending on the sequence and size of the CDR. Given the amino acid sequence of the variable region of an antibody, those skilled in the art can routinely determine which residues contain a particular CDR.

[0282] Kabat defines a numbering system applicable to the variable region sequence of any antibody. Those skilled in the art can explicitly assign this "Kabat numbering" system to any variable region sequence without relying on any experimental data outside the sequence itself. For a given antibody, the Kabat number of a residue can be determined by comparing homologous regions of the antibody sequence with "standard" Kabat numbered sequences. As used herein, "Kabat number" refers to the numbering system proposed by Kabat et al., 1983, Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest". Unless otherwise stated, the numbering of specific amino acid residue positions within the antibody variable region follows the Kabat numbering system. Constant regions are numbered according to the EU numbering index.

[0283] The CDR also contains "specificity-determining residues," or "SDRs," which are residues that contact the antigen. SDRs are contained within the CDR region, referred to as the abbreviation CDR or a-CDR. Unless otherwise stated, HVR residues and other residues (e.g., FR residues) in the variable domain are numbered in this document according to the aforementioned Kabat system.

[0284] The “sequence identity percentage (%)” for the polypeptide sequences identified herein is defined as the percentage of amino acid residues in the candidate sequence that are identical to amino acid residues in the specific polypeptide sequence after sequence alignment and the introduction of vacancies (if necessary) to achieve the maximum sequence identity percentage, without considering any conserved substitutions as part of sequence identity. Those skilled in the art can determine suitable parameters for determining alignment, including any algorithms required to achieve maximum alignment on the full-length sequences being compared.

[0285] According to the invention, the sequence identity of the variable or constant region sequence with the corresponding sequence described herein is at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%. Due to the % specification, this may be purely arithmetic, and amino acids or nucleic acids can no longer be specified as integers. For example, the identity calculation will result in 1.5 amino acids being the same or different. In this case, these values ​​are rounded up, so nucleic acids and amino acids are always given as integers.

[0286] As used herein, the terms “subject” or “patient” or “individual” refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human being, most preferably a person with abnormal cells (including malignant cells in a tumor).

[0287] "Isolated" nucleic acids refer to nucleic acid molecules that have been separated from components of their natural environment. Isolated nucleic acids include nucleic acid molecules that are normally found in cells, but which are located outside chromosomes or at chromosomal locations different from their natural chromosomal locations.

[0288] "Isolated nucleic acid encoding anti-oxMIF" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of an antibody, including such nucleic acid molecules in a single vector or isolated vector, and such nucleic acid molecules present at one or more locations in a host cell.

[0289] "No significant cross-reactivity" means that a molecule (e.g., an antibody) does not recognize or specifically bind to an antigen that is different from the actual target antigen (e.g., an antigen closely related to the target antigen, specifically a reduced MIF), particularly when compared to the target antigen. For example, an antibody may bind less than about 10% and at least less than about 5% of an antigen that is different from the actual target antigen, or may bind said antigen that is different from the actual target antigen in amounts less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1%, preferably less than about 2%, 1%, or 0.5%, and most preferably less than about 0.2% or 0.1%. Binding can be determined by any method known in the art, such as, but not limited to, ELISA or surface plasmon resonance.

[0290] The recombinant production of the antibodies of the present invention preferably employs an expression system, such as an expression construct or vector comprising a nucleotide sequence encoding the antibody form.

[0291] The term "expression system" refers to a nucleic acid molecule containing operablely linked desired coding and control sequences, enabling a host transformed or transfected with these sequences to produce the encoded protein. For transformation, the expression system can be contained on a vector; however, the associated DNA can also be integrated into the host chromosome. Alternatively, the expression system can be used for in vitro transcription / translation.

[0292] As used herein, “expression vector” is defined as the DNA sequence required for the transcription of a cloned recombinant nucleotide sequence (i.e., a recombinant gene) and the translation of its mRNA into a suitable host organism. An expression vector contains an expression cassette and typically also includes an origin of replication or genome integration site for autonomous replication in the host cell, one or more selection markers (e.g., amino acid synthesis genes or genes conferring antibiotic resistance, such as bleomycin, kanamycin, G418, or hygromycin), numerous restriction enzyme cleavage sites, a suitable promoter sequence, and a transcription terminator; these components are operatively linked together. The terms “plasmid” and “vector” as used herein include both autonomously replicating nucleotide sequences and genome-integrating nucleotide sequences.

[0293] Specifically, the term refers to a vector through which a DNA or RNA sequence (e.g., a foreign gene), such as a nucleotide sequence encoding the antibody form of the present invention, can be introduced into a host cell, thereby transforming the host and promoting the expression (e.g., transcription and translation) of the introduced sequence. Plasmids are the preferred vectors of the present invention.

[0294] Vectors typically contain DNA containing transfer factors, into which foreign DNA is inserted. A common method for inserting one DNA fragment into another involves using enzymes called restriction enzymes, which cut the DNA at specific sites (specific sets of nucleotides) called restriction sites.

[0295] A "cassette" refers to a DNA coding sequence or DNA fragment that encodes the expression product and can be inserted into a vector at a defined restriction site. The cassette restriction site is designed to ensure that the cassette is inserted into the correct reading frame. Typically, foreign DNA is inserted into one or more restriction sites on the vector DNA and then carried into the host cell by the vector along with the transfer vector DNA. A DNA fragment or sequence with inserted or added DNA, such as an expression vector, can also be called a "DNA construct." A common type of vector is a "plasmid," which is typically a freestanding double-stranded DNA molecule that readily accepts additional (foreign) DNA and can be readily introduced into a suitable host cell. The vectors of this invention typically contain coding DNA and an expression control sequence, such as promoter DNA, and have one or more restriction sites suitable for the insertion of foreign DNA. Coding DNA is a DNA sequence that encodes a specific amino acid sequence of a particular polypeptide or protein (e.g., the antibody form of this invention). Promoter DNA is a DNA sequence that initiates, regulates, mediates, or controls the expression of coding DNA. Promoter DNA and coding DNA can originate from the same gene or different genes, and can also originate from the same or different organisms. The recombinant cloning vector of the present invention typically includes one or more replication systems for cloning or expression, one or more markers for selection in the host (e.g., antibiotic resistance), and one or more expression cassettes.

[0296] Procedures for linking DNA sequences, such as those providing or encoding the factors of the present invention and / or proteins of interest, promoters, terminators and other sequences, and inserting them into a suitable vector containing information required for integration or host replication are known to those skilled in the art, for example, as described in Sambrook et al., 2012.

[0297] The host cell is specifically understood as a cell, recombinant cell, or cell line transfected with an expression construct (e.g., a vector according to the present invention).

[0298] As used herein, the term "host cell line" refers to an established clone of a specific cell type that has acquired the ability to proliferate over a long period of time. The term "host cell line" also refers to a cell line used to express endogenous or recombinant genes to produce polypeptides (e.g., the recombinant antibody form of this invention).

[0299] "Production host cell" or "production cell" is generally understood to be a cell line or cell culture prepared for cultivation in a bioreactor to obtain the product of the production process (the recombinant antibody form of the present invention). The host cell type according to the present invention can be any prokaryotic or eukaryotic cell.

[0300] As used in this article, the term “recombinant” should mean “prepared by genetic engineering” or “the result of genetic engineering,” such as the specific use of a heterologous sequence integrated into a recombinant vector or recombinant host cell.

[0301] Antibodies can be produced using any known and established expression system and recombinant cell culture technique, 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 this invention can be produced in transgenic organisms (e.g., goats, plants, or transgenic mice, i.e., engineered mouse strains possessing large fragments of human immunoglobulin loci and defective in mouse antibody production). Antibodies can also be produced through chemical synthesis.

[0302] According to one specific implementation scheme, the host cells are production cell lines selected from the group consisting of CHO, PerC6, CAP, HEK, HeLa, NSO, SP2 / O, hybridoma, and Jurkat. More specifically, the host cells are obtained from CHO cells.

[0303] The host cells of the present invention are specifically cultured or maintained in serum-free cultures (e.g., containing other components, such as plasma proteins, hormones and growth factors, as a substitute for serum).

[0304] The host cell is the preferred choice when established, adapted, and fully cultured under serum-free conditions and optionally in a medium free of any animal-derived proteins / peptides.

[0305] The anti-oxMIF antibody of the radioimmunoconjugate described in this article can be recovered from the culture medium using standard protein purification methods.

[0306] The term "pharmaceutical formulation" refers to a formulation in a form that enables the bioactivity of the active ingredient contained therein to be effective and does not contain any additional components that would have unacceptable toxicity to a subject administering the formulation.

[0307] A "pharmaceutically acceptable carrier" refers to a component of a pharmaceutical preparation that is non-toxic to the subject, other than the active ingredient. Examples of pharmaceutically acceptable carriers include water, saline, phosphate-buffered saline, amino acids (e.g., glycine or histidine), glucose, glycerol, ethanol, and combinations thereof. In many cases, isotonic agents, such as sugars, polyols (e.g., mannitol, sorbitol), or sodium chloride, are preferably included in the composition. Other examples of pharmaceutically acceptable substances are wetting agents or small amounts of excipients, such as humectants or emulsifiers, preservatives, or buffers, which can improve the shelf life or efficacy of antibodies.

[0308] As used herein, “treatment,” “treat,” “treating,” or “therapy” refers to a clinical intervention that attempts to alter the natural course of a disease in an individual being treated, and may be performed for prevention or in the course of clinicopathology. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and mitigating or improving prognosis. In some embodiments, the antibodies of this invention are used to delay the development of disease or slow its progression.

[0309] The anti-oxMIF antibody of the radioimmunoconjugate of the present invention and the pharmaceutical composition comprising it can be administered in combination with one or more other therapeutic agents, diagnostic agents, or preventive agents. Depending on the disease to be treated, other therapeutic agents include other anti-vegetative agents, antitumor agents, anti-angiogenic agents, chemotherapeutic agents, steroids, or checkpoint inhibitors.

[0310] The pharmaceutical compositions of the present invention can be in various forms, such as liquid solutions (e.g., injectable and infusionable solutions), dispersions or suspensions, and liposomes. Various other delivery systems are also known for administering the radioimmunoconjugates described herein, such as those encapsulated in liposomes, microparticles, microcapsules, receptor-mediated endocytosis (see, for example, Wu and Wu, 1987).

[0311] The amount of the radioimmunoconjugate composition of the present invention effective in the treatment, prevention, or control of cancer, or in the diagnosis of cancer, can be determined using standard research techniques. For example, the dose of the composition effective in the treatment, prevention, or control of cancer, or in the diagnosis of cancer, can be determined by administering the composition to an animal model (e.g., an animal model disclosed herein or known to those skilled in the art). Additionally, in vitro testing may optionally be employed to help determine the optimal dose range. The selection of a preferred effective dose can be determined by a person skilled in the art based on considerations of several factors known to those skilled in the art (e.g., through clinical trials). These factors include the disease to be treated or prevented, the symptoms involved, the patient's weight, the patient's immune status, and other factors known to the person skilled in the art that reflect the accuracy of the administered pharmaceutical composition. The precise dose used in the formulation will also depend on the route of administration and the severity of the cancer, and should be determined based on the physician's judgment and the individual patient's situation. The effective dose can be inferred from dose-response curves derived from in vitro or animal model testing systems.

[0312] In one specific implementation, the therapeutically effective amount of the radioimmunoconjugate is in the range of about 25 mCi to 250 mCi, 50 mCi to 200 mCi, 75 mCi to 175 mCi, or 100 mCi to 150 mCi.

[0313] Radioimmunoconjugates can be administered in combination with known chemotherapeutic agents. Examples of chemotherapeutic agents include, but are not limited to, BCNU, cisplatin, gemcitabine, hydroxyurea, paclitaxel, temozomide, topotecan, fluorouracil, vincristine, vinblastine, procarbazine, dacarbazine, altretamine, cisplatin, methotrexate, mecaptopurine, thioguanine, fludarabine phosphate, cladribine, pentostatin, fluorouracil, cytarabine, azacitidine, and vinblastine. The following are listed: lastine, vincristine, etoposide, teniposide, irinotecan, docetaxel, doxorubicin, daunorubicin, dactinomycin, idarubicin, plicamycin, adriamycin, mitomycin, bleomycin, tamoxifen, flutamide, leuprolide, goserelin, aminoglutethimide, anastrozole, amsacrine, asparaginase, mitoxantrone, mitotane, and aifostine.

[0314] Preferred formulations depend on the intended route of administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusionable solutions, such as compositions similar to those used for passive immunization in humans. Preferred routes of administration are parenteral (e.g., intradermal, intravenous, subcutaneous, epidural, intraperitoneal, intramuscular). In a preferred embodiment, the radioimmunoconjugate is administered via intravenous infusion or injection. Those skilled in the art will understand that the route and / or method of administration will vary depending on the desired outcome.

[0315] In one specific implementation, it may be necessary to apply the radioimmunoconjugate locally to the area requiring treatment; this can be achieved, for example, but not limited to, local infusion, injection, catheter, suppository, or implant during surgery, said implant being a porous, non-porous, or gel-like material, including membranes such as silicone rubber membranes or fibers.

[0316] In another embodiment, a radioimmunoconjugate or any composition containing a radioimmunoconjugate can be delivered in a controlled-release system. In one embodiment, a pump can be used. In another embodiment, a polymeric material can be used. In yet another embodiment, the controlled-release system can be placed near the therapeutic target (e.g., the brain), thus requiring only a portion of the systemic dose.

[0317] Radioimmunoconjugates can be administered once, but multiple administrations are preferred.

[0318] The present invention also relates to compositions comprising radioimmunoconjugates for the treatment and diagnosis of subjects requiring treatment for MIF-related conditions, particularly (hyper)proliferative disorders. In some embodiments, the subject requiring treatment is a human being.

[0319] As used in this article, the term "cancer" refers to proliferative diseases, particularly non-solid and solid cancers. Examples of cancer include, but are not limited to, tumors, lymphomas, blastomas, sarcomas, and leukemias. More specifically, examples of these cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma of the lung, peritoneal cancer, myeloma (e.g., multiple myeloma), hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma / glioma (e.g., anaplastic astrocytoma, glioblastoma multiforme, anaplastic oligodendroglioma), Ewing's sarcoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer, or uterine cancer. Cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, melanoma, squamous cell carcinoma (SCC) (e.g., head and neck cancer, esophageal cancer, and oral cancer), colorectal adenocarcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, astrocytoma, neuroblastoma, cervical cancer, endometrial carcinoma, and malignant seminoma, including any of the above-mentioned refractory types of cancer, or a combination of one or more of the above-mentioned cancers.

[0320] The radioimmunoconjugates of this invention can be used to treat and / or diagnose hyperproliferative diseases, such as carcinomas or cancers, involving any tissue or organ, including but not limited to brain, lung, squamous cell, bladder, stomach, pancreas, breast, head, neck, liver, kidney, ovary, prostate, colorectal, esophageal, gynecological, nasopharyngeal, or thyroid cancer, melanoma, lymphoma, leukemia, or multiple myeloma. In particular, the radioimmunoconjugates of this invention can be used to treat and / or diagnose ovarian cancer, pancreatic cancer, colon cancer, lung cancer, and hematologic malignancies.

[0321] In one specific implementation, the antibody for radioimmunoconjugates, which is highly suitable for the treatment and / or diagnosis of cancer (particularly for the treatment of solid tumors), is a recombinant anti-oxMIF antibody or its antigen-binding fragment having reduced aggregation potential and reduced hydrophobicity, including

[0322] i.) The following variable structural domains:

[0323] (a) A light chain variable domain comprising SEQ ID NO:32 and at least one of the amino acid substitutions M30L, F49Y, A51G, P80S, and W93F, or a functional variant of SEQ ID NO:32 having 1, 2, 3, 4, or 5 amino acid substitutions, further comprising a conserved tyrosine residue at position 36, and further comprising at least one of the amino acid substitutions M30L, F49Y, A51G, P80S, and W93F; and one of the following

[0324] (b) A heavy-chain variable structural domain containing SEQ ID NO:29; or

[0325] (c) A heavy chain variable domain comprising at least one of SEQ ID NO:29 and an amino acid substitution L5Q or W97Y, or comprising SEQ ID NO:29 having 1, 2, 3, 4 or 5 amino acid substitutions, and further comprising at least one of an amino acid substitution L5Q or W97Y.

[0326] Furthermore, in another preferred aspect, the anti-oxMIF antibody of the radioimmunoconjugate comprises a modification that reduces FcγR binding, which can be identified, for example, by an alanine scan of the Fc. Specifically, it comprises an Fc variant domain having the wild-type human IgG1 constant region of SEQ ID NO:37, which contains one, two, or three amino acid substitutions at any of positions L234, L235, G236, G237, N297, L328, or P329, and wherein the radioimmunoconjugate exhibits reduced FcγR binding compared to a radioimmunoconjugate containing the wild-type IgG1 Fc region. In another embodiment, the radioimmunoconjugate comprises an Fc variant domain (SEQ ID NO:38) with reduced FcγR binding, which contains one, two, or three amino acid substitutions at any of positions I253, H310, or H435, and wherein the radioimmunoconjugate exhibits reduced affinity for human FcRn compared to a radioimmunoconjugate containing the wild-type IgG1 Fc region.

[0327] The radioimmunoconjugates of this invention can be used as diagnostic or detectable reagents. They can be used to monitor or predict the development or progression of cancer as part of clinical testing procedures, such as determining the efficacy of a specific treatment. Furthermore, these radioimmunoconjugates can be used to monitor or predict the development or progression of cancerous diseases.

[0328] In another embodiment, after initial application of the radioimmunoconjugate of the present invention, cancer cells can be imaged, and the relative amount or number of cancer cells can be determined by any available means. The present invention includes diagnostic methods for detecting cancer and / or assessing the effect of therapeutic agents on cancer cells in a patient's organs or body sites.

[0329] This method involves administering a composition containing a detectable amount of a radioisotope-conjugated anti-oxMIF antibody to the patient before and after treatment. Following the administration of the treatment agent, an additional amount of the detectable anti-oxMIF antibody may be administered to determine the relative amount of remaining cancer cells after treatment. Comparison of images before and after treatment can be used as a means of assessing treatment efficacy, with a reduction in the number of cancer cells imaged after treatment indicating an effective treatment regimen.

[0330] As used herein, the term "detectable amount" refers to an amount of radioimmunoconjugate bound to oxMIF administered to a patient sufficient to detect the binding of the labeled antibody to one or more malignant cancer cells in the tumor. As used herein, the term "imaging effective amount" refers to an amount of radioimmunoconjugate administered to a patient sufficient to image the binding of the anti-oxMIF antibody to one or more malignant cancer cells in the tumor.

[0331] The methods of this invention include the radioimmunoconjugate of this invention, which, in conjunction with non-invasive imaging techniques (e.g., gamma imaging, such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT)), for the identification and quantification of abnormal cells in vivo, including malignant cells in tumors. The term "in vivo imaging" refers to any method that allows the detection of the aforementioned radioimmunoconjugate. For gamma imaging, radiation emitted from the examined tumor or region is measured and expressed as total binding, or as the ratio of total binding in one tissue to (e.g., divided by) total binding in another tissue or the whole body of the same subject during the same in vivo imaging process. Total binding in vivo is defined as the total signal detected in a tumor or tissue by an in vivo imaging technique without the need for correction by a second injection of the same amount of labeled compound and a large excess of unlabeled but chemically identical compound.

[0332] For in vivo imaging purposes, the type of detection instrument available is a primary factor in selecting a given label. For example, radioisotopes are particularly suitable for in vivo imaging in the methods described herein. The type of instrument used will guide the selection of the radioisotope. For instance, the selected radioisotope must have a decay type detectable by the given type of instrument. Another consideration relates to the half-life of the radioisotope. The half-life should be long enough to remain detectable at the point of maximum absorption by the target, but also short enough that the host is not exposed to harmful radiation. Isotope-labeled radioimmunoconjugates can be detected using gamma imaging, where gamma radiation emitted at an appropriate wavelength is detected. Gamma imaging methods include, but are not limited to, positron emission tomography (PET) or single-photon emission computed tomography (SPECT). Preferably, for SPECT detection, the selected radiolabel will lack particle emission but will produce a large number of photons. For PET detection, the radiolabel will be a positron-emitting radioisotope, which will be detected by a PET camera.

[0333] In this invention, radioimmunoconjugates can be used for in vivo detection and imaging of tumors. These compounds will be used in conjunction with non-invasive imaging techniques such as positron emission tomography (PET), single-photon emission computed tomography (SPECT), or Cherenkov luminescence imaging (CLI). According to this invention, the immunoconjugates can be labeled (compounded) with any acceptable radioisotope.

[0334] According to another implementation, the radioimmunoconjugate is used to determine the location of cancer cells, tumor microenvironment cells, or tumors in a subject with this need, including administering the radioimmunoconjugate to the subject and detecting the radioimmunoconjugate by in vivo SPECT and PET imaging.

[0335] According to another embodiment, the radioimmunoconjugate is used to image one or more cancer cells, tumor microenvironment cells, organs or tissues in a subject, including administering the radioimmunoconjugate to the subject and detecting the radioimmunoconjugate by in vivo PET or SPECT imaging.

[0336] According to another embodiment, the radioimmunoconjugate is used to determine and (relatively) quantify oxMIF on cancer cells, tumor microenvironment cells, organs, or tissues in a subject, including administering the radioimmunoconjugate to the subject and detecting the radioimmunoconjugate by in vivo PET or SPECT imaging.

[0337] Generally, the dose of radioimmunoconjugates will vary depending on factors such as the patient's age, condition, sex, and severity of disease, contraindications (if any), concomitant therapies, and other variables, and will be adjusted by a person skilled in the art. The dose for therapeutic administration can vary from about 25 mCi to 250 mCi, while the radiation dose for diagnostic purposes can range from 0.5 to 10 mCi, for example, about 1 to 5 mCi. Individual doses can be determined by a technician.

[0338] Application to patients can be local or systemic, and can be administered intravenously, intra-arterially, intradurally (via cerebrospinal fluid), or intracranially. Depending on the body part being examined, it can also be administered intradermally or intraperitoneally.

[0339] After the radioimmunoconjugate has bound to the abnormal cells for a sufficiently long period, such as approximately 30 minutes to 48 hours, or up to 72 hours, or even longer, the study area of ​​the subject is examined using conventional imaging techniques, such as SPECT, planar scintillation imaging, PET, and emerging imaging techniques. As mentioned above, the exact protocol must vary depending on the specific factors of the patient, as well as the body site examined, the method of administration, and the type of marker used; determining the specific procedure is routine for those skilled in the art. For tumor imaging, preferably, the amount of bound radioimmunoconjugate (total binding or specific binding) is measured and compared with the amount of radioimmunoconjugate bound to the tumor after chemotherapy (as a ratio).

[0340] In another embodiment, the radioimmunoconjugate of the present invention can be used for diagnosis and prognosis by using tissue and fluid remote from the site of the primary tumor (as well as by using tissue and fluid of the primary tumor and / or tissue and fluid surrounding the tumor). The radioimmunoconjugate of the present invention can be used to determine oxMIF levels in biological samples using classical immunohistochemical methods known to those skilled in the art (e.g., see Jalkanen et al. (1985) J. Cell. Biol. 101, 976-985; and Jalkanen et al. (1987) J. Cell. Biol. 105, 3087-3096).

[0341] In another embodiment, the present invention provides a diagnostic kit, including an immunoassay kit and an imaging kit, for use in the above-described immunoassay and imaging methods.

[0342] This invention also includes the following items:

[0343] 1. A radioimmunoconjugate comprising a recombinant anti-oxMIF antibody or an antigen-binding fragment thereof, said radioimmunoconjugate comprising:

[0344] (a1) A light chain variable domain comprising SEQ ID NO:32 having at least one amino acid substitution of M30L, F49Y, A51G, P80S, or W93F, or

[0345] (a2) A light chain variable domain comprising SEQ ID NO:32 having substitutions of 1, 2, 3, 4, or 5 amino acids, and further comprising

[0346] At position 36, the conserved tyrosine, and

[0347] Amino acid substitution for at least one of M30L, F49Y, A51G, P80S, and W93F; and

[0348] (b1) A heavy-chain variable structural domain containing SEQ ID NO:29; or

[0349] (b2) A heavy chain variable domain comprising SEQ ID NO:29 and amino acid substitutions L5Q and / or W97Y, or

[0350] (b3) A heavy chain variable domain comprising SEQ ID NO:29 having at least one amino acid substitution L5Q or W97Y, and 1, 2, 3, 4 or 5 other amino acid substitutions;

[0351] The amino acid positions are determined according to Kabat numbering.

[0352] Compared to antibodies or antigen-binding fragments thereof containing SEQ ID NO:32 and SEQ ID NO:29 lacking amino acid substitutions, the antibodies or antigen-binding fragments thereof have reduced aggregation potential and reduced hydrophobicity.

[0353] 2. The radioimmunoconjugate of Project 1, wherein the light chain variable domain comprises an amino acid substituted W93F, and the heavy chain variable domain comprises an amino acid substituted W97Y.

[0354] 3. The radioimmunoconjugate described in item 1 or 2, wherein the radioisotope is selected from the group consisting of: 11 C 13 N、 15 O, 18F, 32 P, 64 Cu、 67 Cu、 67 Ga、 89 Zr、 90 Y、 99 mTc, 103 Pd, 105 Rh、 109 Pd, 111 Ag、111 In、 123 I, 124 I, 125 I, 131 I, 140 La、 149 Tb, 149 Pm, 153 Sm、 159 Gd, 165 Dy、 166 Dy、 166 Ho、 169 Yb、 175 Yb、 177 Lu、 227 Th、 186 Re、 188 Re、 192 Ir、 193 mPt, 195 mPt, 198 Au、 199 Au、 211 At、 212 Pb, 212 Bi、 213 Bi、 225 Ac、 223 Ra、 227 Th; specifically, the radioactive isotope is 67 Ga、 89 Zr、 111 In、 124 I, 131 I, 177 Lu and 225 Ac.

[0355] 4. The radioimmunoconjugate of item 1 or 2, comprising an Fc variant domain having a constant domain of wild-type human IgG1 of SEQ ID NO:37, comprising at least one amino acid substitution at any one of positions L234, L235, G236, G237, N297, L328 or P329, and wherein, compared with a radioimmunoconjugate comprising the Fc region of wild-type IgG1, the radioimmunoconjugate exhibits reduced binding to Fcγ-receptors, particularly reduced binding to FcγRI, FcγRII, and FcγRIII.

[0356] 5. The radioimmunoconjugate of any one of items 1 to 4, comprising an Fc variant domain of a human IgG1 constant domain, containing one, two or three amino acid substitutions at any one of positions I253, H310, and H435, and wherein the radioimmunoconjugate exhibits reduced affinity for human FcRn compared to a radioimmunoconjugate comprising a wild-type IgG1 Fc region.

[0357] 6. The radioimmunoconjugate of any one of items 1 to 5, comprising an amino acid sequence selected from the group consisting of SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36.

[0358] 7. The radioimmunoconjugate of any one of items 1 to 5, comprising SEQ ID NO:29 and SEQ ID NO:34; SEQ ID NO:30 and SEQ ID NO:33, SEQ ID NO:30 and SEQ ID NO:34, SEQ ID NO:31 and SEQ ID NO:34, SEQ ID NO:31 and SEQ ID NO:36, or SEQ ID NO:31 and SEQ ID NO:33, and any one of SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:40.

[0359] 8. The radioimmunoconjugates described in any one of items 1 to 7, selected from the group consisting of: IgG, IgG fusion protein, Fv, scFv, scFv fusion protein, biantibody, biantibody fusion protein, Fab, Fab fusion protein, scFab, scFab fusion protein, Fab' and F(ab')2, Fab'-SH, Fcab, Fcab fusion protein, fusion protein of two or more single-chain antibodies, microantibodies, small immunoprotein (SIP) forms, and nanobodies and nanoantibody fusion proteins.

[0360] 9. The radioimmunoconjugate of any one of items 1 to 8, further comprising a chelating group selected from the group consisting of DOTA and deferoxamine B (DFO) and DFO*.

[0361] 10. The radioimmunoconjugate described in any one of items 1 to 9, used in the preparation of a drug.

[0362] 11. A pharmaceutical composition comprising the radioimmunoconjugate described in any one of items 1 to 9, optionally further comprising a drug carrier or adjuvant.

[0363] 12. The pharmaceutical composition of item 11, wherein the pharmaceutical composition is formulated for intravenous administration.

[0364] 13. The pharmaceutical composition of item 11 or 12, for the treatment of patients with cancer, particularly for the treatment of tumors, especially hematologic malignancies or solid tumors, and even more particularly for the treatment of colorectal cancer, ovarian cancer, breast cancer, prostate cancer, pancreatic cancer and lung cancer.

[0365] 14. An isolated nucleic acid encoding a recombinant anti-oxMIF antibody or an antigen-binding fragment thereof that is a radioimmunoconjugate as described in any one of items 1 to 8.

[0366] 15. An expression vector comprising the nucleic acid molecule described in item 14.

[0367] 16. A host cell comprising the nucleic acid described in item 12 or 13 or the expression vector described in item 13.

[0368] 17. A method for producing the radioimmunoconjugate of any one of items 1 to 9, comprising expressing a nucleic acid encoding an anti-oxMIF antibody in a host cell, and labeling the antibody with a radioisotope.

[0369] 18. Use of any one of items 1 to 9 for detecting oxMIF levels in cancer subjects.

[0370] 19. Use of any one of items 1 to 9 for the diagnosis of cancer in a subject.

[0371] 20. A method for in vitro diagnosis of cancer, wherein the radioimmunoconjugate described in any one of items 1 to 9 is used to detect tumor cells in a sample.

[0372] 21. A method for diagnosing cancer in vivo, wherein the radioimmunoconjugate described in any one of items 1 to 9 is used to detect tumor cells in a subject.

[0373] 22. A kit for producing the radioimmunoconjugate of any one of items 1 to 9, comprising two or more vials, one vial containing a conjugate containing a chelating agent linked to an anti-oxMIF antibody, and a second vial containing a radioisotope, specifically selected from... 177 Lu and 89 Zr.

[0374] 23. The kit described in item 21, wherein the contents of one or more vials are in lyophilized or solution form.

[0375] 24. A method for treating cancer, wherein the radioimmunoconjugate described in any one of items 1 to 9 or the pharmaceutical composition described in item 11 or 12 is used.

[0376] Example

[0377] The embodiments described herein are illustrative of the invention and are not intended to limit the invention. Many modifications and variations can be made to the techniques described and illustrated herein without departing from the spirit and scope of the invention. Therefore, it should be understood that these embodiments are merely illustrative and do not limit the scope of the invention.

[0378] Example 1

[0379] 177 Production of Lu-C0008-Radioimmunoconjugate

[0380] Materials and methods:

[0381] C0008 was incubated with SCN-Bn-DOTA at a molar ratio of 1:20 in HEPES 0.3M pH 9. The labeled mixture was stirred at 37°C for 2 hours. After the reaction, excess SCN-Bn-DOTA was removed by multiple centrifugations at 3000 g in 0.9% NaCl using a 30 kDa MWCO Amicon ultracentrifuge filter (regenerated cellulose, Millipore). At the end of purification, the conjugate was assessed for chemical purity by SE-HPLC. SE-HPLC was performed on a Superdex 200 10 / 300GL column, eluting with phosphate buffer containing arginine at a flow rate of 0.6 mL / min. Furthermore, the purity and integrity of DOTA-C0008 were assessed by SDS-PAGE under reducing and non-reducing conditions (4–15% linear gradient polyacrylamide gel; Bio-Rad Laboratories).

[0382] The number of DOTA molecules linked to each antibody was determined using the method of Meares et al., 1984, and rapid thin-layer chromatography (ITLC). Small aliquots of the DOTA-conjugated antibody were then subjected to tracer-level radioactivity. 111 In and stable indium ( 115 Incubate together with a mixture of (In) 111 / 115 In was added in excess of DOTA-conjugated antibody by 2, 4, 6, and 8 times (labeling conditions are shown in Table 1). Unconsumed indium was then conjugated with excess EDTA to prevent the formation of insoluble indium hydroxide and non-specific binding of indium to the antibody. For analysis, 1 μL of radioactive solution was placed on ITLC test paper and eluted with 0.1 M citrate buffer at pH 5. Autoradiography of the TLC plates was obtained after exposure on a phosphorus screen (Typhoon IP, Amersham) using an ImageQuant TL, based on volume integral. The number of DOTA molecules linked to each DOTA-C0008 was determined by labeling efficiency and the amount of protein added to the solution. 111 / 115 The molar ratio of In ions was estimated.

[0383] Table 1: Labeling scheme for determining the number of chelates grafted onto C0008

[0384]

[0385] In order to use a specific activity of 15 mCi / mg (555 MBq / mg) 177 Lu radiolabeled DOTA-C0008 at 4500 μCi (166.5 MBq) in 0.04 N HCl. 177 Lu chloride (ITG Germany) and 12 μL of 0.25M ammonium acetate at pH 5.4 were added to 300 μg of DOTA-C0008. The reaction mixture was incubated at 37°C for 10 minutes, followed by incubation of the radiolabeled conjugate with 1 mM DTPA (final concentration) for an additional 5 minutes to recombine excess. 177 Lu. After adding DTPA, the chelation efficiency was evaluated by ITLC, eluted in 0.1M citrate buffer at pH 5, and autoradiography of the TLC plate (Typhoon IP, Amersham) was performed using ImageQuant TL based on volume integral.

[0386] result: Incubation of antibody C0008 with SCN-Bn-DOTA and subsequent purification yielded a high-purity DOTA-C0008 conjugate, as measured by SE-HPLC. Figure 1 A) and SDS PAGE Figure 1 As shown in B). The number of DOTA molecules linked to each antibody in the obtained DOTA-C0008 conjugate was analyzed, and the ITLC results are shown in [Figure B]. Figure 2 As shown. Under the selected conditions, an average of approximately 5.8 DOTA molecules were obtained per antibody. The DOTA-C0008 conjugate was then successfully labeled with lutetium-177 and analyzed by ITLC (…). Figure 3 A) and autoradiography ( Figure 3 B) The obtained radioimmunoconjugate showed a specific activity of 16 mCi / mg (592 MBq / mg) and a total radiochemical purity of 97.5%.

[0387] in conclusion: A method was established using 177 Lu is an effective method for radiolabeling anti-oxMIF antibody C0008. The resulting radioimmunoconjugate exhibits sufficient purity and specific activity.

[0388] Figure 1The purity and integrity analysis of DOTA-C0008 is shown; A: DOTA-C0008 SE-HPLC chromatogram (λ=280nm). B: Coomassie staining SDS PAGE: 1: Molecular weight standard; 2: C0008 non-reduced; 3: C0008 reduced; 4: Concentrated C0008 (labeled batch) non-reduced; 5: Concentrated C0008 (labeled batch) reduced; 6: DOTA-C0008 non-reduced; 7: DOTA-C0008 reduced.

[0389] Figure 2 The number of DOTA molecules grafted onto C0008 is shown by ITLC determination: ITLC separation of DOTA-C0008, DOTA-C0008 with tracer amounts of radioactive indium ( 111 In) and stable indium ( 115 Incubate together with a mixture of (In) 111 / 115 In an overdose of 2-fold (lane 6), 4-fold (lane 7), 6-fold (lane 8), or 8-fold (lane 9) of DOTA conjugate antibody.

[0390] Figure 3 The following image shows the radiolabeled C0008 analyzed by ITLC and autoradiography: A: ITLC of radiolabeled C0008 (lane 3: 177 The labeling reaction of Lu with DOTA-C0008; Lane 4: 177 B: Autoradiography of the ITLC film, lane 3 (after DTPA addition) 177 Lu-DOTA-C0008; Peak 1 reflects free... 177 Lu-DTPA, peak 2 reflects 177 Lu-DOTA-C0008).

[0391] Example 2

[0392] 177 Stability of Lu-DOTA-C0008 in formulation buffer

[0393] Materials and methods:

[0394] preparation 177 Lu-DOTA-C0008 solution was prepared to achieve a final concentration of 700 μg / mL suitable for stability studies. 177 The properties of Lu-DOTA-C0008 solution are shown in Table 2.

[0395] Table 2: 177 Characteristics of Lu-DOTA-C0008 solution

[0396]

[0397] The formulation buffer stored at 4°C was analyzed by ITLC and SE-HPLC at different time points. 177 Lu-DOTA-C0008. Radiochemical purity was determined by ITLC, eluting with 0.1 M citrate at pH 5 to quantify the percentage of radioactivity released over time by the radioimmunoconjugate. SE-HPLC was performed on a Superdex 200, 10 / 300GL column, eluting with arginine-containing phosphate buffer at a flow rate of 0.6 mL / min to identify aggregates and contaminants (i.e.,...). 177 Lu-DTPA or 177 (Lu-DOTA). Chromatograms were obtained using a Radiomatic 150TR flow scintillation analyzer.

[0398] Furthermore, the radioactivity of the radioimmunoconjugate was measured by gamma counting to provide information about... 177 Information on the solubility of Lu-DOTA-C0008 in formulation buffer. For this purpose, 5 μL was directly counted from a 1 / 20 dilution in the formulation buffer.

[0399] result: 177 ITLC analysis of the Lu-DOTA-C0008 solution showed that the radiolabeled immunoconjugate released almost no radioactivity over time. After 7 days, the radioactivity purity of the radiolabeled antibody solution reached 96.25%. When maintained at 4°C in formulation buffer (Table 3), the radiolabeled antibody released approximately 1.2% of the initial bound radioactivity (Table 2). 177 Lu-DOTA-C0008 was eluted in 21.6 minutes, and its contaminants ( 177 Lu-DTPA and 177 The Lu-DOTA derivative was eluted in 32.8 minutes. From day 0 to day 3, HMW aggregates increased slightly, but no additional aggregates were detected after day 3 (Table 5). After 7 days, the quantitative solution showed 93% intact antibody as analyzed by SE-HPLC. The SE-HPLC results were consistent with the ITLC results. Over time, the observed radioactivity was very consistent with the theoretical radioactive decay of lutetium-177 (values ​​in parentheses in Table 4).

[0400] in conclusion: When using ITLC and SE-HPLC for detection 177 Lu-DOTA-C0008 is stable in formulation buffer for up to 7 days.

[0401] Table 3: Buffer solution for formulation at 4℃ 177ITLC radioactivity purity of Lu-DOTA-C0008

[0402] D0 97.40％ D1 97.35％ D2 96.81％ D3 96.54％ D7 96.25％

[0403] *Antibody Linkage 177 The percentage of Lu corresponds to intact protein, protein fragments, and aggregates.

[0404] Table 4: Buffer solution for formulation at 4℃ 177 Radioactivity concentration of Lu-DOTA-C0008

[0405] D0 10.86 mCi / mL D3 7.86 mCi / mL (7.8 mCi / mL) D7 5.00 mCi / mL (5.2 mCi / mL)

[0406] Table 5: 177 Stability of Lu-DOTA-C0008 in formulation buffer (SE-HPLC)

[0407]

[0408] HMW: High molecular weight impurities, such as polymers and oligomers; LMW: Low molecular weight impurities, such as... 177 Lu-DTPA or 177 Lu-DOTA derivatives

[0409] Example 3

[0410] 177 Stability of Lu-DOTA-C0008 in mouse plasma

[0411] Materials and methods : 177 The plasma stability of Lu-DOTA-C0008 was assessed using fresh mouse plasma (prepared with lithium heparin tubes) at 37°C. 177 The concentration of Lu-DOTA-C0008 in plasma was 70 μg / mL. Stability in plasma was evaluated by ITLC (eluting in 0.1 M citrate at pH 5), autoradiography, and SDS-PAGE on days 0, 1, 2, 3, and 7.

[0412] result: After incubation in mouse plasma at 37°C for 7 days, the radiolabeled antibody solution exhibited approximately 80% radioactivity purity, correspondingly releasing approximately 20% of the initial bound radioactivity (Table 6). The released radioactivity corresponds to... 177 Lu-DOTA-C0008 derivatives and free 177 Lu, as shown in the ITLC results ( Figure 4 SDS-PAGE analysis ( Figure 5 The results showed that the radiolabeled antibodies did not significantly degrade when incubated in plasma. Furthermore, no adverse reactions were observed.177 Trans chelation of Lu with other plasma proteins ( Figure 5 ).

[0413] in conclusion: The stability results in plasma were acceptable, with approximately 20% of the plasma released after 7 days at 37°C. 177 Lu activity. The small amount of radioactive products observed during stability studies likely correspond to... 177 Lu-DOTA-C0008 derivatives and free 177 Lu. As shown below, with 177 Compared to Lu-DOTA-C0008, the reduced hydrophobicity of the newly designed molecule C0083 resulted in the obtained 177 The plasma stability of Lu-DOTA-C0083 radioimmunoconjugate is greatly enhanced.

[0414] Table 6: 177 Stability of Lu-DOTA-C0008 in mouse plasma (ITLC)

[0415] D0 97.65％ D1 87.20％ D2 83.94％ D3 82.18％ D7 79.06％

[0416] Figure 4 Showing 177 Plasma stability (autoradiography) of Lu-DOTA-C0008: 177 Lu-DOTA-C0008 incubated in mouse plasma for 7 days showed autoradiography; 1: 177 Lu-DOTA-C0008 derivatives and free 177 Lu; 2: 177 Lu-DOTA-C0008.

[0417] Figure 5 Showing 177 Plasma stability of Lu-DOTA-C0008 (SDS-PAGE): in mouse plasma 177 Reducing SDS-PAGE of Lu-DOTA-C0008 (Left: Coomassie staining; Right: Autoradiography). Lanes 1 and 10: Molecular weight standards; Lane 2: DOTA-C0008; Lane 3: In buffer solution. 177 Lu-DOTA-C0008(T0); Lane 5: 177 Lu-DOTA-C0008 (T0 hours in plasma); Lane 6: 177 Lu-DOTA-C0008 (1 day in plasma), Lane 7: 177 Lu-DOTA-C0008 (2 days in plasma); Lane 8: 177 Lu-DOTA-C0008 (3 days in plasma); Lane 9:177 Lu-DOTA-C0008 (7 days in plasma).

[0418] Example 4

[0419] Biodistribution of C0008 in Balb / c mice bearing CT26 tumors

[0420] Materials and Methods: The biodistribution of C0008 in female Balb / c mice carrying subcutaneous tumors of the syngeneic colon cancer cell line CT26 was investigated. Ten female Balb / c mice received 3 x 10 μL of C0008 in PBS. 5 CT26 cells were administered via unilateral subcutaneous injection (100 μl / animal). This was used when the individual tumor volume reached 150-300 mm. 3 Mice received a single intravenous dose of 5 mg / kg of IRDye 800CW-labeled C0008. Two mice served as untreated “no signal” controls.

[0421] Following the manufacturer's instructions, C0008 was labeled with the IRDye 800CW protein labeling kit (high MW) from LI-COR Biosciences. After labeling and before injection of the labeled C0008 into mice, the protein concentration and labeling efficiency of the IRDye 800CW-labeled antibody were determined using nanodrop technology, and mice were administered the drug based on the post-labeling protein concentration. (LI-COR...) In vivo imaging of the administered C0008-labeled antibody was performed at the following time points in the Trilogy imaging system: 1 hour, 6–8 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, and 168 hours post-administration. Image analysis was performed to quantify the relative fluorescence intensity (RFU) of the antibody in the tumor (RFU = RFU tumor area - RFU background / mm). 2 *Tumor area).

[0422] result: Figure 6 A shows significant intratumoral distribution of intravenously administered IRDye 800CW-labeled C0008, peaking at 24 hours and persisting in the tumor for up to 7 days. No signal was detected in untreated control mice. Figure 6 B).

[0423] in conclusion: Biodistribution studies have shown that oxMIF antibodies are suitable as radiopharmaceuticals.

[0424] Figure 6Biodistribution of IRDye 800CW-labeled C0008 in female Balb / c mice carrying subcutaneous CT26 tumors is shown: in vivo infrared imaging of mice carrying subcutaneous CT26 tumors. Infrared images of mice taken at 1, 6, 24, 48, 72, 96, and 168 hours after injection of IRDye 800CW-labeled C0008 (5 mg / kg) or B: untreated mice (scale bars applicable to A and B); C: tumor penetration and retention of C0008 quantified by digital image analysis.

[0425] Example 5:

[0426] Table 7: Variant IDs and sequence combinations of antibodies used in radioimmunoconjugates

[0427]

[0428]

[0429] The newly designed anti-oxMIF antibody exhibits reduced aggregation tendency and hydrophobicity.

[0430] Materials and methods To assess hydrophobicity and aggregation, the newly designed antibody was analyzed by gel filtration (SEC) and hydrophobic interaction chromatography (HIC) using two different SEC columns and running buffer conditions, and compared with the anti-oxMIF antibody C0008.

[0431] For SEC, the sample was diluted to 0.1 mg / ml in 5x phosphate-buffered saline (5xPBST) containing 0.02% Tween, and 50 μl of the sample was loaded onto a Superdex 200 increase 10 / 300GL (GE Healthcare) gel filter column at a flow rate of 0.75 mL / min. Separation and equilibration were performed in 5xPBST 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). Alternatively, the sample was diluted to 0.2 mg / ml in 1x phosphate-buffered saline (1xPBS), and 100 μl of the sample was loaded onto an Enrich 650 (Bio-Rad) gel filter column at a flow rate of 1 mL / min. Separation and equilibration were performed in 1xPBS 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 the retention volume of the main peak (Vr, ml), and any aggregates present are manually classified.

[0432] 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 individually loaded 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 from 0–100% B (buffer B: 50 mM phosphate, 20% isopropanol; pH 7.0) along a 20 column volume (CV) gradient. Protein peaks were monitored using absorbance at 280 nm, and spectra were analyzed using the Unicornemulation software package (GE Healthcare). For each peak, results were reported as maximum peak volume (Vr) (mL).

[0433] result: The retention volume of the control antibody C0008 was close to the bed volume of the volume exclusion column (approximately 24 ml Superdex 200, approximately 18 ml Enrich 650), which corresponds to a molecular weight much smaller than that of human IgG. Figure 7 A and 7B). The unusually long retention times are primarily due to hydrophobic interactions with the stationary phase surface. In addition to the high retention volume, C0008 also contains a large amount of IgG dimers and aggregates. All newly designed antibodies showed reduced retention volumes (Vr), indicating decreased interactions with the column and thus reduced molecular hydrophobicity (Table 8, ). Figure 7 A and 7B). Compared with molecular weight standards, C0083 and C0090 exhibited the lowest retention volumes (Table 8, Figure 7 B), where Vr corresponds to the molecular weight of monomeric human IgG. Furthermore, in samples containing the newly designed antibodies C0073, C0078, C0083, and C0090, both antibody dimers and aggregations were significantly reduced. Figure 7 (A and 7B). In particular, C0083 and C0090 show only single monomer peaks.

[0434] Using HIC column retention volumes, antibodies were sorted from the minimum to the maximum retention volume (from lowest to highest retention volume) for hydrophobic IgG. Figure 7 C (Table 9), the results confirmed the data from the SEC analysis. The newly designed antibody C0083 (retention volume 12.37 ml) had the lowest hydrophobicity, while the control antibody C0008 (retention volume 14.92 ml) had the highest hydrophobicity.

[0435] in conclusion: The SEC and HIC analyses clearly show that the optimized anti-oxMIF antibody has reduced hydrophobicity and aggregation tendency.

[0436] Table 8: Volume Exclusion Chromatography

[0437] C0008 5xPBS Superdex 200 24.20 ++ C0073 5xPBS Superdex 200 14.00 (+) C0076 5xPBS Superdex 200 21.10 + C0077 5xPBS Superdex 200 15.77 + C0078 5xPBS Superdex 200 14.02 (+) C0081 5xPBS Superdex 200 16.03 + C0083 5xPBS Superdex 200 13.01 - C0008 1xPBS Enrich 650 18.06 + C0083 1xPBS Enrich 650 12.39 - C0090 1xPBS Enrich 650 12.38 -

[0438] Table 9: Chromatography of Hydrophobic Interactions

[0439]

[0440]

[0441] Figure 7 The displayed chromatograms demonstrate the aggregation and reduced hydrophobicity of the newly designed anti-oxMIF antibody. (A) Elution curves of C0008 (control antibody, gray area) and the newly designed antibody compared on a Superdex 200increase 10 / 300GL gel filter column using 5xPBST as the mobile phase. (B) Elution curves of C0008 (control antibody, gray area) and the newly designed antibody compared on an Enrich 650 gel filter column using 1xPBS as the mobile phase. (C) Elution curves of C0008 (control antibody, gray area) and the newly designed antibody compared on a HiTrap butyl HPHIC column.

[0442] Example 6:

[0443] The newly designed anti-oxMIF antibody binds to immobilized MIF (K D (Measurement)

[0444] Materials and methods: Recombinant human MIF was diluted in PBS at 1 μg / ml, immobilized on ELISA plates, and incubated 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 plates. Finally, the bound antibody was detected using protein L-HRP conjugate and tetramethylbenzidine (TMB) as substrates. The colorimetric reaction was terminated with 3MH2SO4, and the OD was measured at 450 nm. Data from different experiments were normalized relative to the maximum OD (=100%) of anti-oxMIF antibody C0008 in each experiment, and EC50 was determined by GraphPad Prism using a 4-parameter fit. 50 Value (showing the average of the two experiments).

[0445] Results and conclusions: The binding of the newly designed antibody to immobilized MIF (oxMIF) was measured over a wide concentration range, and the resulting binding curves are shown below. Figure 8 As shown. Anti-oxMIF antibody C0008 was used as a reference for oxMIF binding. Binding curves and calculated EC50 values ​​are shown. 50 Value (representing K) DThis clearly shows that, compared to C0008 (EC) 50 Compared to 0.7 nM, 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 range). Figure 3 A). Compared to C0076, C0077 showed a 5-fold decrease in affinity for oxMIF due to a point mutation (Y36F) in VL. Figure 8 Although the hydrophobicity of C0077 is significantly reduced ( Figure 7 A).

[0446] Figure 8 The binding curves (K) of the newly designed anti-oxMIF antibody with immobilized MIF are shown. D (Assay). C0008 is used as a reference antibody.

[0447] Table 10: K of anti-oxMIF antibodies D value

[0448]

[0449]

[0450] Example 7:

[0451] The newly designed anti-oxMIF antibody binds differentially to oxMIF and redMIF.

[0452] Materials and methods: Anti-oxMIF antibody at a concentration of 15 nM was immobilized in microplates and incubated overnight at 4 °C. After blocking, 50 ng / ml of redMIF or the oxMIF substitute NTB-MIF was incubated in the wells (Schinagl et al., 2018). Captured oxMIF was detected using biotinylated polyclonal rabbit antiMIF antibody and streptavidin-HRP conjugate. Plates were stained with tetramethylbenzidine (TMB), and the colorimetric reaction was terminated by adding 30% H₂SO₄. OD was measured at 450 nm. Data from different experiments were normalized relative to the maximum OD (=100%) of anti-oxMIF antibody C0008 in each experiment, and the average values ​​from two to three experiments are shown.

[0453] Results and conclusions: The newly designed antibody binds to soluble oxMIF and redMIF as follows: Figure 9As shown in the figure. The results clearly demonstrate that the newly designed antibodies C0073, C0076, C0078, C0083, and C0090 exhibit strong binding to oxMIF but not to redMIF; therefore, the antibodies with reduced hydrophobicity and aggregation retain their ability to distinguish between oxMIF and redMIF. The newly designed antibodies show similar OD to antibody C0008, which has been described as being able to distinguish between oxMIF and redMIF (Thiele et al., 2015). Figure 9 Compared to C0076, C0077 showed a significantly reduced binding to oxMIF due to a point mutation in VL (Y36F). Figure 9 C0077 exhibits significantly reduced hydrophobicity. Figure 7 A), but failed to maintain binding properties.

[0454] Figure 9 The novel antibody was shown to bind differentially to oxMIF and redMIF. C0008 was used as a reference antibody.

[0455] Example 8:

[0456] Improved production of the newly designed antibody C0083

[0457] To compare expression yields, the newly designed anti-oxMIF antibodies C0083 and C0008 were generated by transient expression in ExpiCHO cells (Thermo Fisher). Briefly, 50–200 ml of exponentially growing ExpiCHO cells were transiently transfected using the ExpiFectamine CHO kit (Thermo Scientific) and cultured 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 the supernatant was diluted 1:1 with 40 mM sodium phosphate and 300 mM sodium chloride (pH 7.2). The diluted supernatant was filtered 0.2 μm and applied to a Mab Select Prism A column (Cytiva) using the NGCQUEST 10 Chromatography System (Bio-Rad) at room temperature. The column was washed with 10 column volumes of 20 mM sodium phosphate and 150 mM sodium chloride (pH 7.2), and the antibody was eluted with 10 column volumes of 100 mM glycine (pH 3.5). The antibody was immediately neutralized to pH 5 and prepared in 1xPBS (pH 7.2) using a Bio-Scale P-6 desalting filter, and concentrated to approximately 1 mg / mL using an Amicon Ultra-15 centrifuge (Merck Millipore). Protein concentration was determined using nanodrop technology and their respective extinction coefficients. The antibody titer (mg / L) was calculated by dividing the amount of antibody purified after neutralization using a Mab Select Prism A column (the final antibody yield) by the cell culture volume.

[0458] result Compared to C0008, the three independent production results of C0083 clearly demonstrate that the newly designed antibody C0083 was produced at a higher level in large quantities (unpaired t-test, p = 0.03). Figure 10 ).

[0459] in conclusion Compared to C0008, after optimization in reducing the hydrophobicity and aggregation tendency of the antibody, a significantly higher antibody titer was obtained (117 mg / L C0083 vs. 93 mg / mL C0008).

[0460] Figure 10 The improved production of the newly designed anti-oxMIF antibody C0083 compared to the control antibody C0008 is shown.

[0461] Example 9:

[0462] Biodistribution of the newly designed anti-oxMIF antibody C0083 in Balb / c mice bearing CT26 tumors

[0463] Materials and methods This study investigated the biodistribution of a newly designed anti-oxMIF antibody, C0083, in female Balb / c mice carrying subcutaneous tumors of the syngeneic colon cancer model CT26. Female Balb / c mice received 3 x 10⁻⁶ oz. PBS. 5 CT26 cells were administered via unilateral subcutaneous injection (100 μl / animal). This was used when the individual tumor volume reached 150-300 mm. 3 Mice were assigned to the treatment group and received a single intravenous dose of 5 mg / kg of IRDye 800CW-labeled C0083.

[0464] Following the manufacturer's instructions, C0083 was labeled with the LI-COR Biosciences IRDye 800CW Protein Labeling Kit (high MW). After labeling and before injection of the labeled antibody into mice, the protein concentration and labeling efficiency of the IRDye 800CW labeled antibody were determined using nanodroplet technology, and the mice were administered the antibody based on the post-labeling protein concentration. At LI-COR... In vivo imaging of the administered labeled antibody was performed at the following time points in the Trilogy imaging system: 1 hour, 6–8 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, and 168 hours post-dose. Image analysis was performed to quantify the relative fluorescence intensity (RFU) of the antibody in the tumor (RFU = RFU tumor area - RFU background / mm). 2 *Tumor area)

[0465] result Significant intratumoral distribution of C0083 labeled with intravenously administered IRDye 800CW was determined. Figure 11 AB), its peak is in 24 hours, and the tumor is retained for more than 7 days.

[0466] in conclusion The results clearly demonstrate that C0083 exhibits improved tumor penetration and preservation properties compared to C0008. Figure 6 A / Figure 11 C). Furthermore, biodistribution studies have shown that C0083 is suitable for use as a radioimmunoconjugate.

[0467] Figure 11This study demonstrates the tumor penetration of newly designed anti-oxMIF antibodies C0083 and C0008 in mice carrying subcutaneous CT26 tumors using in vivo infrared imaging. A: Infrared images of mice taken at 1, 6, 24, 48, 72, 96, and 168 hours after injection of 5 mg / kg of IRDye 800CW-labeled C0083; B: Tumor penetration and retention of C0083 quantified by digital image analysis; C: Tumor penetration and retention of IRDye 800CW-labeled C0083 compared to C0008 quantified by digital image analysis.

[0468] Example 10

[0469] The newly designed C0083 177 Production of Lu-radioimmunoconjugates

[0470] Materials and methods :

[0471] C0083 was incubated with SCN-Bn-DOTA at a molar ratio of 1:10 in 0.06 M HEPES at pH 9. The labeled mixture was stirred at 37 °C for 2 hours. After the reaction, excess SCN-Bn-DOTA was removed by multiple centrifugations at 3000 g in 0.9% NaCl using a 30 kDa MWCO Amicon ultracentrifuge filter (regenerated cellulose, Millipore). At the end of purification, the chemical purity of the conjugate was assessed by SE-HPLC. SE-HPLC was performed on a Superdex 200 10 / 300GL column, eluting with phosphate buffer containing arginine at a flow rate of 0.6 mL / min. In addition, the purity and integrity of DOTA-C0083 were assessed by SDS-PAGE (4–15% linear gradient polyacrylamide gel; Bio-Rad Laboratories) under reducing and non-reducing conditions.

[0472] The number of DOTA molecules linked to each antibody was determined using the method of Meares et al. (1984) and rapid thin-layer chromatography (ITLC). Small aliquots of the DOTA-conjugated antibody were then subjected to tracer amounts of radioactive... 177 Lu and stable lutetium ( 175 Incubate together with a mixture of Lu) 175 / 177Lu was added in excess of DOTA antibody by 2, 4, 6, and 8 times (Table 11). Unconsumed lutetium was then conjugated with excess EDTA to prevent the formation of insoluble lutetium hydroxide and non-specific binding of lutetium to the antibody. For analysis, 1 μL of radioactive solution was placed on ITLC test paper and eluted with 0.1 M citrate buffer at pH 5. Autoradiography of the TLC plate was obtained after exposure on a phosphorus screen (Typhoon IP, Amersham) using an ImageQuant TL according to volume integral. The number of DOTA molecules linked to each DOTA-C0083 was determined by labeling efficiency and the amount of protein added to the solution. 175 / 177 The estimate is based on the molar ratio of Lu ions.

[0473] Table 11: Marking scheme used to determine the number of DOTA grafted to C0083

[0474]

[0475] In order to use a specific activity of 275 MBq / mg 177 Lu radiolabeled DOTA-C0083 will be used in 0.04N HCl at a concentration of 81.4 MBq. 177 Lu chloride (ITG Germany) and 20 μL of 0.25M ammonium acetate at pH 5.4 were added to 300 μg of DOTA-C0083. The reaction mixture was incubated at 37°C for 10 minutes, followed by incubation of the radiolabeled conjugate with 1 mM DTPA (final concentration) for an additional 5 minutes to recombine excess. 177 Lu. After adding DTPA, the chelation efficiency was evaluated by ITLC, eluted in 0.1M citrate buffer at pH 5, and autoradiography of the TLC plate (Typhoon IP, Amersham) was performed using ImageQuant TL based on volume integral.

[0476] result Co-incubation of C0083 with SCN-Bn-DOTA, followed by purification, yielded a DOTA-C0083 conjugate with a chemical purity of approximately 97%, as shown by SE-HPLC. Figure 12 A) and SDS PAGE Figure 12 As shown in B). The number of DOTA molecules linked to each antibody in the obtained DOTA-C0083 conjugate was analyzed, and the ITLC results are shown in Figure B). Figure 13 As shown. Under the selected conditions, each antibody yielded an average of approximately one DOTA molecule. The DOTA-C0083 conjugate was then successfully labeled with lutetium-177, as determined by ITLC autoradiography. Figure 14The obtained radioimmunoconjugate showed a specific activity of 7.5 mCi / mg (277 MBq / mg) and a total radiochemical purity of 97.8%.

[0477] in conclusion A method was established using 177 An efficient method for radiolabeling a newly designed anti-oxMIF antibody, C0083, using Lu. Compared to C0008, the resulting radioimmunoconjugate exhibits superior purity and specific activity. Furthermore, SEC analysis showed that both the DOTA-C0083 conjugate and its naked antibody (C0083) exhibited reduced hydrophobicity compared to C0008. Figure 12 A is held for 19.1 minutes. Figure 1 A (retention time: 20.8 minutes).

[0478] Figure 12 The purity and integrity analysis of DOTA-C0083 is shown; A: DOTA-C0083 SE-HPLC chromatogram (λ=280nm). B: Coomassie staining SDS PAGE: 1: Molecular weight standard; 2: C0083 non-reduced; 3: C0083 reduced; 5: DOTA-C0083 non-reduced; 6: DOTA-C0083 reduced.

[0479] Figure 13 The results show the number of DOTA molecules grafted onto C0083 determined by ITLC: ITLC separated C0083-DOTA, and C0083-DOTA was traced to radioactivity in 0.04N HCl. 177 Lu and stable lutetium ( 175 Incubate together with a mixture of Lu) 177 / 175 In is 2 times (lane 1), 4 times (lane 2), 6 times (lane 3) or 8 times (lane 4) more than the DOTA antibody.

[0480] Figure 14 The analysis of radiolabeled data by ITLC and autoradiography is shown. 177 Lu-DOTA-C0083: Autoradiography of ITLC thin films (after DTPA addition) 177 Lu-DOTA-C0083; Peak 1 / 2 reflects free... 177 Lu-DTPA and 177 Lu-DOTA-C0083 derivative, peak 3 reflects 177 Lu-DOTA-C0083).

[0481] Example 11

[0482] 177 Stability of Lu-DOTA-C0083 in formulation buffer

[0483] Materials and methods :

[0484] preparation 177 Lu-DOTA-C0083 solution was prepared to achieve a final concentration of 200 μg / mL suitable for stability studies. 177 The properties of Lu-DOTA-C0083 solution are shown in Table 12.

[0485] Table 12: 177 Characteristics of Lu-DOTA-C0083 solution

[0486]

[0487] The formulation buffer stored at 4°C was analyzed by ITLC and SE-HPLC at different time points. 177 Lu-DOTA-C0083. Radiochemical purity was determined by ITLC, eluting with 0.1 M citrate at pH 5 to quantify the percentage of radioactivity released over time by the radioimmunoconjugate. SE-HPLC was performed on a Superdex 200, 10 / 300GL column, eluting with arginine-containing phosphate buffer at a flow rate of 0.6 mL / min to determine aggregation and contaminants (i.e.,...). 177 Lu-DTPA or 177 (Lu-DOTA). Chromatograms were obtained using a Radiomatic 150TR flow scintillation analyzer.

[0488] Furthermore, the radioactivity of the radioimmunoconjugate was measured by gamma counting to provide information about... 177 Information on the solubility of Lu-DOTA-C0083 in formulation buffer. For this purpose, 5 μL was directly counted from a 1 / 20 dilution in the formulation buffer.

[0489] result : 177 ITLC analysis of Lu-DOTA-C0083 showed that the radiolabeled immunoconjugate released almost no radioactivity over time. After 7 days, the radiolabeled antibody solution exhibited 94% radioactivity purity. When stored in formulation buffer at 4°C (Table 13), the radiolabeled antibody released approximately 4% of the initial bound radioactivity (Table 13). 177 Lu-DOTA-C0083 was eluted in 19.1 minutes, and its contaminants ( 177 Lu-DTPA and 177The Lu-DOTA derivative was eluted in 28.6 minutes. From day 0 to day 7, the LMW material increased slightly, but no increase in aggregates was detected (Table 15). After 7 days, the administered solution showed 93% intact antibody as analyzed by SE-HPLC. Therefore, the SE-HPLC results were consistent with the ITLC results. Over time, the monitored radioactivity was very consistent with the theoretical radioactive decay of lutetium-177 (values ​​in parentheses in Table 14).

[0490] in conclusion When tested with ITLC and SE-HPLC, the newly designed anti-oxMIF antibody C0083 showed... 177 The Lu-DOTA conjugate is stable for up to 7 days in formulation buffer.

[0491] Table 13: Buffer solutions for formulations at 4°C 177 ITLC radioactivity purity of Lu-DOTA-C0083

[0492] D0 97.76％ D1 97.34％ D3 96.15％ D7 93.69％

[0493] *Antibody Linkage 177 The percentage of Lu corresponds to intact protein, protein fragments, and aggregates.

[0494] Table 14: Buffer solutions for formulations at 4°C 177 Radioactivity concentration of Lu-DOTA-C0083

[0495] D0 1.41 mCi / mL D3 1.03 mCi / mL (1.01 mCi / mL) D7 0.68 mCi / mL (0.67 mCi / mL)

[0496] Table 15: 177 Stability of Lu-DOTA-C0083 in formulation buffer (SE-HPLC)

[0497]

[0498] HMW: High molecular weight impurities, such as aggregates and oligomers; LMW: Low molecular weight impurities, such as... 177 Lu-DTPA or 177 Lu-DOTA derivatives

[0499] Example 12

[0500] 177 Stability of Lu-C0083 in mouse plasma

[0501] Materials and methods Evaluation was conducted using fresh mouse plasma (prepared with lithium heparin tubes) at 37°C. 177 Plasma stability of Lu-DOTA-C0083. 177The concentration of Lu-DOTA-C0083 in plasma was 20 g / mL. Stability in plasma was evaluated by ITLC (eluting in 0.1 M citrate at pH 5), autoradiography, and SDS-PAGE on days 0, 1, 3, and 7.

[0502] result After incubation in mouse plasma at 37°C for 7 days, the radiolabeled antibody solution exhibited approximately 96% radioactivity purity, correspondingly releasing only 1% of the initial bound radioactivity (Table 16). The released radioactivity corresponds to... 177 Lu-DOTA-C0083 derivatives and free 177 Lu, as shown in the ITLC results ( Figure 15 SDS-PAGE analysis showed that the radiolabeled antibody did not significantly degrade when incubated in plasma. Figure 16 Furthermore, no observations were made. 177 Trans chelation of Lu with other plasma proteins ( Figure 16 ).

[0503] in conclusion : 177 Lu-DOTA C0083 exhibits excellent stability in plasma, releasing only 1% after 7 days at 37°C. 177 Lu activity, and no degradation products. 177 Compared to Lu-DOTA-C0008, the reduced hydrophobicity of the newly designed molecule C0083 also led to the obtained 177 The plasma stability of the Lu-DOTA-C0083 radioimmunoconjugate was significantly enhanced (after 7 days). 177 Lu-DOTA-C0083 and 177 The initial binding radioactive release of Lu-DOTA-C0008 (Example 3) was 1% and 20%, respectively.

[0504] Table 16: 177 Stability of Lu-DOTA-C0083 in mouse plasma (ITLC)

[0505] D0 97.36％ D1 97.01％ D3 96.01％ D7 96.26％

[0506] Figure 15 Showing 177 Plasma stability (autoradiography) of Lu-DOTA-C0083: 177 Autoradiography of Lu-DOTA-C0083 after 7 days of incubation in mouse plasma; 1: 177 Lu-DOTA-C0083 derivatives and free 177 Lu; 2: 177 Lu-DOTA-C0083.

[0507] Figure 16 Showing 177 Plasma stability of Lu-DOTA-C0083 (SDS-PAGE): in mouse plasma 177 Reducing SDS-PAGE of Lu-DOTA-C0083 (Left: Coomassie staining; Right: Autoradiography). Lane 1: Molecular weight standard; Lane 2: In buffer solution 177 Lu-DOTA-C0083(T0); Lane 3: 177 Lu-DOTA-C0083 (T0 hours in plasma); Lane 4: 177 Lu-DOTA-C0083 (1 day in plasma), Lane 5: 177 Lu-DOTA-C0083 (3 days in plasma); Lane 6: 177 Lu-DOTA-C0083 (7 days in plasma).

[0508] Example 13: The newly designed anti-oxMIF antibody with Fc silencing exhibits reduced aggregation tendency and hydrophobicity.

[0509] To assess hydrophobicity and aggregation, the newly designed Fc-silencing antibodies C0115 and C0118 were analyzed by gel filtration (SEC) of an SEC column and by hydrophobic interaction chromatography (HIC), and compared with the control anti-oxMIF antibody C0008 and its parental antibodies C0083 and C0090 without Fc silencing.

[0510] For SEC, the sample was diluted to 1 mg / ml in 1x phosphate-buffered saline (1x PBS), and 100 μl of sample was loaded onto an Enrich 650 (Bio-Rad) gel filter column at a flow rate of 1.25 ml / min. Separation and equilibration were performed in 1x PBS at room temperature. Protein peaks were monitored using absorbance at 280 nm, and spectra were analyzed using the ChromLab software package (Bio-Rad). Results were reported as the retention volume (Vr, ml) of the main peak, and aggregates present were manually fractionated.

[0511] For hydrophobic interaction chromatography (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 6.9). Highly purified antibody samples (approximately 100 μg) were individually 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 22 °C. Peak separation was performed from 0–100% B (buffer B: 50 mM phosphate, 20% isopropanol; pH 7.0) using a 20 column volume (CV) gradient. Protein peaks were monitored using absorbance at 280 nm, and spectra were analyzed using the ChromLab software package (Bio-Rad).

[0512] result The retention volume of the control antibody C0008 was close to the empty volume of the volume exclusion column (approximately 15 ml Enrich 650), which corresponds to a molecular weight much smaller than that of human IgG. Figure 18 A). The unusually long retention time is primarily due to hydrophobic interactions with the stationary phase surface. In addition to the high retention volume, C0008 also exhibits a large number of IgG dimers and aggregates. Compared to molecular weight standards, the newly designed antibodies C0115 and C0118 show retention volumes corresponding to the molecular weight of monomeric human IgG (Table 17). Figure 18 A). Furthermore, in samples containing the newly designed antibodies C0115 and C0118, antibody dimers and aggregations were significantly reduced ( Figure 18 A). Compared to their parental antibodies C0083 and C0090 (which have wild-type Fc), the Fc silencing mutations in the newly designed antibodies C0115 and C0118 did not alter their SEC profiles. Figure 18 A and Example 6 Figure 7 B).

[0513] HIC column retention volume is a measure of hydrophobicity, where antibodies with lower retention volumes are less hydrophobic than antibodies with higher retention volumes. Figure 18 B). The newly designed Fc silencing antibodies C0115 and C0118 exhibit low hydrophobicity (retention volume of 15-16 ml), while the control antibody C0008 (retention volume of approximately 21 ml) exhibits very high hydrophobicity. The Fc silencing mutations in the newly designed antibodies C0115 and C0118 did not alter their HIC profiles compared to their parental antibodies C0083 and C0090 (which possess wild-type Fc).

[0514] in conclusion The SEC and HIC analyses clearly show that, compared with the control anti-oxMIF antibody C0008, the newly designed Fc-silencing antibody has improved biochemical properties, especially reduced hydrophobicity and aggregation tendency.

[0515] Table 17: Volume Exclusion Chromatography

[0516]

[0517] Figure 18 The chromatograms shown demonstrate the aggregation and reduced hydrophobicity of the newly designed Fc-silencing anti-oxMIF antibodies. (A) Elution curves of C0008 (control antibody, gray area) and the newly designed Fc-silencing antibodies C0115 and C0118 on an Enrich 650 gel filter column using 1xPBS as the mobile phase; (B) Elution curves of C0008 (control antibody, gray area) and the newly designed Fc-silencing antibodies C0115 and C0118 and their parental antibodies C0083 and C0090 (without Fc silencing) on ​​a HiTrap butyl HP HIC column.

[0518] Example 14: Binding of newly designed Fc-silencing anti-oxMIF antibodies C0115 and C0118 to immobilized MIF (K D (Assay), anti-oxMIF antibodies C0115 and C0118 conjugated or unconjugated with DFO*.

[0519] Recombinant human MIF was diluted in PBS at 1 μg / ml, immobilized on ELISA plates, and incubated overnight at 4°C (MIF was converted to oxMIF according to Thiele et al., 2015). After blocking, serial dilutions of anti-oxMIF antibodies with and without the DFO* conjugate (described in Example 19) were added to the plates. Finally, the bound antibodies were detected using goat anti-human IgG (Fc)-HRP conjugate and tetramethylbenzidine (TMB) as substrates. The colorimetric reaction was terminated with 3M H2SO4, and the OD was measured at 450 nm. If data from different experiments were pooled, the data were normalized relative to the maximum OD (=100%) of the anti-oxMIF antibody C0008 for each experiment, and the EC was determined by GraphPad Prism using a 4-parameter fit. 50 Values ​​(showing the average of the two experiments + / - SEM).

[0520] Results and Conclusions The binding of the newly designed antibody to immobilized MIF (oxMIF) was measured over a wide concentration range, and the resulting binding curves are shown below. Figure 19 As shown. Anti-oxMIF antibody C0008 was used as a reference for oxMIF binding. Binding curves and calculated EC50 values ​​are shown. 50 Value (representing apparent K) D This clearly demonstrates that, compared to C0008, the newly designed Fc silencing antibodies C0115 and C0118 retain their low nanomolar affinity for oxMIF. Figure 19 A, Table 18). Furthermore, DFO* conjugation of the newly designed Fc silencing antibodies C0115 and C0118 did not alter their affinity ( Figure 19 B). This indicates that data obtained using unconjugated antibodies, as well as those labeled with IRDye800CW, can be directly transferred to radioimmunoconjugates, such as DFO*-Zr89-labeled antibodies.

[0521] Figure 19 The binding curves (K) of the newly designed Fc-silenced anti-oxMIF antibody with immobilized oxMIF are shown. D (Assay). Anti-oxMIF antibody was detected by anti-human IgG(Fc)-HRP conjugate, with C0008 used as a reference antibody.

[0522] Table 18: K values ​​of newly designed Fc-silencing anti-oxMIF antibodies C0115 and C0118 compared to C0008 D Value comparison.

[0523] C0008 0.19 C0115 0.28 C0118 0.26

[0524] Example 15: Differential binding of newly designed Fc-silencing anti-oxMIF antibodies C0115 and C0118 to oxMIF and redMIF.

[0525] Materials and methods: As described in Example 8, the binding of C0115 and C0118 with oxMIF and redMIF was analyzed.

[0526] Results and Conclusions The binding of newly designed Fc silencing antibodies C0115 and C0118 to soluble oxMIF and redMIF is as follows: Figure 20 As shown in the figure. The results clearly demonstrate that the newly designed Fc-silencing antibodies C0115 and C0118 exhibit strong binding to oxMIF but not to redMIF. C0115 and C0118 show OD values ​​very similar to the reference antibody C0008, which has been described as capable of distinguishing between oxMIF and redMIF (Thiele et al., 2015). No binding to isotype IgG was observed. Therefore, the newly designed antibodies with reduced hydrophobicity and aggregation, as well as Fc silencing, retain their ability to distinguish between oxMIF and redMIF.

[0527] Figure 20 The study demonstrated the differential binding of the newly designed Fc silencing antibody to oxMIF and redMIF. C0008 was used as a reference antibody, and isotype IgG was used as a negative control.

[0528] Example 16: Newly designed Fc-silencing anti-oxMIF antibodies C0115 and C0118 with A2780 MIF - / - Non-specific binding to cells is reduced.

[0529] A2780MIF cells were generated by CRISPR / Cas9 gene editing of the human MIF gene in the A2780 ovarian cancer cell line. - / - Cell lines. In short, based on GenCRISPR... TM Following general rules for designing targeted guide RNAs (gRNAs), the target gene sequence was analyzed and the target site was located. A guide RNA (gRNA) was designed to specifically recognize the 5' region of the MIF gene (TTGGTGTTTACGATGAACATCGG, SEQ ID NO:48), and the gRNA sequence was cloned into the PX459 (lentiviral vector addgene) vector containing the *S. pyogenes* Cas9 (SpCas9) nuclease. A2780 cells were transiently transfected via electroporation, and the cells were seeded in 96-well plates using limiting dilutions to generate isogenetic monoclonal clones. Isogenetic monoclonal clones were identified by Sanger sequencing, in which the endogenous MIF gene was effectively mutated, resulting in a corresponding reduction (or elimination) of MIF protein expression. The final clones showed a 10 bp deletion at position +2 after the start codon of the human MIF gene. A2780 MIF - / - The absence of endogenous human MIF protein in the cell line was confirmed by Western blotting using a polyclonal anti-human MIF antibody.

[0530] A2780 MIF was separated using a cell-splitting agent (Corning, catalog number 25-056-Cl). - / - Cells were washed with staining buffer (PBS + 5% BSA) and divided into 2 × 10⁶ cells per well. 5Cells were seeded into 96-well U-shaped plates. Cells were stained with the immobilizing viability dye eFluor 780 (Invitrogen, diluted 1:2000 in PBS) at 4°C for 20 minutes, followed by washing with staining buffer. Cells were resuspended in 50 μl of staining buffer, and 50 μl of serially diluted Fc-silencing anti-oxMIF antibodies C0115 and C0118 or their parental antibodies C0083 and C0090, or control anti-oxMIF antibody C0008 or isotype IgG (final concentration 37 nM–9.4 nM) were added. After incubation at 4°C for 40 minutes, cells were washed with staining buffer and resuspended in 100 μl of secondary antibody (goat anti-human IgG (H+L)-Alexa Fluor 488, diluted 1:100). After incubation at 4°C for 30 minutes, the cells were washed with staining buffer, resuspended in PBS + 2% BSA, and collected on a Cytoflex-S flow cytometer (Beckman Coulter).

[0531] Data was analyzed using FlowJo, and the geometric mean of live cells (mean fluorescence intensity of AF488) was plotted against antibody concentration in GraphPad Prism.

[0532] Results and Conclusions :from Figure 21 As can be clearly seen from A and 21B, the newly designed Fc-silencing anti-ox MIF antibody C0118 and its parent antibody C0090 (A) and C0118 and its parent antibody C0083 (B) are not similar to A2780 MIF. - / - Cells bind to each other because their geometric mean is very close to that of the negative control isotype IgG. Conversely, the anti-oxMIF antibody C0008 exhibits similarity to A2780 MIF. - / - The cells showed significant binding, even though they were shown not to express MIF. Therefore, the reduced hydrophobicity led to a significant decrease or elimination of the newly designed anti-oxMIF antibody with A2780 MIF. - / - Non-specific binding, while the anti-oxMIF control antibody C0008 binds non-specifically to the cell surface due to its hydrophobicity.

[0533] Figure 21 The newly designed anti-oxMIF antibody was demonstrated in A2780 MIF as measured by FACS. - / - Reduced nonspecific binding on cells. The novel Fc-silencing anti-oxMIF antibody C0118 and its parental antibodies C0090(A) and C0115 and their parental antibody C0083(B), and the control antibody C0008, along with isotype IgG as a negative control, were used to target A2780 MIF. - / -Cells were stained; the geometric mean of live cells (average fluorescence intensity of AF488) was plotted against antibody concentration.

[0534] Example 17: The ADCC effector function of the newly designed Fc-silencing anti-oxMIF antibodies C0115 and C0118 was significantly reduced, as determined by reporter gene analysis.

[0535] As mentioned above, efforts can be made to reduce the ADCC potential of antibodies through point mutations in the Fc moiety (i.e., L234A / L235A). When the Fc moiety of a target-binding antibody binds to the Fc receptor on the cell surface of an effector cell, multiple cross-linking occurs between the two cell types, leading to activation of the ADCC pathway. This is detrimental to the use of target-neutralizing antibodies to prevent functional Fc-related adverse events.

[0536] Using engineered Jurkat cells stably expressing the human FcγRIIIA receptor, V158 (high-affinity allotype), and NFAT effector elements that drive firefly luciferase expression as effector cells, the luciferase produced by NFAT pathway was activated to quantify antibody biological ADCC activity.

[0537] The ADCC reporter gene assay was performed primarily as recommended by the manufacturer (Promega #G7010).

[0538] To generate highly responsive target cells, HCT116 cells were transfected with the huMIF-pDisplay plasmid (Invitrogen), selected with genimycin, and sorted by FACS to produce a cell line stably expressing membrane-anchored monomeric human MIF (HCT116-pMIF), i.e., MIF is displayed as a monomeric protein, where the oxMIF epitope can be accessed by anti-oxMIF antibodies (Schinagl et al., 2018). This cell line showed increased presentation of oxMIF on the cell surface, thus providing a more sensitive tool for in vitro analysis.

[0539] In short, HCT116-pMIF target cells in 100 μl of assay medium (RPMI 1640 medium supplemented with Pen / Strept / L-glutamine and 4% low IgG FBS) were incubated at 1 × 10⁻⁶ ppm. 4Cells were seeded into 96-well flat-bottomed white plates and incubated overnight at 37°C / 5% CO2. The next day, the medium was replaced with 25 μl of fresh assay medium. Serial dilutions of 25 μl of newly designed anti-oxMIF antibodies C0115, C0118, or their parental antibodies C0083, C0090 (final concentration 0.01-100 nM) with silenced Fc cells and 25 μl of freshly thawed effector cells (FcγRIIIA receptor effector cells of the high-response genotype V158) were added to the plates at an effector cell to target cell ratio of approximately 6:1. After incubation at 37°C / 5% CO2 for 6 hours, the assay plates were equilibrated to room temperature, and Bio-Glo luciferase reagent was added. After 10-20 minutes of incubation, luminescence (RLU) was measured using a Tecan multi-well plate reader (0.5-second integration time).

[0540] result :from Figure 22 It is evident that the newly designed antibodies C0115 and C0118 containing the Fc silencing mutation did not show any effector cell activation in the ADCC reporter gene assay, while the parental antibodies C0083 and C0090 with wild-type Fc induced strong FcyRIIIA-mediated effector cell activation.

[0541] in conclusion The newly designed variants C0115 and C0118, containing Fc silencing mutations (L234A / L235A), did not exhibit any ADCC initiation in reporter gene bioassays. Therefore, they showed significantly reduced ADCC effector function.

[0542] Figure 22 The results show that the ADCC effector function of the newly designed Fc silencing antibodies C0115 and C0118 was significantly reduced by reporter gene assays. ADCC reporter gene bioassays were performed using engineered Jurkat effector cells stably expressing FcyRIIIA and HCT116-pMIF target cells, and the newly designed Fc silencing antibodies C0115 and / or C0118 were compared with their parental antibodies C0083 and C0090, which possess wild-type Fc. Mean values ​​and SEM (n=2) are shown.

[0543] Example 18: Biodistribution of the newly designed Fc-silencing anti-oxMIF antibody C0115 and control antibody C0008 in nude Balb / c mice bearing HCT116 tumors.

[0544] Materials and methodsIn a human colon adenocarcinoma cell line HCT116 xenograft mouse model, the biodistribution of a newly designed Fc-silenced anti-oxMIF antibody C0115 was investigated by head-to-head comparison with the reference anti-oxMIF antibody C0008. Female Balb / c nude mice received 5x10 [units of antibiotics] in 50% PBS and 50% Matrigel. 6 HCT116 cells were administered via unilateral subcutaneous injection, with a total injection volume of 100 μl. This was used when the individual tumor volume reached 150-300 mm². 3 Mice were assigned to the treatment group and received a single intravenous dose of 5 mg / kg of IRDye 800CW-labeled C0115 or C0008.

[0545] Following the manufacturer's instructions, C0115 and C0008 were labeled using the LI-COR Biosciences IRDye 800CW Protein Labeling Kit (high MW). After labeling and before injection of the labeled antibodies into mice, the protein concentration and labeling efficiency of the IRDye 800CW labeled antibodies were determined using nanodroplet technology, and mice were administered the antibodies based on the post-labeling protein concentration. At LI-COR... In the Trilogy imaging system, in vivo imaging of the administered labeled antibody was performed at the following time points: 1 hour, 6–8 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, and 168 hours post-drug administration. Image analysis was performed to quantify the relative fluorescence intensity (RFU) of the antibody in the tumor (RFU / area = RFU / tumor area / mm²). 2 Tumor area - RFU background / mm 2 (Background area)

[0546] Results and Conclusions : Figure 23 The study showed that intravenously administered IRDye 800CW-labeled C0115 and C0008 exhibited significant intratumoral distribution and tumor retention for up to 7 days. Figure 23 A and quantitative image analysis ( Figure 23 B) It is evident that, compared to the reference anti-oxMIF antibody C0008 (which showed a peak at approximately 24 hours), the newly designed Fc-silencing anti-oxMIF antibody C0115 exhibited significantly enhanced and increased tumor uptake within 7 days. Therefore, biodistribution studies indicate that C0115 is more suitable than C0008 for use as a radioimmunoconjugate.

[0547] Figure 23In vivo infrared imaging of mice carrying subcutaneous HCT116 tumors shows tumor penetration and retention of the newly designed Fc-silenced anti-oxMIF antibody C0115 and the reference antibody C0008. (A) Infrared images of mice taken at 1 h, 6 h, 24 h, 48 h, 72 h, 96 h, and 168 h after injection of IRDye800CW-labeled antibodies C0115 (top) and C0008 (bottom) at doses of 5 mg / kg; (B) Tumor penetration and retention of C0115 and C0008 quantified by digital image analysis.

[0548] Example 19: Coupling with DFO* 89 Biodistribution of novel Zr-labeled Fc-silencing antioxMIF antibodies C0115 and C0118 in Balb / c or Balb / c nude mice bearing CT26 or HCT116 tumors

[0549] Materials and methods Using a mouse model with human colon adenocarcinoma cell line HCT116 xenograft and a mouse model with mouse colon adenocarcinoma cell line CT26 allograft, a newly designed Fc-silencing anti-oxMIF antibody was studied. 89 Zr-DFO*-C0115 and 89 Biodistribution of Zr-DFO*-C0118.

[0550] Antibodies C0115 and C0118 were conjugated with the bifunctional chelating agent DFO*-NCS (ABX advanced biochemical compounds GmbH). The antibody buffer was replaced with 0.9% NaCl, 50 mM NaHCO3, pH 9 (5.0 mg / mL). Subsequently, the antibodies were reacted with 3 molar equivalents (excess) of DFO*-NCS in DMSO (1 mg / mL) at 37°C for 45 min. After the reaction, the antibodies were purified using a HiTrap column (desalted column, GE 17-1408-01, 5 mL). During purification, the buffer was replaced with a (w / o) Ca-free buffer. 2+ and Mg 2+ DPBS.

[0551] use 89 Zr (Perkin Elmer) was radiolabeled with DFO*-Ab. First, approximately 100 MBq of... 89 Zr (in 1M oxalic acid) was mixed with 180 μl of 2M Na₂CO₃ solution and reacted at room temperature (RT) for 3 minutes. The solution was then diluted with 1 mL of HEPES (pH 7). 89The Zr solution was neutralized to pH 7, and 2 mg of DFO* antibody was added to the mixture, allowing it to react at room temperature for 60 minutes. The radiolabeled antibody was purified by centrifugation and filtration, and the buffer was replaced with 0.01 M PBS at pH 6.5 containing 50 mM arginine. Radiochemical purity (RCP) was analyzed directly by iTLC after radiolabeling, using 0.05 MDTPA as eluent, and a radio-HPLC (Agilent Infinity II, 1260) with a UV detector (280 nm), a Posiram radiodetector (Lablogic), a volume exclusion column SEC-2000 (Phenomenex, set to 30 °C), and 0.1 M sodium phosphate at pH 6.8 as the mobile phase.

[0552] Female Balb / c nude mice were treated with 5x10 [units of solution] in 50% PBS and 50% matrix gel. 6 HCT116 cells were administered via unilateral subcutaneous injection, with a total injection volume of 100 μl. Female Balb / c mice received 3 x 10⁻⁶ HCT116 cells in 100 μl PBS. 5 unilateral subcutaneous injection of CT26 cells. This is indicated when the individual tumor volume reaches 150-250 mm. 3 Mice were then assigned to the treatment group and received 10-11 MBq of medication. 89 Zr-DFO*-C0115 and 89 Single intravenous dose of Zr-DFO*-C0118 antibody. The radioactive dose was determined by measuring the syringe before and after administration using a cross-calibrated dose calibrator (VDC-405, Veenstra instruments).

[0553] Animals were anesthetized with isoflurane and underwent whole-body PET scans (60 minutes) 4, 7, or 10 days after injection of BioPET small animal PET / CT (Sedecal, Madrid, Spain), followed by CT imaging as an anatomical reference. Images were reconstructed using a 2DOSEM algorithm based on CT attenuation correction. Image analysis was performed using PMOD software (version 3.7, PMOD Technologies LLC, Zürich, Switzerland).

[0554] Results and Conclusions Both antibodies were successfully modified with DFO* and retained their affinity for oxMIF. Figure 19 B). The radiochemical yield was approximately 80% and the calculated specific activity was 19.7 MBq / mg. 89Zr-DFO*-C0115. The injected radioactivity was 11.0 ± 0.5 MBq (mean ± standard deviation, n = 6), and the injected mass was 0.56 ± 0.03 mg (mean ± standard deviation, n = 6). It was synthesized with a radiochemical yield of approximately 72% and a calculated specific activity of 54.0 MBq / mg. 89 Zr-DFO*-C0118. The injected radioactivity was 11.0 ± 0.2 MBq (mean ± standard deviation, n = 6), and the injected mass was 0.20 ± 0.01 mg (mean ± standard deviation, n = 6). From Figure 24 It is evident that the two newly designed Fc-silencing anti-oxMIF antibodies accumulated in tumors in both animal models and remained there for at least 10 days. Therefore, biodistribution studies indicate that the newly designed Fc-silencing antibodies C0115 and C0118 are suitable for use as diagnostic radioimmunoconjugates.

[0555] Figure 24 This demonstrates in vivo PET / CT imaging of mice carrying subcutaneous CT26 or HCT116 tumors, coupled with DFO*. 89 Tumor penetration and retention of novel Zr-labeled anti-oxMIF antibodies C0115 and C0118. (Injection) 89 PET and CT images of mice taken 4, 7, and 10 days after administration of the Zr-DFO*-labeled antibody at a dose of approximately 10 MBq per mouse; representative digital vertical cross-sections of the mice are shown, with white arrows marking tumors.

[0556] References

[0557] Bloom BR and Bennet,B.,1966,Mechanism of a reaction in vitro associated with delayed-type hypersensitivity,Science 153,80-82.

[0558] Brechbiel M.W.(2008).Bifunctional chelates for metal nuclides.Thequarterly journal of nuclear medicine and molecular imaging:officialpublication of the Italian Association of Nuclear Medicine(AIMN)[and]theInternational Association of Radiopharmacology(IAR),[and]Section of theSociety of...,52(2),166–173.

[0559] Brinkmann U.and Kontermann R.E.,2017,The making of bispecificantibodies,MABS,9,2,182-212.

[0560] M,et al.,1987,Comparison of the effector functions ofhuman immunoglobulins using a matched set of chimeric antibodies,J.Exp.Med.166 1351-1361.

[0561] Chomet,M.,et al.,(2021).Head-to-head comparison of DFO*and DFOchelators:selection of the best candidate for clinical 89Zr-immuno-PET.European Journal of Nuclear Medicine and Molecular Imaging,48(3),694–707.https: / / doi.org / 10.1007 / s00259-020-05002-7.

[0562] Schinagl,A.,Kerschbaumer,R.J.,Sabarth,N.,Douillard,P.,Scholz,P.,Voelkel,D.,Hollerweger,J.C.,Goettig,P.,Brandstetter,H.,Scheiflinger,F.,&Thiele,M.(2018).Role of the Cysteine 81 Residue of Macrophage MigrationInhibitory Factor as a Molecular Redox Switch.Biochemistry,57(9),1523–1532.https: / / doi.org / 10.1021 / acs.biochem.7b01156.

[0563] Clynes,et al.,1998,Fc receptors are required in passive and activeimmunity to melanoma,Proc.Nat'l Acad.Sci.USA95 652-656.

[0564] David,J.R.,1966,Delayed hypersensitivity in vitro:its mediation bycell-free substances formed by lymphoid cell-antigen interaction,Proc.Natl.Acad.Sci.U.S.A.56,72-77.

[0565] Carter L.M.et al.,2018,Preclinical optimization of antibody-basedradiopharmaceuticals for cancer imaging and radionuclide therapy-Model,vector,and radionuclide selection,J Labelled Comp Radiopharm.61(9),611-635.

[0566] Cragg,M.S.,et al.,2003,Complement-mediated lysis by anti-CD20 mAbcorrelates with segregation into lipid rafts,Blood 101 1045-1052.

[0567] Cragg,M.S.,and Glennie,M.J.,2004,Antibody specificity controls invivo effector mechanisms of anti-CD20 reagents,Blood 103(2004)2738-2743.

[0568] Estep P.et al.,2015,An alternative assay to hydrophobic interactionchromatography for high-throughput characterization of monoclonal antibodies,MAbs,7(3),553-561.

[0569] Ewert S.et al.,2003,Structure-based improvement of the biophysicalproperties of immunoglobulin VH domains with a generalizable approach,Biochemistry,18,42(6),1517-28.

[0570] Hellstrom,L,et al.,1986,Antitumor effects of L6,an IgG2a antibodythat reacts with most human carcinomas,Proc.Nat'l Acad.Sci.USA83 7059-7063.

[0571] Hellstrom,L,et al.,1985,Strong antitumor activities of IgG3antibodies to a human melanoma-associated ganglioside,Proc.Nat'lAcad.Sci.USA82(1985)1499-1502.

[0572] Hu S.Z.et al.,1996,Minibody:Anovel engineered anti-carcinoembryonicantigen antibody fragment(single-chain Fv-CH3)which exhibits rapid,high-leveltargeting of xenografts,Cancer Research,56,3055-3061.

[0573] Huang C-Y.,et al.,2012,Optimizing radioimmunoconjugate delivery inthe treatment of solid tumor,Cancer Treat Rev.,38(7):854-60.

[0574] Hussain F.et al.,2013,Human anti-macrophage migration inhibitoryfactor antibodies inhibit growth of human prostate cancer cells in vitro andin vivo,Mol Cancer Ther.Jul;12(7):1223-34.

[0575] Ianello A.and Ahmad A.,2005,Role of antibody-dependent cell-mediatedcytotoxicity in the efficacy of therapeutic anti-cancer monoclonalantibodies,Cancer and Metastasis Review,24,487-499.

[0576] Jalkanen M.et al.,1985,Heparan sulfate proteoglycans from mousemammary epithelial cells:localization on the cell surface with a monoclonalantibody,J.Cell.Biol.101,976-985.

[0577] Jalkanen M.et al.1987,Cell surface proteoglycan of mouse mammaryepithelial cells is shed by cleavage of its matrix-binding ectodomain fromits membrane-associated domain,J.Cell.Biol.105,3087-3096.

[0578] Kabat et al.,Sequences of Proteins ofImmunological Interest,FifthEdition,NIH Publication 91-3242,Bethesda MD(1991),vols.1-3.

[0579] Kerschbaumer R.J.,et al.2012 Neutralization of macrophage migrationinhibitory factor(MIF)by fully human antibodies correlates with theirspecificity for theβ-sheet structure of MIF.J Biol Chem.Mar 2;287(10):7446-55.doi:10.1074 / jbc.M111.329664.Epub 2012 Jan 11.PMID:22238348;PMCID:PMC3293543.

[0580] Krebs et al.,"Lewin′s Genes XI",Jones&Bartlett Learning,(2017).

[0581] Liu S.,2008,Bifunctional coupling agents for radiolabeling ofbiomolecules and target-specific delivery of metallic radionuclides.Adv DrugDeliv Rev.Sep;60(12):1347-70.doi:10.1016 / j.addr.2008.04.006.Epub 2008 Apr23.PMID:18538888;PMCID:PMC2539110.

[0582] Mahalingam D.et al.,2015,ASCO Abstract ID2518.

[0583] Mahalingam D.et al.2020,Phase I study of Imalumab(BAX69),a fullyhuman recombinant antioxidized macrophage migration inhibitory factorantibody in advanced solid tumours,Br J Clin Pharmacol.86(9):1836-1848.

[0584] Meares et al.1984,Conjugation of antibodies with bifunctionalchelating agents:isothiocyanate and bromoacetamide reagents,methods ofanalysis and subsequent addition of metal ions.Anal.Biochem.142,68-78.

[0585] Morrison,S.L.,et al.,1984,Chimeric human antibody molecules:mouseantigen-binding domains with human constant region domains,Proc.Natl.Acad.Sci.81,6851-6855.

[0586] Muda M.et al.,2011,Therapeutic assessment of SEED:a new engineeredantibody platform designed to generate mono-and bispecific antibodies,ProteinEng.Des.Sel.,24(5),447-54.

[0587] Murphy&Weaver,"Janeway′s Immunobiology"(9th Ed.,or more recenteditions),Taylor&Francis Inc,2017.

[0588] Natsume A.et al.,2009,“Improving effector functions of antibodies forcancer treatment:Enhancing ADCC and CDC”,Drug,Design,development and Therapy,3,7-16.

[0589] Petkova,S.B.,et al.,2006,Enhanced half-life of genetically engineeredhuman IgG1antibodies in a humanized FcRn mouse model:potential application inhumorally mediated autoimmune disease,Int'l.Immunol.18(12)1759-1769.

[0590] Phaeton R.,et al.,2016,Beta emitters rhenium-188 and lutetium-177 areequally effective in radioimmunotherapy of HPV-positive experimental cervicalcancer.Cancer Med.,Jan;5(1):9-16.doi:10.1002 / cam4.562.

[0591] Ravetch,and Kinet,1991,Fc receptors,Annu.Rev.Immunol.9,457-492.

[0592] Sambrook et al,"Molecular Cloning:ALaboratory Manual"(4th Ed.),Vols.1-3,Cold Spring Harbor Laboratory Press(2012).

[0593] Schinagl,A.,et al.,2018,Role of the Cysteine 81 Residue of MacrophageMigration Inhibitory Factor as a Molecular Redox Switch.Biochemistry,57(9),1523–1532.https: / / doi.org / 10.1021 / acs.biochem.7b01156.

[0594] Schinagl.A.et al.,2016,Oxidized macrophage migration inhibitoryfactor is a potential new tissue marker and drug target in cancer,Oncotarget.Nov 8;7(45):73486-73496.

[0595] Spiess C.et al.,2015,Alternative molecular formats and therapeuticapplications for bispecific antibodies,Mol.Immunol.,67,95-106.

[0596] Thiele M.et al.,(2015),Afully human anti-oxMIF antibody penetratescolorectal metastasis and accumulates in cancer tissue,Annals of Oncology,26,ii16.

[0597] Thiele,M.et al.,(2015).Selective Targeting of a Disease-RelatedConformational Isoform of Macrophage Migration Inhibitory Factor AmelioratesInflammatory Conditions.Journal of Immunology,195(5),2343–2352.https: / / doi.org / 10.4049 / jimmunol.1500572.

[0598] Van der Kant,R.et al.,2017,Prediction and Reduction of theAggregation of Monoclonal Antibodies,J.Mol.Biol.,429(8),1244-1261.

[0599] Warram J.M.et al,2014,Antibody-based imaging strategies for cancer,Cancer Metastasis Rev,33(2-3),809-22.

[0600] Wei Li et al.,2016,Antibody Aggregation:Insights from Sequence andStructure,Antibodies,5(3),19.

[0601] Willuda J.et al.,1999,High thermal stability is essential for tumortargeting of antibody fragments:engineering of a humanized anti-epithelialglycoprotein-2(epithelial cell adhesion molecule)single-chain Fv fragment,Cancer Res.,15;59(22),5758-67.

[0602] Wu GYand Wu CH, 1987, Receptor-mediated in vitro gene transformation by a soluble DNA carrier system, J. Biol. Chem. 262: 4429-4432 (1987)).

[0603] Zeglis BM and Lewis,JS2011,Apractical guide to the construction of radiometallated bioconjugates for positron emission tomography,Dalton trans.,40(23),6168-95. SEQUENCE LISTING <110> Oncoonne Research & Development Co., Ltd. <120> AntioxMIF radioimmunoconjugate <130> P23JM1WN00602AT <160> 48 <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1-H1 Bax 8 <400> 1 Ile Tyr Thr Met Asp 1 5 <210> 2 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2-H1 Bax 8 <400> 2 Tyr Ile Ser Pro Ser Gly Gly Asn Thr Ser Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 3 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR3-H1 Bax 8 <400> 3 Arg Gln Tyr Val Leu Arg Tyr Phe Asp Trp Ser Ala Asp Ala Phe Asp 1 5 10 15 Ile <210> 4 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR1-L1 Bax 8 <400> 4 Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2-L1 Bax 8 <400> 5 Ala Ala Ser Ser Leu Gln Ser 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR3-L1 Bax 8 <400> 6 Gln Gln Ser Tyr Ser Thr Pro Trp Thr 1 5 <210> 7 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1-H1 Imalumab <400> 7 Ile Tyr Ser Met Asn 1 5 <210> 8 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2-H1 Imalumab <400> 8 Ser Ile Gly Ser Ser Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 9 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR3-H1 Imalumab <400> 9 Ser Gln Trp Leu Tyr Gly Met Asp Val 1 5 <210> 10 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR1-L1 Imalumab <400> 10 Arg Ser Ser Gln Arg Ile Met Thr Tyr Leu Asn 1 5 10 <210> 11 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2-L1 Imalumab <400> 11 Val Ala Ser His Ser Gln Ser 1 5 <210> 12 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR3-L1 Imalumab <400> 12 Gln Gln Ser Phe Trp Thr Pro Leu Thr 1 5 <210> 13 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1-H1 Bax 74 <400> 13 Lys Tyr Tyr Met Ile 1 5 <210> 14 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2-H1 Bax 74 <400> 14 Trp Ile Gly Pro Ser Gly Gly Phe Thr Phe Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 15 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> CDR3-H1 Bax 74 <400> 15 Gly Thr Pro Asp Tyr Gly Gly Asn Ser Leu Asp His 1 5 10 <210> 16 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR1-L1 Bax 74 <400> 16 Arg Ala Ser Gln Ser Ile Gly Thr Tyr Leu Ser 1 5 10 <210> 17 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2-L1 Bax 74 <400> 17 Ala Thr Ser Arg Leu Gln Ser 1 5 <210> 18 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR3-L1 Bax 74 <400> 18 Gln Gln Thr Tyr Ser Thr Pro Leu Thr 1 5 <210> 19 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1-H1 Bax 94 <400> 19 Ile Tyr Ala Met Asp 1 5 <210> 20 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2-H1 Bax 94 <400> 20 Gly Ile Val Pro Ser Gly Gly Phe Thr Lys Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> twenty one <211> 18 <212> PRT <213> Artificial Sequence <220> <223> CDR3-H1 Bax 94 <400> twenty one Val Asn Val Ile Ala Val Ala Gly Thr Gly Tyr Tyr Tyr Tyr Gly Met 1 5 10 15 Asp Val <210> twenty two <211> 12 <212> PRT <213> Artificial Sequence <220> <223> CDR1-L1 Bax 94, Bax A10 <400> twenty two Arg Ala Ser Gln Gly Val Ser Ser Ser Ser Leu Ala 1 5 10 <210> twenty three <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2-L1 Bax 94 <400> twenty three Gly Thr Ser Ser Arg Ala Thr 1 5 <210> twenty four <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR3-L1 Bax 94 <400> twenty four Gln Gln Tyr Gly Arg Ser Leu Thr 1 5 <210> 25 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2-L1 Bax 152 <400> 25 Gly Ala Ser Asn Arg Ala Thr 1 5 <210> 26 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1-H1 BaxA10 <400> 26 Trp Tyr Ala Met Asp 1 5 <210> 27 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2-H1 Bax A10 <400> 27 Gly Ile Tyr Pro Ser Gly Gly Arg Thr Lys Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 28 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> CDR1-L1 Bax A10, Bax94 <400> 28 Arg Ala Ser Gln Gly Val Ser Ser Ser Ser Leu Ala 1 5 10 <210> 29 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Sequence VH (wt) <400> 29 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> 30 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> VH L5Q <400> 30 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> 31 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> VH L5Q W97Y <400> 31 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> 32 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Sequence VL (wt) <400> 32 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> 33 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL F49Q A51G <400> 33 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> 34 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL F49Y / A51G / W93F <400> 34 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> 35 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL M30L / F49Y / A51G / W93F <400> 35 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> 36 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL M30L / F49Y / A51G / P80S / W93F <400> 36 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> 37 <211> 329 <212> PRT <213> Artificial Sequence <220> <223> CH1-CH3 wt <400> 37 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> 38 <211> 329 <212> PRT <213> Artificial Sequence <220> <223> CH1-CH3, L234A / L235A <400> 38 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 Ala Ala 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> 39 <211> 329 <212> PRT <213> Artificial Sequence <220> <223> CH1-CH3, L234A / L235A / I253A <400> 39 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 Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ala 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> 40 <211> 329 <212> PRT <213> Artificial Sequence <220> <223> CH1-CH3, L234A / L235A / H310A / H435Q <400> 40 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 Ala Ala 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 Ala 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 Gln Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly 325 <210> 41 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> CL wt <400> 41 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> 42 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> C0090 LC <400> 42 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> 43 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> C0090 HC <400> 43 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> 44 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> MIF Catalytic Motif <400> 44 Cys Ala Leu Cys 1 <210> 45 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> oxMIF epitope <400> 45 Glu Pro Cys Ala Leu Cys Ser 1 5 <210> 46 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> C0083 / C0115 LC <400> 46 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> 47 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> C0115 / C0118 HC <400> 47 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 Ala Ala 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> 48 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> MIF gene <400> 48 ttggtgttta cgatgaacat cgg 23

Claims

1. A radioimmunoconjugate comprising a recombinant anti-oxMIF antibody or its antigen-binding fragment, characterized in that, The recombinant anti-oxMIF antibody or its antigen-binding fragment comprises the following: The light chain variable domain of SEQ ID NO: 34, the light chain constant domain of SEQ ID NO: 41, the heavy chain variable domain of SEQ ID NO: 31, and the heavy chain constant domain of SEQ ID NO: 38; or The light chain variable domain of SEQ ID NO: 36, the light chain constant domain of SEQ ID NO: 41, the heavy chain variable domain of SEQ ID NO: 31, and the heavy chain constant domain of SEQ ID NO: 38; or The light chain variable domain of SEQ ID NO: 34, the light chain constant domain of SEQ ID NO: 41, the heavy chain variable domain of SEQ ID NO: 29, and the heavy chain constant domain of SEQ ID NO: 37; or The light chain variable domain of SEQ ID NO: 34, the light chain constant domain of SEQ ID NO: 41, the heavy chain variable domain of SEQ ID NO: 30, and the heavy chain constant domain of SEQ ID NO: 37; or The light chain variable domain of SEQ ID NO: 34, the light chain constant domain of SEQ ID NO: 41, the heavy chain variable domain of SEQ ID NO: 31, and the heavy chain constant domain of SEQ ID NO: 37; or The light chain variable domain of SEQ ID NO: 36, the light chain constant domain of SEQ ID NO: 41, the heavy chain variable domain of SEQ ID NO: 31, and the heavy chain constant domain of SEQ ID NO: 37; The recombinant anti-oxMIF antibody or its antigen-binding fragment is labeled with a radioactive isotope; The amino acid positions are based on the Kabat number; Compared to antibodies or antigen-binding fragments thereof containing SEQ ID NO:32 and SEQ ID NO:29 lacking amino acid substitutions, the antibodies or antigen-binding fragments thereof have reduced aggregation potential and reduced hydrophobicity.

2. The radioimmunoconjugate according to claim 1, wherein the radioisotope is selected from the group consisting of: 11 C 13 N、 15 O, 18F, 32 P, 64 Cu、 67 Cu、 67 Ga、 89 Zr、 90 Y、 99 mTc, 103 Pd, 105 Rh、 109 Pd, 111 Ag、 111 In、 123 I, 124 I, 125 I, 131 I, 140 La、 149 Tb, 149 Pm, 153 Sm、 159 Gd, 165 Dy、 166 Dy、 166 Ho、 169 Yb、 175 Yb、 177 Lu、 227 Th、 186 Re、 188 Re、 192 Ir、 193 mPt, 195 mPt, 198 Au、 199 Au、 211 At、 212 Pb, 212 Bi、 213 Bi、 225 Ac、 223 Ra、 227 Th.

3. The radioimmunoconjugate according to claim 2, wherein the radioisotope is selected from the group consisting of: 67 Ga、 89 Zr、 111 In、 124 I, 131 I, 177 Lu and 225 Ac.

4. The radioimmunoconjugate according to claim 1, further comprising a chelating group.

5. The radioimmunoconjugate according to claim 4, wherein the chelating group is selected from DOTA, deferoxamine B, and DFO*, and the DFO* has the following structure: 。 6. Use of the radioimmunoconjugate of any one of claims 1 to 5 in the preparation of a medicament for treating cancer, wherein the cancer is selected from colorectal cancer, ovarian cancer, breast cancer, prostate cancer, pancreatic cancer, or lung cancer.

7. A pharmaceutical composition comprising the radioimmunoconjugate according to any one of claims 1 to 5.

8. The pharmaceutical composition according to claim 7, wherein the pharmaceutical composition further comprises a drug carrier or adjuvant.

9. The pharmaceutical composition according to claim 7, wherein the pharmaceutical composition is formulated for intravenous administration.

10. Use of the pharmaceutical composition of any one of claims 7 to 9 or the radioimmunoconjugate of any one of claims 1 to 5 in the preparation of a medicament for treating a patient suffering from cancer, wherein the cancer is selected from colorectal cancer, ovarian cancer, breast cancer, prostate cancer, pancreatic cancer, or lung cancer.

11. A method for producing the radioimmunoconjugate of any one of claims 1 to 5, comprising expressing a nucleic acid encoding an anti-oxMIF antibody in a host cell, and labeling the antibody with a radioisotope.

12. Use of the radioimmunoconjugate of any one of claims 1 to 5 in the preparation of a diagnostic reagent for detecting oxMIF levels in a subject’s cancer, wherein the cancer is selected from colorectal cancer, ovarian cancer, breast cancer, prostate cancer, pancreatic cancer, or lung cancer.

13. Use of the radioimmunoconjugate of any one of claims 1 to 5 in the preparation of a diagnostic reagent for diagnosing cancer in a subject, wherein the cancer is selected from colorectal cancer, ovarian cancer, breast cancer, prostate cancer, pancreatic cancer, or lung cancer.

14. The use according to claim 13, wherein the diagnostic reagent is used to diagnose cancer in a subject.

15. The use according to claim 14, wherein the diagnostic reagent is used for in vivo detection of tumor cells.

16. The use according to claim 15, wherein the diagnostic reagent is used for in vitro diagnosis of cancer.

17. The use according to claim 16, wherein the diagnostic reagent is used to detect tumor cells in a sample.

18. A kit for producing the radioimmunoconjugate of any one of claims 1 to 5, comprising two or more vials, one vial containing a conjugate containing a chelating agent linked to an anti-oxMIF antibody, the second vial containing a radioisotope, specifically selected from 177Lu and 89Zr, wherein the contents of one or more vials are optionally in lyophilized or solution form.

Images