Compositions and methods related to modulating macrophage migration inhibitory factor (MIF)-CD74 signaling and related treatments for neuroinflammatory conditions

Abstract

The present disclosure provides methods for preventing, treating, or delaying progression of neuroinflammatory CNS conditions, e.g., Alzheimer's disease, by administration of an agent that inhibits macrophage migration inhibitory factor (MIF)-CD74 signaling. In some embodiments the agent to be administered is a MIF-binding protein such as a MIF antibody. In other embodiments disclosed herein the agent is a targeting polynucleotide against MIF or CD74.

Description

[0001] COMPOSITIONS AND METHODS RELATED TO MODULATING MACROPHAGE MIGRATION INHIBITORY FACTOR (MIF)-CD74 SIGNALING AND RELATED TREATMENTS FOR NEUROINFLAMMATORY CONDITIONS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of and priority to US provisional no. 63 / 551,708 filed February 9, 2024, the disclosure of which is incorporated herein by reference in its entirety.

[0004] FIELD OF THE INVENTION

[0005] The specification relates generally to the field of therapeutic methods for neuroinflammatory conditions. More particularly, the specification relates to treatment of Alzheimer’s disease and mild cognitive impairment.

[0006] BACKGROUND OF THE INVENTION

[0007] Most neurodegenerative disorders are characterised by progressive pathological aggregation of specific proteins or peptides such as Amyloid 0(1-42) and microtubule-associated protein tau (“Tau”). In addition, however, such disorders are characterized by persistent neuroinflammatory state associated with reactive gliosis, loss of synapses, and neuronal death, particularly in the hippocampus and cortex. Attempts to halt or reverse these processes in preclinical models and human patients have included immunotherapies directed against proteins known to form pathological aggregates such as Amyloid 0 and Tau, but to date these have shown very limited or no efficacy.

[0008] Thus, there is an ongoing and dire need to identify therapeutic methods that treat neurodegenerative disorders such Alzheimer’s disease (AD) and other neurological conditions characterized by chronic neuroinflammation.

[0009] SUMMARY

[0010] To identify druggable targets relevant to neurodegenerative disorders, the inventors searched for genes the expression of which was upregulated in an AD- associated microglial TREM2(' / ')background and, as a parallel approach, utilized a CRISPRi functional genomics approach to identify targets that modulated microglial uptake of A0 fibrils. Unexpectedly, these independent approaches identified macrophage migration inhibitory factor (MIF)-CD74 signaling as a key pathway underlying neuroinflammation associated with AD, and more particularly with the increased ability of microglia to clear neurotoxic Ap fibrils when MIF-CD74 signaling is reduced.

[0011] Accordingly, in one aspect, provided herein is a method for preventing, treating, or delaying progression of Alzheimer’s disease or mild cognitive impairment in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an agent that inhibits MIF-CD74 signaling. In another aspect, provided herein is a method for treating a condition associated with neuroinflammation, e.g., AD, autism spectrum disorder, schizophrenia, or Parkinson’s disease. Also provided is the use of an agent that inhibits MIF-CD74 signaling in the manufacture of a medicament for preventing, treating, or delaying progression of Alzheimer’s disease or mild cognitive impairment.

[0012] In some embodiments, the macrophage migration inhibitory factor (MIF) is oxidized MIF (oxMIF), a structural isoform. In some embodiments, the agent that inhibits MIF-CD74 signaling comprises a MIF-binding protein. In some embodiments, the agent that inhibits MIF-CD74 signaling comprises a CD74-binding protein.

[0013] In some embodiments, where the agent is a CD74-binding protein, the CD74- binding protein comprises at least one immunoglobulin variable region that binds specifically to CD74. In some embodiments the CD74-binding protein comprising at least one immunoglobulin variable domain region comprises a nanobody.

[0014] In other embodiments the CD74-binding protein comprising at least one immunoglobulin variable domain region comprises an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL), wherein the CD74-binding protein binds specifically, through an antigen binding site formed by the VH and VL, to an epitope of human CD74 protein.

[0015] In some embodiments, where the agent is a MIF-binding protein, the MIF- binding protein comprises at least one immunoglobulin variable region that binds specifically to MIF. In some embodiments the MIF-binding protein comprising at least one immunoglobulin variable domain region comprises a nanobody.

[0016] In other embodiments the MIF-binding protein comprising at least one immunoglobulin variable domain region comprises an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL), wherein the MIF-binding protein binds specifically, through an antigen binding site formed by the VH and VL, to an epitope of human MIF protein.

[0017] In some embodiments of the foregoing methods or uses: (i) the amino acid sequence of the VH comprises:

[0018] (a) VH-CDR1 comprising SEQ ID NO:5 with up to two (e.g., 0, 1, or 2) amino acid substitutions, VH-CDR2 comprising SEQ ID NO:6 with up to four (e.g., 0, 1, 2, 3, 4) amino acid substitutions, and VH-CDR3 comprising SEQ ID NO:7 with up to three (e.g., 0, 1, 2, 3) amino acid substitutions;

[0019] (b) VH-CDR1 comprising SEQ ID NO: 13 with up to two (e.g., 0, 1, or 2) amino acid substitutions, VH-CDR2 comprising SEQ ID NO: 14 with up to four (e.g., 0, 1, 2, 3, 4) amino acid substitutions, and VH-CDR3 comprising SEQ ID NO:15 with up to four (e.g., 0, 1, 2, 3, 4) amino acid substitutions;

[0020] (c) VH-CDR1 comprising SEQ ID NO:21 with up to two (e.g., 0, 1, or 2) amino acid substitutions, VH-CDR2 comprising SEQ ID NO:22 with up to four (e.g., 0, 1, 2, 3, 4) amino acid substitutions, and VH-CDR3 comprising SEQ ID NO:23 with up to three (e.g., 0, 1, 2, 3) amino acid substitutions;

[0021] (d) VH-CDR1 comprising SEQ ID NO:29 with up to two (e.g., 0, 1, or

[0022] 2) amino acid substitutions, VH-CDR2 comprising SEQ ID NO:30 with up to four (e.g., 0, 1, 2, 3, 4) amino acid substitutions, and VH-CDR3 comprising SEQ ID NO:31 with up to four (e.g., 0, 1, 2, 3, 4) amino acid substitutions;

[0023] (e) VH-CDR1 comprising SEQ ID NO:37 with up to two amino acid substitutions, VH-CDR2 comprising SEQ ID NO:38 with up to four (e.g., 0, 1, 2, 3, 4) amino acid substitutions, and VH-CDR3 comprising SEQ ID NO:39 with up to four (e.g., 0, 1, 2, 3, 4) amino acid substitutions; or

[0024] (f) VH-CDR1 comprising SEQ ID NO:45 with up to two amino acid substitutions, VH-CDR2 comprising SEQ ID NO:46 with up to four (e.g., 0, 1, 2, 3, 4) amino acid substitutions, and VH-CDR3 comprising SEQ ID NO:47 with up to four (e.g., 0, 1, 2, 3, 4) amino acid substitutions; and

[0025] (ii) the amino acid sequence of the VL comprises:

[0026] (a) VL-CDR1 comprising SEQ ID NO:8 with up to three (e.g., 0, 1, 2, 3) amino acid substitutions, VL-CDR2 comprising SEQ ID NO:9 with up to two (e.g., 0, 1, or 2) amino acid substitutions, and VL-CDR3 comprising SEQ ID NO: 10 with up to three (e.g., 0, 1, 2, 3) amino acid substitutions;

[0027] (b) VL-CDR1 comprising SEQ ID NO:16 with up to three (e.g., 0, 1, 2,

[0028] 3) amino acid substitutions, VL-CDR2 comprising SEQ ID NO: 17 with up to two (e.g., 0, 1, or 2) amino acid substitutions, and VL-CDR3 comprising SEQ ID NO: 18 with up to three (e.g., 0, 1, 2, 3) amino acid substitutions;

[0029] (c) VL-CDR1 comprising SEQ ID NO:24 with up to three amino acid substitutions, VL-CDR2 comprising SEQ ID NO:25 with up to two (e.g., 0, 1, or 2) amino acid substitutions, and VL-CDR3 comprising SEQ ID NO:26 with up to three (e.g., 0, 1, 2, 3) amino acid substitutions;

[0030] (d) VL-CDR1 comprising SEQ ID NO:32 with up to three (e.g., 0, 1, 2, 3) amino acid substitutions, VL-CDR2 comprising SEQ ID NO:33 with up to two (e.g., 0, 1, or 2) amino acid substitutions, and VL-CDR3 comprising SEQ ID NO:34 with up to two amino acid substitutions;

[0031] (e) VL-CDR1 comprising SEQ ID NO:40 with up to three (e.g., 0, 1, 2, 3) amino acid substitutions, VL-CDR2 comprising SEQ ID NO:41 with up to two (e.g., 0, 1, or 2) amino acid substitutions, and VL-CDR3 comprising SEQ ID NO:42 with up to two (e.g., 0, 1, or 2) amino acid substitutions; or

[0032] (f) VL-CDR1 comprising SEQ ID NO:48 with up to three (e.g., 0, 1, 2, 3) amino acid substitutions, VL-CDR2 comprising SEQ ID NO:49 with up to two (e.g., 0, 1, or 2) amino acid substitutions, and VL-CDR3 comprising SEQ ID NO:50 with up to two amino acid substitutions. In some embodiments:

[0033] (i) the amino acid sequence of the VH comprises: VH-CDR1 comprising SEQ ID NO:5 with up to two amino acid substitutions, VH-CDR2 comprising SEQ ID NO:6 with up to four amino acid substitutions, and VH-CDR3 comprising SEQ ID NO:7 with up to three amino acid substitutions; and

[0034] (ii) the amino acid sequence of the VL comprises:VL-CDRl comprising SEQ ID NO:8 with up to three amino acid substitutions, VL-CDR2 comprising SEQ ID NO:9 with up to two amino acid substitutions, and VL-CDR3 comprising SEQ ID NO: 10 with up to three amino acid substitutions. In some embodiments the total number of substitutions in the VH CDRS comprises up to six amino acid substitutions, and the total number of substitutions in the VL CDRS comprises up to six amino acid substitutions.

[0035] In some embodiments the VH comprises the amino acid sequence corresponding to any one of SEQ ID NOs:ll, 19, 27, 35, 43, or 51; and the VL comprises the amino acid sequence of any one of SEQ ID NOs:12, 20, 28, 36, 44, or 52; and wherein the VH comprises up to six amino acid substitutions and the VL comprises up to six amino acid substitutions. In some embodiments the VH comprises the amino acid sequence corresponding to SEQ ID NO: 11 and the VL comprises the amino acid sequence corresponding to SEQ ID NO: 12; wherein the VH comprises up to six amino acid substitutions and the VL comprises up to six amino acid substitutions. In some embodiments: (i) the amino acid sequence of: VH-CDR1 corresponds to SEQ ID NO:5, VH- CDR2 corresponds to SEQ ID NO:6, and VH-CDR3 corresponds to SEQ ID NO:7; and

[0036] (ii) the amino acid sequence of: VL-CDR1 corresponds to SEQ ID NO:8, VL- CDR2 corresponds to SEQ ID NO:9, and VL-CDR3 corresponds to SEQ ID NO: 10.

[0037] In some embodiments the VH comprises the amino acid sequence corresponding to SEQ ID NO: 11 and the VL comprises the amino acid sequence corresponding to SEQ ID NO:12.

[0038] In some embodiments of any of the methods or uses disclosed herein the VH and the VL of the MIF-binding protein are in a single polypeptide chain. In some embodiments, where the VH and the VL of the MIF-binding protein are in a single polypeptide chain, the MIF-binding protein is: (i) a single chain Fv fragment (scFv);

[0039] (ii) a dimeric scFv (di-scFv); or

[0040] (iii) at least one of (i) and / or (ii) linked to a Fc or a heavy chain constant domain (CH)2 and / or CH3.

[0041] In other embodiments the VH and the VL are in separate polypeptide chains. In some embodiments, where the VH and the VL of the MIF-binding protein are in separate polypeptide chains, the MIF-binding protein is:

[0042] (i) a divalent antibody;

[0043] (ii) a Fab’;

[0044] (iii) a diabody;

[0045] (iv) a triabody; or

[0046] (v) a tetrabody.

[0047] In other embodiments of the methods or uses disclosed herein, the agent that inhibits MIF-CD74 signaling comprises a CD74-targeting polynucleotide that reduces an expression level of CD74 in a cell population in the subject, whereby MIF-CD74 signaling is inhibited in the cell population. In some embodiments the CD74-targeting polynucleotide is selected from the group consisting of: guide RNA (gRNA), an siRNA, a shRNA, a microRNA (miRNA), and an antisense oligonucleotide (ASO). In some embodiments the CD74-targeting polynucleotide is a gRNA.

[0048] In some embodiments of any of the foregoing methods or uses the route of administration or formulation of the medicament is intravenous, intrathecal, intraarterial, intra-cerebral, intracerebroventricular, intranasal, or subcutaneous.

[0049] In some embodiments the subject is a human subject.

[0050] In some embodiments the above-mentioned methods for prevention, treatment, or inhibition also include a step of scanning ultrasound transient permeabilization of the blood brain barrier in the subject.

[0051] BRIEF DESCRIPTION OF THE DRAWINGS

[0052] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, and accompanying drawings, where:

[0053] FIG. 1 shows qPCR validation of specificity of microglial gRNA knockdown. Microglia were infected with lentiviral guide RNAs at various MOIs, incubated for 3-5 days, lysed, and transcripts were quantified via qPCR. Guide RNAs resulted in genespecific inhibition of RNA levels, as shown in heat map. Notably, CD74 gRNA lentiviruses knock down specifically CD74 transcript levels. FIG. 2 is a schematic showing convergence of dual screening approaches to identify MIF-CD74 interaction as a critical pathway in microglial function and dysfunction in neuroimmune cortical organoids (NICOs). (Left) Single cell RNA- sequencing of TREM2 heterozygous and homozygous knock-out iPSC-derived microglia identify CD74 as elevated in knock-outs relative to wild-type control. (Right) Microglial phagocytosis CRISPR inhibition screen of 30 genes identifies CD74 transcriptional inhibition as the top target in increasing Amyloid Beta Phagocytosis. iPSC-derived microglia were transduced with guide-RNA specific lentiviruses, replated into 384well format, incubated with Amyloid Beta-pHrodo fibrils and imaged every 30 minutes for 24 hours to measure Amyloid phagocytosis via pHrodo signal. Total overlap of pHrodo (Green) and Microglia (Red) (“phagocytosis score”) is divided by total microglial signal to account for differences in transduction efficiency. Phagocytosis scores are shown as line graphs.

[0054] FIGS. 3A-3B show disease-associated microglia (DAM) related data. FIG. 3A illustrates TREM2-deficient microglia are disease-associated microglia (DAM) markerdeficient and express increased levels of homeostatic markers relative to wild-type control microglia. FIG. 3B is a heatmap of a panel of DAM genes (left) and homeostatic genes (right) is shown for isogenic (top), heterozygous TREM2-KO (middle), and homozygous TREM2-KO microglia (bottom).

[0055] FIGS. 4A-4B demonstrate that TREM2-deficient microglia exhibit decreased migration in NICOs relative to wild-type control microglia in NICOs. FIG. 4A shows a fluorescence image cross-section of fluorescently labelled WT-control microglia (“ISO”) and TREM2 homozygous KO (“TREM2 HOM”)-note concentration of microglia close to the surface of the cortical organoid in TREM2 HOM vs more evenly dispersed microglia in ISO microglia. FIG. 4B is a graphic showing quantification of microglial density within cortical organoids shows decreased migration in association with loss of TREM2 (one or both alleles) consistent with a role for TREM2 in microglial migration within the brain.

[0056] FIGS. 5A-5C illustrate dCas9-KRAB-mediated knockdown of CD74 in microglia introduced into Cortical Organoids (COs). FIG. 5 A is an illustration of the development process for dCAS9-KRAB NICOs to enable target validation. FIG. 5B is a bar graph showing dCas9-KRAB knockdown of CD74 in TREM2-deficient microglia results in decreased expression of homeostatic marker P2RY12 (top panel) and an increase in DAM inflammatory marker SPP1 expression (bottom panel). FIG. 5C top left and right panel show the effects of CD74 knock down on microglial motility within cortical organoids, the bottom panel quantifies the mean microglial motility within cortical organoids.

[0057] FIGS. 6A-6B illustrate the effect of antibody-targeting of MIF on microglial phagocytosis of Amyloid 0. FIG. 6A shows the results of a microglial amyloid-0 phagocytosis screen for a control antibody versus Donanemab biosimilar versus Imalumab. FIG. 6B is a bar graph quantifying microglial phagocytosis mediated by dose-dependent Imalumab at 0.4, 2, 10, and 50pg / mL.

[0058] FIGS. 7A-7B demonstrate the effect of targeting of MIF-CD74 on microglial phagocytosis of Amyloid 0. FIG. 7A quantifies via phagocytosis score, the effect of various doses of anti-CD74 antibody Milatuzumab on microglial phagocytosis of Amyloid 0 (Milatuzumab: 0.4 - 50 pg / mL). FIG. 7B quantifies via phagocytosis score, the effect of various doses of MIF targeting small molecule BTZO-1 on microglial phagocytosis of Amyloid 0 (BTZO-1: .05-5 pM).

[0059] FIGS. 8A-8B demonstrates the effects of various anti-CD74 and / or anti-MIF actors on microglial Amyloid 0. FIG. 8A is a bar graph quantifying the effects of various anti- CD74 and anti-MIF antibodies and molecules on microglial Amyloid 0 phagocytosis in a dose-dependent manner. FIG. 8B are line graphs of phosphate-buffered saline (top left), an IgGl control (top right), Donanemab (bottom left) and Imalumab (bottom right) quantifying their effects on microglial Amyloid 0 phagocytosis in a dosedependent manner.

[0060] DETAILED DESCRIPTION

[0061] Interpretational conventions

[0062] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (z.e. one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter.

[0063] Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

[0064] The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equi valent products, compositions and methods are clearly within the scope of the present disclosure.

[0065] Any example of the present disclosure herein shall be taken to apply mutatis mutandis to any other example of the disclosure unless specifically stated otherwise.

[0066] Any example of the present disclosure related to the use of a MIF-binding protein will be taken to apply mutatis mutandis to a MIF-binding antibody.

[0067] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (for example, in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, biochemistry, and bioinformatics).

[0068] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature.

[0069] The description and definitions of variable regions and parts thereof, immunoglobulins, antibodies and fragments thereof herein may be further clarified by the discussion in Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991, Bork et al., J Mol. Biol. 242, 309- 320, 1994, Chothia and Lesk J. Mol Biol. 796:901 -917, 1987, Chothia et al. Nature 342, 877-883, 1989 and / or or Al-Lazikani et al., J Mol Biol 273, 927-948, 1997.

[0070] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0071] Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0072] As used herein the term “derived from” shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source. Reference herein to a range of, e.g., residues, will be understood to be inclusive. For example, reference to “a region comprising amino acids 56 to 65” will be understood in an inclusive manner, i.e., the region comprises a sequence of amino acids as numbered 56, 57, 58, 59, 60, 61, 62, 63, 64 and 65 in a specified sequence.

[0073] Definitions

[0074] For the purposes of nomenclature only and not limitation, an exemplary sequence of a human Macrophage migration inhibitory factor (MIF) is set out in the Uniprot database as P14174 (and set out in SEQ ID NO:1). The sequence of MIF protein from other species can be determined using sequences provided herein and / or in publicly available databases and / or determined using standard techniques.

[0075] Reference to human Macrophage migration inhibitory factor may be abbreviated to human MIF or simply “MIF”.

[0076] The term “oxidized MIF” or “oxMIF” refers to an oxidized conformational isoform of MIF, as described in Thiele et al., 2015, J Immunol, 195:2343-2352. In some preferred embodiments, a MIF-binding protein, e.g., a MIF antibody such as imalumab selectively binds to oxMIF.

[0077] The term “reduced MIF” or “redMIF” refers to a reduced conformational isoform of MIF, as described in Thiele et al., supra.

[0078] The term “isolated protein” or “isolated polypeptide” is a protein or polypeptide that by virtue of its origin or source of derivation is not associated with naturally- associated components that accompany it in its native state; is substantially free of other proteins from the same source. A protein may be rendered substantially free of naturally associated components or substantially purified by isolation, using protein purification techniques known in the art. By “substantially purified” is meant the protein is substantially free of contaminating agents, e.g., at least about 70% or 75% or 80% or 85% or 90% or 95% or 96% or 97% or 98% or 99% free of contaminating agents.

[0079] The term “recombinant” shall be understood to mean the product of artificial genetic recombination. Accordingly, in the context of a recombinant protein comprising an antibody antigen binding domain, this term does not encompass an antibody naturally occurring within a subject’s body that is the product of natural recombination that occurs during B cell maturation. However, if such an antibody is isolated, it is to be considered an isolated protein comprising an antibody antigen binding domain. Similarly, if nucleic acid encoding the protein is isolated and expressed using recombinant means, the resulting protein is a recombinant protein comprising an antibody antigen binding domain. A recombinant protein also encompasses a protein expressed by artificial recombinant means when it is within a cell, tissue or subject, e.g., in which it is expressed.

[0080] The term “protein” shall be taken to include a single polypeptide chain, i.e., a series of contiguous amino acids linked by peptide bonds or a series of polypeptide chains covalently or non-covalently linked to one another (i.e., a polypeptide complex). For example, the series of polypeptide chains can be covalently linked using a suitable chemical or a disulfide bond. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, Van der Waals forces, and hydrophobic interactions.

[0081] The term “polypeptide” or “polypeptide chain” will be understood from the foregoing paragraph to mean a series of contiguous amino acids linked by peptide bonds.

[0082] As used herein, the term “antigen binding domain” shall be taken to mean a region of an antibody that is capable of specifically binding to an antigen, i.e., a VH or a VL or an Fv comprising both a VH and a VL. The antigen binding domain need not be in the context of an entire antibody, e.g., it can be in isolation (e.g., a domain antibody) or in another form, e.g., as described herein, such as a scFv.

[0083] For the purposes for the present disclosure, the term “antibody” includes a protein capable of specifically binding to one or a few closely related antigens by virtue of an antigen binding domain contained within a Fv. This term includes four chain antibodies (e.g., two light chains and two heavy chains), recombinant or modified antibodies (e.g., chimeric antibodies, humanized antibodies, human antibodies, CDR- grafted antibodies, primatized antibodies, de-immunized antibodies, synhumanized antibodies, half-antibodies, bispecific antibodies). An antibody generally comprises constant domains, which can be arranged into a constant region or constant fragment or fragment crystallizable (Fc). Exemplary forms of antibodies comprise a four-chain structure as their basic unit. Full-length antibodies comprise two heavy chains (~50 to 70 kD) covalently linked and two light chains (~23 kDa each). A light chain generally comprises a variable region (if present) and a constant domain and in mammals is either a K light chain or a X light chain. A heavy chain generally comprises a variable region and one or two constant domain(s) linked by a hinge region to additional constant domain(s). Heavy chains of mammals are of one of the following types a, 8, E, y, or p.. Each light chain is also covalently linked to one of the heavy chains. For example, the two heavy chains and the heavy and light chains are held together by inter-chain disulfide bonds and by non-covalent interactions. The number of inter-chain disulfide bonds can vary among different types of antibodies. Each chain has an N- terminal variable region (VH or VL wherein each are —110 amino acids in length) and one or more constant domains at the C- terminus. The constant domain of the light chain (CL which is ~110 amino acids in length) is aligned with and disulfide bonded to the first constant domain of the heavy chain (CHI which is 330 to 440 amino acids in length). The light chain variable region is aligned with the variable region of the heavy chain. The antibody heavy chain can comprise 2 or more additional CH domains (such as, CH2, CH3 and the like) and can comprise a hinge region between the CHI and CH2 constant domains. Antibodies can be of an immunoglobulin type selected from among e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGi, IgGi, IgGa, IgG4, IgAi and IgAi) or subclass. In some embodiments, the antibody is a murine (mouse or rat) antibody or a primate (such as, human) antibody. In some embodiments the antibody heavy chain is missing a C-terminal lysine residue. In some embodiments, the antibody is humanized, synhumanized, chimeric, CDR-grafted or deimmunized.

[0084] The terms “full-length antibody,” “intact antibody” or “whole antibody” are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antigen-binding fragment of an antibody. Specifically, whole antibodies include those with heavy and light chains including an Fc region. The constant domains may be wild-type sequence constant domains (e.g., human wild-type sequence constant domains) or amino acid sequence variants thereof.

[0085] As used herein, “variable region” refers to the portions of the light and / or heavy chains of an antibody as defined herein that is capable of specifically binding to an antigen and, includes amino acid sequences of complementarity determining regions (CDRs); i.e., CDR1, CDR2, and CDR3, and framework regions (FRs). For example, the variable region comprises three or four FRs (e.g., FR1, FR2, FR3 and optionally FR4) together with three CDRs. VH refers to the variable region of the heavy chain. VL refers to the variable region of the light chain.

[0086] As used herein, the term “complementarity determining regions” (syn. CDRs; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of an antibody variable region the presence of which are major contributors to specific antigen binding. Each variable region domain (VH or VL) typically has three CDRs identified as CDR1, CDR2 and CDR3. In some embodiments, the amino acid positions assigned to CDRs and FRs are defined according to Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 (also referred to herein as “the Kabat numbering system”). In other embodiments, the amino acid positions assigned to CDRs and FRs are defined according to the Enhanced Chothia Numbering Scheme (bioinf.org.uk / abs / info.html). According to the numbering system of Kabat, VH FRS and CDRs are generally, but not exclusively, positioned as follows: residues 1 to 30 (FR1), 31 to 35 (CDR1), 36 to 49 (FR2), 50 to 65 (CDR2), 66 to 94 (FR3), 95 to 102 (CDR3) and 103 to 113 (FR4). According to the numbering system of Kabat, VL FRs and CDRs are positioned as follows: residues 1 to 23 (FR1), 24 to 34 (CDR1), 35 to 49 (FR2), 50 to 56 (CDR2), 57 to 88 (FR3), 89 to 97 (CDR3) and 98 to 107 (FR4). The present disclosure is not limited to FRs and CDRs as defined by the Kabat numbering system, but includes all numbering systems, including the canonical numbering system or of Chothia and Lesk J. Mol. Biol. 196: 901-917, 1987; Chothia et al., Nature 342: 877-883, 1989; and / or Al-Lazikani et al., J. Mol. Biol. 273 : 927-948, 1997; the numbering system of Honnegher and Plukthun J. Mol. Biol. 309: 657-670, 2001; or the IMGT system discussed in Giudicelli et al., Nucleic Acids Res. 25: 206- 211 1997. In one example, the CDRs are defined according to the Kabat numbering system. Optionally, heavy chain CDR2 according to the Kabat numbering system does not comprise the five C-terminal amino acids listed herein or any one or more of those amino acids are substituted with another naturally occurring amino acid. In this regard, Padlan et al., FASEB J., 9: 133-139, 1995 established that the five C-terminal amino acids of heavy chain CDR2 are not generally involved in antigen binding.

[0087] “Framework regions” (FRs) are those variable region residues other than the CDR residues.

[0088] As used herein, the term “Fv” shall be taken to mean any protein, whether comprised of multiple polypeptides or a single polypeptide, in which a VL and a VH associate and form a complex having an antigen binding domain, i.e., capable of specifically binding to an antigen. The VH and the VL which form the antigen binding domain can be in a single polypeptide chain or in different polypeptide chains. Furthermore, an Fv of the disclosure (as well as any protein of the disclosure) may have multiple antigen binding domains which may or may not bind the same antigen. This term shall be understood to encompass fragments directly derived from an antibody as well as proteins corresponding to such a fragment produced using recombinant means. In some examples, the VH is not linked to a heavy chain constant domain (CH) 1 and / or the VL is not linked to a light chain constant domain (CL). Exemplary Fv containing polypeptides or proteins include a Fab fragment, a Fab’ fragment, a F(ab’) fragment, a scFv, a diabody, a triabody, a tetrabody or higher order complex, or any of the foregoing linked to a constant region or domain thereof, e.g., CH2 or CH3 domain, e.g., a minibody. A “Fab fragment” consists of a monovalent antigen-binding fragment of an immunoglobulin and can be produced by digestion of a whole antibody with the enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a heavy chain or can be produced using recombinant means. A “Fab’ fragment” of an antibody can be obtained by treating a whole antibody with pepsin, followed by reduction, to yield a molecule consisting of an intact light chain and a portion of a heavy chain comprising a VH and a single constant domain. Two Fab’ fragments are obtained per antibody treated in this manner. A Fab’ fragment can also be produced by recombinant means. A “F(ab’)2 fragment” of an antibody consists of a dimer of two Fab’ fragments held together by two disulfide bonds and can be obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction. A “Fabi” fragment is a recombinant fragment comprising two Fab fragments linked using, for example a leucine zipper or a CH3 domain. A “single chain Fv” or “scFv” is a recombinant molecule containing the variable region fragment (Fv) of an antibody in which the variable region of the light chain and the variable region of the heavy chain are covalently linked by a suitable, flexible polypeptide linker.

[0089] A “single-domain antibody” (sdAb), also known as a nanobody® consists of a single monomeric variable antibody domain, typically a VH domain. In some embodiments a nanobody is based on a camelid antibody. In other embodiments a nanobody comprises one or more CDRs derived from a mouse or human antibody grafted onto a nanobody scaffold.

[0090] The term ‘ fixed dose combination1as used herein has its conventional meaning and refers to a combination of defined doses of two or more drugs or active ingredients presented in a single dosage unit (e.g. a tablet or a capsule) and administered as such. The term ‘free dose combination ’ as used herein has its conventional meaning and refers to a combination of two drugs or active ingredients administered simultaneously but as two distinct dosage.

[0091] As used herein, the term “binds” in reference to the interaction of a human MIF- binding protein or an antigen binding domain thereof with an antigen means that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the antigen. For example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody binds to epitope “A”, the presence of a molecule containing epitope “A” (or free, unlabeled “A”), in a reaction containing labeled “A” and the protein, will reduce the amount of labeled “A” bound to the antibody.

[0092] As used herein, the term “specifically binds” or “binds specifically” shall be taken to mean that a MIF-binding protein of the disclosure reacts or associates more frequently, more rapidly, with greater duration and / or with greater affinity with a particular antigen than it does with alternative antigens. For example, a human MIF- binding protein binds to proteins or polypeptides comprising the amino acid sequence set forth in SEQ ID NO:1. Reference to “binding” provides explicit support for the term “specific binding” and vice versa.

[0093] As used herein, the term “does not detectably bind” shall be understood to mean that a human MIF-binding protein, e.g., an antibody, binds to a candidate antigen at a level less than 10%, or 8% or 6% or 5% above background. The background can be the level of binding signal detected in the absence of the protein and / or in the presence of a negative control protein (e.g., an isotype control antibody) and / or the level of binding detected in the presence of a negative control antigen. The level of binding is detected using biosensor analysis (e.g. Biacore) in which the antigen (e.g., a polypeptide) is immobilized and contacted with a human MIF-binding protein.

[0094] As used herein, the term “does not significantly bind” shall be understood to mean that the level of binding of a human MIF-binding protein of the disclosure to a polypeptide is not statistically significantly higher than background, e.g., the level of binding signal detected in the absence of the human MIF-binding protein and / or in the presence of a negative control protein (e.g., an isotype control antibody) and / or the level of binding detected in the presence of a negative control polypeptide. For example, the level of binding is detected using biosensor analysis (e.g. Biacore) in which the antigen (e.g., a polypeptide) is immobilized and contacted with a human MIF-binding protein. The dissociation constant (Kd), association constant (Ka) and / or affinity constant (KD) of an immobilized MIF-binding protein for a MIF polypeptide comprising the amino acid sequence corresponding to SEQ ID NO:1 or the cognate epitope thereof. The “Kd” or “Ka” or “KD” for a MIF-binding protein is in one example measured by a radiolabeled or fluorescently labeled MIF binding assay. In the case of a “Kd”, this assay equilibrates the MIF-binding protein with a minimal concentration of labeled MIF protein in the presence of a titration series of unlabeled MIF protein or peptide. Following washing to remove unbound MIF protein, the amount of label is determined and is indicative of the Kd of the protein.

[0095] According to another example the Kd, Ka or KD is measured by using surface plasmon resonance assays, e.g., using BIAcore surface plasmon resonance (BIAcore, Inc., Piscataway, NJ) with immobilized MIF protein or immobilized MIF-binding protein.

[0096] In some examples, the MIF-binding protein has a similar KD or an improved KD (i.e., a KD value lower than) than antibody BAX69 (AKA “Imalumab”) described in U.S. Patent No. 8,668,909, which is herein incorporated by reference. In some embodiments the MIF-binding protein to be used in the methods disclosed herein is ON203, human, affinity-matured antibody against MIF, as described in Rossmueller et al., 2023, Molecular Cancer Therapeutics, 22(5):555-569, which is herein incorporated by reference.

[0097] The in vivo half-life of a MIF-binding protein of the disclosure can also be measured by pharmacokinetic studies, e.g., according to the method described by Kim et al, Eur J of Immunol 24:542, 1994. According to this method radiolabeled MIF- binding protein is injected intravenously into mice and its plasma concentration is periodically measured as a function of time, for example at 3 minutes to 72 hours after the injection. The clearance curve thus obtained should be biphasic, that is, an alpha phase and beta phase. For the determination of the in vivo half-life of the protein, the clearance rate in beta-phase is calculated and compared with that of the wild type or unmodified protein.

[0098] As used herein, phrases referring to “reduced binding” or “binding being at a lower level” in relation to an antigen will be understood to mean that a human MIF- binding protein, e.g., antibody, binds to an antigen with an affinity at least about 1.5 fold or 2 fold or 5 fold or 10 fold or 20 fold or 50 fold or 100 fold or 200 fold less than a control epitope or antigen.

[0099] A human MIF-binding protein or antibody may be considered to “preferentially bind” to a polypeptide if it binds that polypeptide with a dissociation constant (KD) that is less than the protein’s or antibody’s KD for another polypeptide. In some embodiments, a human MIF-binding protein or antibody is considered to preferentially bind to a polypeptide if it binds the polypeptide with an affinity (i.e., KD) that is at least about 1.5-fold or 2 fold or 5 fold or 10 fold or 20 fold or 50 fold or 100 fold or 200 fold more than the protein’s or antibody’s KD for another polypeptide.

[0100] For the purposes of clarification and as will be apparent to the skilled artisan based on the exemplified subject matter herein, reference to “affinity” in this specification is a reference to KD of a human MIF-binding protein or antibody.

[0101] For the purposes of clarification and as will be apparent to the skilled artisan based on the description herein, reference to an “affinity of at least about” will be understood to mean that the affinity is equal to the recited value or higher (i.e., the value recited as the affinity is lower), i.e., an affinity of 2 nM is greater than an affinity of 3 nM. Stated another way, this term could be “an affinity of X or less”, wherein X is a value recited herein.

[0102] An “IC50 of at least about” will be understood to mean that the IC50 is equal to the recited value or lower (i.e., the value recited as the IC50 is lower), i.e., an IC50 of 2 pg / ml is greater than an IC50 of 1 pg / ml. Stated another way, this term could be “an IC50 of X or less”, wherein X is a value recited herein. As used herein, the term “epitope” (syn. “antigenic determinant”) shall be understood to mean a region of human MIF protein to which a human MIF-binding protein comprising an antigen binding domain of an antibody binds. This term is not necessarily limited to the specific residues or structure to which the human MIF- binding protein makes contact. For example, this term includes a region spanning amino acids contacted by the human MIF-binding protein and 5-10 (or more) or 2-5 or 1-3 amino acids outside of this region.

[0103] The term “competitively inhibits” shall be understood to mean that a test human MIF-binding protein of the disclosure (or an antigen-binding domain thereof) reduces or prevents binding of a reference antibody or human MIF-binding protein to human MIF protein, e.g., a protein comprising SEQ ID NO:1. This may be due to the human MIF-binding protein (or antigen binding domain) and antibody binding to the same or an overlapping epitope. It will be apparent from the foregoing that the human MIF- binding protein need not completely inhibit binding of the antibody, rather it need only reduce binding by a statistically significant amount, for example, by at least about 10% or 20% or 30% or 40% or 50% or 60% or 70% or 80% or 90% or 95%. For example, the human MIF-binding protein reduces binding of the antibody by at least about 30%, for example by at least about 50%, such as, by at least about 70%, for example by at least about 75%, even more preferably, by at least about 80% or 85% e.g., by at least about 90%. Methods for determining competitive inhibition of binding are known in the art and / or described herein.

[0104] As used herein, the term “neutralize” shall be taken to mean that a human MIF- binding protein is capable of blocking, reducing or preventing human MIF protein from binding to CD74.

[0105] As used herein, the terms “preventing”, “prevent” or “prevention” include administering an agent that inhibits MIF-CD74 signaling (e.g., a human MIF-binding protein disclosed herein) to thereby stop or hinder the development of at least one symptom or biomarker of AD, MCI, or another neuroinflammation-driven neurological condition. This term also encompasses treatment of a subject in remission to prevent or hinder relapse. Prevention need not be a complete inhibition of the onset of a condition being targeted, but could include a delay in the onset of the condition or particular symptoms associated with the onset or progression of the condition, e.g., a 10% delay, a 20% delay, a 30% delay, a 40%, delay, a 50% delay, a 60% delay, an 80% delay, a 90% delay, or another delay of the onset of a condition relative to what be otherwise be expected to be observed for a subject at risk in the absence of a treatment / prevention regime, based on a clinical assessment of the subject. As used herein, the terms “treating”, “treat” or “treatment” include administering an agent that inhibits MIF-CD74 signaling described herein to thereby reduce or eliminate at least one symptom of a specified neurological disease or condition.

[0106] As used herein, the term “subject” shall be taken to mean any mammal including humans. Exemplary subjects include but are not limited to humans and nonhuman primates. For example, the subject is a human.

[0107] Antibodies

[0108] In some embodiments, a human MIF -binding protein as disclosed herein is an antibody.

[0109] Methods for generating antibodies are known in the art and / or described in Harlow and Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988). Generally, in such methods human MIF protein or a polypeptide comprising the sequence set forth in SEQ ID NO:1) or a cell expressing and displaying same (z.e., an immunogen), optionally formulated with any suitable or desired carrier, adjuvant, or pharmaceutically acceptable excipient, is administered to a non-human animal, for example, a mouse, chicken, rat, rabbit, guinea pig, dog, horse, cow, goat or Pig-

[0110] Accordingly also disclosed herein is a non-naturally occurring immunogenic peptide or polypeptide comprising the amino acid sequence according to SEQ ID NO:1. In some embodiments the amino acid sequence of an immunogenic polypeptide consists of the amino acid corresponding to SEQ ID NO:1. In other embodiments disclosed herein is an immunogenic peptide 12 to about 80 amino acids in length and comprising a fragment of SEQ ID NO:1, e.g., 14, 15, 17, 18, 20, 22, 25, 27, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or another number of amino acids from 12 to about 80 amino acids in length.

[0111] The immunogen may be administered intranasally, intramuscularly, subcutaneously, intravenously, intradermally, intraperitoneally, or by another known route. Alternatively, the peptide may be used to screen expression display libraries (e.g. , phage display libraries) expressing VH-VL antigen-binding domains, as described herein. In other embodiments the peptide is used to screen single domain antibody libraries (AKA “nanobody” libraries).

[0112] The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. One or more further immunizations may be given, if required to achieve a desired antibody titer. The process of boosting is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal is bled and the serum isolated and stored, and / or the animal is used to generate monoclonal antibodies (mAbs).

[0113] Monoclonal antibodies are one exemplary form of antibody contemplated by the present disclosure. The term “monoclonal antibody” or “mAb” refers to a homogeneous antibody population capable of binding to the same antigen(s), for example, to the same epitope within the antigen. This term is not intended to be limited with regard to the source of the antibody or the manner in which it is made.

[0114] For the production of mAbs any one of a number of known techniques may be used, such as, for example, the procedure exemplified in US4196265 or Harlow and Lane (1988), supra.

[0115] For example, a suitable animal is immunized with an immunogen under conditions sufficient to stimulate antibody producing cells. Rodents such as rabbits, mice and rats are exemplary animals. Mice genetically engineered to express human antibodies and, for example, do not express murine antibodies, can also be used to generate an antibody of the present disclosure (e.g., as described in W02002 / 066630).

[0116] Following immunization, somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the mAb generating protocol. These cells may be obtained from biopsies of spleens, tonsils or lymph nodes, or from a peripheral blood sample. The B cells from the immunized animal are then fused with cells of an immortal myeloma cell, generally derived from the same species as the animal that was immunized with the immunogen.

[0117] Hybrids are amplified by culture in a selective medium comprising an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary agents are aminopterin, methotrexate and azaserine.

[0118] The amplified hybridomas are subjected to a functional selection for antibody specificity and / or titer, such as, for example, by flow cytometry and / or immunoassay (e.g. radioimmunoassay, enzyme immunoassay, cytotoxicity assay, plaque assay, dot immunoassay, and the like).

[0119] Alternatively, ABL-MYC technology (NeoClone, Madison WI 53713, USA) is used to produce cell lines secreting Mabs (e.g., as described in Largaespada et al, J. Immunol. Methods. 197: 85-95, 1996).

[0120] Antibodies can also be produced or isolated by screening a display library, e.g., a phage display library, e.g., as described in US6300064 and / or US5885793.

[0121] For example, a phage display library is screened with a polypeptide comprising SEQ ID NO: 1 or a peptide fragment thereof. In a further example, human MIF protein comprising SEQ ID NO:1 or a fragment thereof is utilized as a “bait” antigen and contacted with the BAX69 antibody described herein. A phage display library is then brought into contact with the bait antigen and phage expressing proteins that can compete with the BAX69 for binding are then selected.

[0122] In some embodiments antibodies disclosed herein present disclosure are synthetic. Such synthetic antibodies include, but are not limited to, chimeric antibodies, humanized antibodies, human antibodies, synhumanized antibodies, primatized antibodies, or de-immunized antibodies.

[0123] Deimmunized, Chimeric, CDR Grafted, Humanized, Synhumanized, Primatized, Human and Composite human MIF-binding proteins

[0124] The human MIF protein-binding proteins of the present disclosure may be CDR grafted proteins which include CDRs from an antibody from a non-human species (e.g., mouse or rat or non-human primate) grafted onto or inserted into FRs from a human antibody or which include CDRs from an antibody from one type of antibody (e.g. , one type of human antibody) grafted onto or inserted into FRs from another type of antibody (e.g., another type of human antibody). This term also encompasses a composite human MIF-binding protein comprising, for example, one or more CDR grafted variable regions and one or more, e.g., human variable regions, chimeric variable regions, synhumanized variable regions or primatized variable regions.

[0125] The human MIF-binding proteins of the present disclosure may be a humanized protein.

[0126] The term “humanized protein” shall be understood to refer to a protein comprising a human-like variable region, which includes CDRs from an antibody from a non-human species (e.g., mouse, rat, rabbit, or non-human primate) grafted onto or inserted into FRs from a human antibody (this type of antibody is falls within the class of “CDR-grafted antibody”). Humanized human MIF protein-binding proteins also include proteins in which one or more residues of the human protein are modified by one or more amino acid substitutions and / or one or more FR residues of the human protein are replaced by corresponding non-human residues. Humanized proteins may also comprise residues which are found in neither the human antibody or in the non- human antibody. Any additional regions of the protein (e.g., Fc region) are generally human. Humanization can be performed using a method known in the art, e.g., US5225539, US6054297, US7566771 US5585089. The skilled artisan will also be aware that various methods for evaluating the “humanness” of variable region sequences by comparison to human natural antibody repertoires to determine suggested residues for substitution based on positional frequency in corresponding human VH and VL framework and CDR sequences as described for computational methods such as the “BioPhi” platform described in Prihoda et al., (2022), mAbs, (doi: 10.1080 / 19420862.2021.2020203) in combination with human antibody sequence databases such as the Observed Antibody Space (OAS available at opig.stats.ox.ac.uk / webapps / oas / ); or the “AbDiver” platform described in Mlokosiewicz et al., (2022), Bioinformatics, 38(9):2628-2630. In addition, computational methods for optimal human framework selection for grafting of nonhuman CDRs while retaining antigen affinity based on energy-based rankings (“CUMAb”) has been described in Tennenhouse et al. (2023), Nature Biomedical Engineering, (doi: 10.1038 / s41551-023-01079-l). The term “humanized protein” also encompasses a super-humanized protein, e.g., as described in US7732578. This term also encompasses a composite protein comprising, for example, one or more humanized variable regions and one or more, e.g., human variable regions, chimeric variable regions, synhumanized variable regions or primatized variable regions.

[0127] In some embodiments the human MIF -binding proteins disclosed herein are of human origin. A “human protein” or “of human origin” as used herein in reference to a human MIF-binding protein refers to proteins having variable antibody regions found in humans. In embodiments, where an antibody also includes constant antibody regions, such constant antibody regions are also from ones found in humans, e.g. in the human germline or somatic cells or from libraries produced using such regions. The “human” proteins can include amino acid residues not encoded by human sequences, e.g. mutations introduced by random or site directed mutations in vitro (in particular mutations which involve substitutions or mutations in a small number of residues of the protein, e.g. in 1, 2, 3, 4 or 5 of the residues of the protein). These “human proteins” do not necessarily need to be generated as a result of an immune response of a human, rather, they can be generated using recombinant means (e.g., screening a phage display library) and / or by a transgenic animal (e.g., a mouse) comprising nucleic acid encoding human antibody constant and / or variable regions and / or using guided selection (e.g. , as described in US5565332). This term also encompasses affinity matured forms of such antibodies. For the purposes of the present disclosure, a human protein will also be considered to include a protein comprising FRs from a human antibody or FRs comprising sequences from a consensus sequence of human FRs and in which one or more of the CDRs correspond to those disclosed as part of MIF-binding protein, antibody “BAX69” described herein, as well as variants that are substituted as disclosed herein. Accordingly, in some exemplary embodiments human MIF -binding proteins for use in the methods disclosed herein are antibodies comprising VH and VL regions, where:

[0128] (i) the amino acid sequence of the VH comprises:

[0129] (a) VH-CDR1 comprising SEQ ID NO:5 with up to two amino acid substitutions, VH-CDR2 comprising SEQ ID NO:6 with up to four amino acid substitutions, and VH-CDR3 comprising SEQ ID NO:7 with up to three amino acid substitutions;

[0130] (b) VH-CDR1 comprising SEQ ID NO: 13 with up to two amino acid substitutions, VH-CDR2 comprising SEQ ID NO: 14 with up to four amino acid substitutions, and VH-CDR3 comprising SEQ ID NO: 15 with up to four amino acid substitutions;

[0131] (c) VH-CDR1 comprising SEQ ID NO:21 with up to two amino acid substitutions, VH-CDR2 comprising SEQ ID NO:22 with up to four amino acid substitutions, and VH-CDR3 comprising SEQ ID NO:23 with up to three amino acid substitutions;

[0132] (d) VH-CDR1 comprising SEQ ID NO:29 with up to two amino acid substitutions, VH-CDR2 comprising SEQ ID NO:30 with up to four amino acid substitutions, and VH-CDR3 comprising SEQ ID NO:31 with up to four amino acid substitutions;

[0133] (e) VH-CDR1 comprising SEQ ID NO:37 with up to two amino acid substitutions, VH-CDR2 comprising SEQ ID NO:38 with up to four amino acid substitutions, and VH-CDR3 comprising SEQ ID NO:39 with up to four amino acid substitutions; or

[0134] (f) VH-CDR1 comprising SEQ ID NO:45 with up to two amino acid substitutions, VH-CDR2 comprising SEQ ID NO:46 with up to four amino acid substitutions, and VH-CDR3 comprising SEQ ID NO:47 with up to four amino acid substitutions; and

[0135] (ii) the amino acid sequence of the VL comprises:

[0136] (a) VL-CDR1 comprising SEQ ID NO:8 with up to three amino acid substitutions, VL-CDR2 comprising SEQ ID NO:9 with up to two amino acid substitutions, and VL-CDR3 comprising SEQ ID NO: 10 with up to three amino acid substitutions;

[0137] (b) VL-CDR1 comprising SEQ ID NO: 16 with up to three amino acid substitutions, VL-CDR2 comprising SEQ ID NO: 17 with up to two amino acid substitutions, and VL-CDR3 comprising SEQ ID NO: 18 with up to three amino acid substitutions;

[0138] (c) VL-CDR1 comprising SEQ ID NO:24 with up to three amino acid substitutions, VL-CDR2 comprising SEQ ID NO:25 with up to two amino acid substitutions, and VL-CDR3 comprising SEQ ID NO:26 with up to three amino acid substitutions;

[0139] (d) VL-CDR1 comprising SEQ ID NO:32 with up to three amino acid substitutions, VL-CDR2 comprising SEQ ID NO:33 with up to two amino acid substitutions, and VL-CDR3 comprising SEQ ID NO:34 with up to two amino acid substitutions;

[0140] (e) VL-CDR1 comprising SEQ ID NO:40 with up to three amino acid substitutions, VL-CDR2 comprising SEQ ID NO:41 with up to two amino acid substitutions, and VL-CDR3 comprising SEQ ID NO:42 with up to two amino acid substitutions; or

[0141] (f) VL-CDR1 comprising SEQ ID NO:48 with up to three amino acid substitutions, VL-CDR2 comprising SEQ ID NO:49 with up to two amino acid substitutions, and VL-CDR3 comprising SEQ ID NO:50 with up to two amino acid substitutions.

[0142] KEY TO SEQUENCE LISTING (sequences provided in Appendix 1)

[0143] SEQ ID NO 1 : amino acid sequence of Homo sapiens MIF

[0144] SEQ ID NO 2 : amino acid sequence of Homo sapiens CD74

[0145] SEQ ID NO 3 : nucleotide sequence of Homo sapiens CD74

[0146] SEQ ID NO 4 : amino acid sequence of Homo sapiens CD74 soluble ectodomain (sCD74)

[0147] SEQ ID NO 5: amino acid sequence of Imalumab (BAX69) VH-CDR1

[0148] SEQ ID NO 6: amino acid sequence of Imalumab (BAX69) VH-CDR2

[0149] SEQ ID NO 7: amino acid sequence of Imalumab (BAX69) VH-CDR3

[0150] SEQ ID NO 8: amino acid sequence of Imalumab (BAX69) VL-CDR1

[0151] SEQ ID NO 9: amino acid sequence of Imalumab (BAX69) VL-CDR2

[0152] SEQ ID NO 10: amino acid sequence of Imalumab (BAX69) VL-CDR3

[0153] SEQ ID NO 11: amino acid sequence of Imalumab (B AX69) VH full sequence

[0154] SEQ ID NO 12: amino acid sequence of Imalumab (B AX69) VL full sequence

[0155] SEQ ID NO 13: amino acid sequence of BAX8 VH-CDR1

[0156] SEQ ID NO 14: amino acid sequence of BAX8 VH-CDR2

[0157] SEQ ID NO 15: amino acid sequence of BAX8 VH-CDR3 SEQ ID NO 16 amino acid sequence of BAX8 VL-CDR1

[0158] SEQ ID NO 17 amino acid sequence of BAX8 VL-CDR2

[0159] SEQ ID NO 18 amino acid sequence of BAX8 VL-CDR3

[0160] SEQ ID NO 19 BAX8 VH full sequence

[0161] SEQ ID NO:20 BAX8 VL full sequence

[0162] SEQ ID NO:21 amino acid sequence of BAX74 VH-CDR1

[0163] SEQ ID NO:22 amino acid sequence of BAX74 VH-CDR2

[0164] SEQ ID NO:23 amino acid sequence of BAX74 VH-CDR3

[0165] SEQ ID NO:24 amino acid sequence of BAX74 VL-CDR1

[0166] SEQ ID NO:25 amino acid sequence of BAX74 VL-CDR2

[0167] SEQ ID NO:26 amino acid sequence of BAX74 VL-CDR3

[0168] SEQ ID NO:27 amino acid sequence of BAX74 VH full sequence

[0169] SEQ ID NO:28 amino acid sequence of BAX74 VL full sequence

[0170] SEQ ID NO:29 amino acid sequence of BAX94 VH-CDR1

[0171] SEQ ID NO:30 amino acid sequence of BAX94 VH-CDR2

[0172] SEQ ID NO:31 amino acid sequence of BAX94 VH-CDR3

[0173] SEQ ID NO:32 amino acid sequence of BAX94 VL-CDR1

[0174] SEQ ID NO:33 amino acid sequence of BAX94 VL-CDR2

[0175] SEQ ID NO:34 amino acid sequence of BAX94 VL-CDR3

[0176] SEQ ID NO:35 BAX94 VH full sequence

[0177] SEQ ID NO:36 BAX94 VL full sequence

[0178] SEQ ID NO:37 amino acid sequence of BAX152 VH-CDR1

[0179] SEQ ID NO:38 amino acid sequence of BAX152 VH-CDR2

[0180] SEQ ID NO:39 amino acid sequence of BAX152 VH-CDR3

[0181] SEQ ID NO:40 amino acid sequence of BAX152 VL-CDR1

[0182] SEQ ID NO:41 amino acid sequence of BAX152 VL-CDR2

[0183] SEQ ID NO:42 amino acid sequence of BAX152 VL-CDR3

[0184] SEQ ID NO:43 BAX 152 VH full sequence

[0185] SEQ ID NO:44 BAX 152 VL full sequence

[0186] SEQ ID NO:45 amino acid sequence of B AXA 10 VH-CDR1

[0187] SEQ ID NO:46 amino acid sequence of B AXA 10 VH-CDR2

[0188] SEQ ID NO:47 amino acid sequence of B AXA 10 VH-CDR3

[0189] SEQ ID NO:48 amino acid sequence of BAXA10 VL-CDR1

[0190] SEQ ID NO:49 amino acid sequence of B AXA 10 VL-CDR2

[0191] SEQ ID NO:50 amino acid sequence of B AXA 10 VL-CDR3

[0192] SEQ ID NO:51 BAXA10 VH full sequence SEQ ID NO:52 BAXA10 VH full sequence SEQ ID NO:53 CD74 gRNA nucleotide sequence SEQ ID NO:54 ABB gRNA nucleotide sequence SEQ ID NO:55 ADAM8 gRNA nucleotide sequence SEQ ID NO:56 ADRB2 gRNA nucleotide sequence SEQ ID NO:57 AMPD3 Gma nucleotide sequence SEQ ID NO:58 C3 ARI gRNA nucleotide sequence SEQ ID NO:59 CA2 gRNA nucleotide sequence SEQ ID NO:60 CD74 gRNA nucleotide sequence SEQ ID NO:61 CDC42 gRNA nucleotide sequence SEQ ID NO:62 CSF1R gRNA nucleotide sequence SEQ ID NO:63 CYSLTR1 gRNA nucleotide sequence SEQ ID NO:64 FFAR4 gRNA nucleotide sequence SEQ ID NO:65 FUCA2 gRNA nucleotide sequence SEQ ID NO-.66 INPP5D gRNA nucleotide sequence SEQ ID NO:67 JAK3 gRNA nucleotide sequence SEQ ID NO:68 KCNJ2 gRNA nucleotide sequence SEQ ID NO:69 LPAR6 gRNA nucleotide sequence SEQ ID NO:70 hNTl gRNA nucleotide sequence SEQ ID NO:71 NR1H2 gRNA nucleotide sequence SEQ ID NO-.72 P2RX7 gRNA nucleotide sequence SEQ ID NO:73 P2RY6 gRNA nucleotide sequence SEQ ID NO:74 AMPD3 gRNA nucleotide sequence SEQ ID NO:75 PDE6D gRNA nucleotide sequence SEQ ID NO:76 PLA2G7 gRNA nucleotide sequence SEQ ID NO:77 PTAFR gRNA nucleotide sequence SEQ ID NO:78 PTK2B gRNA nucleotide sequence SEQ ID NO:79 SLC6A6 gRNA nucleotide sequence SEQ ID NO:80 SUCNR1 gRNA nucleotide sequence SEQ ID NO:81 TGFBR2 gRNA nucleotide sequence SEQ ID NO:82 TLR7 gRNA nucleotide sequence SEQ ID NO:83 TREM2 gRNA nucleotide sequence Optionally, the VH is linked to a heavy chain constant region, e.g., an IgG4 heavy chain constant region or a stabilized IgG4 constant region. In one example, the heavy chain constant region lacks the C-terminal lysine residue.

[0193] Optionally, the VL is linked to a light chain constant region.

[0194] The MIF-binding proteins of the present disclosure may be synhumanized proteins. The term “synhumanized protein” refers to a protein prepared by a method described in W02007 / 019620. A synhumanized MIF-binding protein includes a variable region of an antibody, wherein the variable region comprises FRs from a New World primate antibody variable region and CDRs from a non-New World primate antibody variable region. For example, a synhumanized MIF-binding protein includes a variable region of an antibody, wherein the variable region comprises FRs from a New World primate antibody variable region and CDRs from a mouse or rat antibody. In some embodiments, a synhumanized MIF-binding protein is a MIF protein-binding antibody in which one or both of the variable regions are synhumanized. This term also encompasses a composite protein comprising, for example, one or more synhumanized variable regions and one or more, e.g., human variable regions or humanized variable regions or chimeric variable regions.

[0195] In some embodiments the MIF-binding proteins disclosed herein are primatized proteins. A “primatized protein” comprises variable region(s) from an antibody generated following immunization of a non-human primate (e.g., a cynomolgus macaque). Optionally, the variable regions of the non-human primate antibody are linked to human constant regions to produce a primatized antibody. Exemplary methods for producing primatized antibodies are described in US6113898. This term also encompasses a composite protein comprising, for example, one or more primatized variable regions and one or more, e.g., human variable regions or humanized variable regions or chimeric variable regions.

[0196] In some embodiments a MIF-binding protein disclosed herein is a chimeric protein. The term “chimeric proteins” refers to proteins in which an antigen-binding domain is from a particular species (e.g., murine, such as mouse or rat) or belonging to a particular antibody class or subclass, while the remainder of the protein is from a protein derived from another species (such as, for example, human or non-human primate) or belonging to another antibody class or subclass. In some embodiments, a chimeric protein is a chimeric antibody comprising a VH and / or a VL from a non-human antibody (e.g., a murine antibody) and the remaining regions of the antibody are from a human antibody. The production of such chimeric proteins is known in the art, and may be achieved by standard means (as described, e.g., in US6331415; US5807715; US4816567 and US4816397). This term also encompasses a composite protein comprising, for example, one or more chimeric variable regions and one or more, e.g., human variable regions or humanized variable regions or chimeric variable regions.

[0197] The present disclosure also contemplates a deimmunized MIF-binding protein, e.g., by a process such as that described in W02000 / 34317 and W02004 / 108158. Deimmunized antibodies and proteins have one or more epitopes, e.g. , B cell epitopes or T cell epitopes removed (z.e., mutated) to thereby reduce the likelihood that a subject will raise an immune response against the antibody or protein. De-immunization, in some embodiments, is facilitated by computational approaches such as those described in Prihoda et al. supra, which evaluate the “humaneness” and suggest residue mutations to increase humanization of a given antibody. For example, a MIF-binding protein disclosed herein is analyzed to identify one or more B or T cell epitopes and one or more amino acid residues within the epitope is mutated to thereby reduce the immunogenicity of the MIF-binding protein.

[0198] It will be apparent to the skilled artisan from the foregoing disclosure that a “composite” protein comprises one form of VH (e.g., human) and another form of VL (e.g., humanized). The present disclosure explicitly encompasses all combinations of forms of VH and VL.

[0199] Antibody Binding Domain-Containing Proteins

[0200] Single Chain Fv (scFv)

[0201] In some embodiments a MIF protein-binding is a scFv. The skilled artisan will be aware that scFvs comprise VH and VL regions in a single polypeptide chain and a polypeptide linker between the VH and VL which enables the scFv to form the desired structure for antigen binding (i.e., for the VH and VL of the single polypeptide chain to associate with one another to form a Fv). For example, the linker comprises in excess of 12 amino acid residues with (Gly4Ser)3 being one of the preferred linkers for a scFv.

[0202] The present disclosure also contemplates a disulfide-stabilized Fv (or diFv or dsFv), in which a single cysteine residue is introduced into a FR of VH and a FR of VL and the cysteine residues linked by a disulfide bond to yield a stable Fv.

[0203] In some embodiments, a MIF-binding protein is a dimeric scFv, i.e., a protein comprising two scFv molecules linked by a non-covalent or covalent linkage, e.g. , by a leucine zipper domain (e.g., derived from Fos or Jun). Alternatively, two scFvs are linked by a peptide linker of sufficient length to permit both scFvs to form and to bind to an antigen, e.g., as described in US20060263367. Diabodies, Triabodies, Tetrabodies In some embodiments, a MIF -binding protein of the disclosure is or comprises a diabody, triabody, tetrabody or higher order protein complex such as those described in W098 / 044001 and / or W094 / 007921.

[0204] For example, a diabody is a protein comprising two associated polypeptide chains, each polypeptide chain comprising the structure VL-X-VH or VH-X-VL, wherein VL is an antibody light chain variable region, VH is an antibody heavy chain variable region, X is a linker comprising insufficient residues to permit the VH and VL in a single polypeptide chain to associate (or form an Fv) or is absent, and wherein the VH of one polypeptide chain binds to a VL of the other polypeptide chain to form an antigen binding domain, i.e., to form a Fv molecule capable of specifically binding to one or more antigens. The VL and VH can be the same in each polypeptide chain or the VL and VH can be different in each polypeptide chain so as to form a bispecific diabody (i.e., comprising two Fvs having different specificity).

[0205] Single-Domain Antibodies

[0206] In some examples, a MIF-binding protein for use in the methods of the disclosure is or comprises a single-domain antibody (“sdAb” which is used interchangeably with the term “domain antibody” or “dAb” or “nanobody®”). A single-domain antibody is a single polypeptide chain comprising all or a portion of the heavy chain variable region of an antibody. In certain examples, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., US6,248,516).

[0207] MIF-Binding Proteins and Sequence Variants

[0208] Described for use in the methods disclosed herein is a MIF-binding protein that comprises one or multiple mutations in one or more CDRs disclosed herein, and binds to an epitope of human MIF protein, whereby interaction of MIF with CD74 is inhibited. In some embodiments the Kd of the MIF-binding protein for the epitope is no greater than about 5 x 10'7M, e.g., between about 5 pM and about 100 nM, e.g., 7 pM, 10 pM, 12 pM, 20 pM, 30 pM, 40 pM, 80 pM, 100 pM, 200 pM, 250 pM, 300 pM, 500 pM, 1 nM, 2 nM, 5 nM, 10 nM, 15 nM, 20, nM, 50 nM, 75 nM, or another Kd from about 5 pM and about 100 nM. The “Kd” or “Ka” or “KD” for a MIF-binding protein is in one example measured by a radiolabeled or fluorescently labeled MIF binding assay. In the case of a “Kd”, this assay equilibrates the MIF-binding protein with a minimal concentration of labeled MIF protein, polypeptide (comprising SEQ ID NO:1), or cognate epitope-containing fragment thereof in the presence of a titration series of unlabeled MIF protein or peptide. Following washing to remove unbound MIF polypeptide or peptide, the amount of label is determined and is indicative of the Kd of the protein.

[0209] According to another example the KD or Ka is measured by using surface plasmon resonance assays, e.g., using BIAcore surface plasmon resonance (BIAcore, Inc., Piscataway, NJ) with immobilized MIF protein comprising SEQ ID NO:1, or immobilized MIF-binding protein.

[0210] In some examples, the MIF-binding protein has a similar KD or an improved KD (z.e. , a KD value lower than) than antibody BAX69 described herein.

[0211] Accordingly, in some embodiments of a MIF-binding protein disclosed herein:

[0212] (i) the amino acid sequence of the VH comprises:

[0213] (a) VH-CDR1 comprising SEQ ID NO:5 with up to two (e.g., 0, 1, or 2) amino acid substitutions, VH-CDR2 comprising SEQ ID NO:6 with up to four (e.g., 0, 1, 2, 3, or 4) amino acid substitutions, and VH-CDR3 comprising SEQ ID NO:7 with up to three (e.g., 0, 1, 2, or 3) amino acid substitutions;

[0214] (b) VH-CDR1 comprising SEQ ID NO: 13 with up to two (e.g., 0, 1, or 2) amino acid substitutions, VH-CDR2 comprising SEQ ID NO: 14 with up to four (e.g., 0, 1, 2, 3, or 4) amino acid substitutions, and VH-CDR3 comprising SEQ ID NO:15 with up to four (e.g., 0, 1, 2, 3, or 4) amino acid substitutions;

[0215] (c) VH-CDR1 comprising SEQ ID NO:21 with up to two (e.g., 0, 1, or 2) amino acid substitutions, VH-CDR2 comprising SEQ ID NO:22 with up to four (e.g., 0, 1, 2, 3, or 4) amino acid substitutions, and VH-CDR3 comprising SEQ ID NO:23 with up to three (e.g., 0, 1, 2, or 3) amino acid substitutions;

[0216] (d) VH-CDR1 comprising SEQ ID NO:29 with up to two (e.g., 0, 1, or 2) amino acid substitutions, VH-CDR2 comprising SEQ ID NO:30 with up to four (e.g., 0, 1, 2, 3, or 4) amino acid substitutions, and VH-CDR3 comprising SEQ ID NO:31 with up to four (e.g., 0, 1, 2, 3, or 4) amino acid substitutions;

[0217] (e) VH-CDR1 comprising SEQ ID NO:37 with up to two (e.g., 0, 1, or

[0218] 2) amino acid substitutions, VH-CDR2 comprising SEQ ID NO:38 with up to four (e.g., 0, 1, 2, 3, or 4) amino acid substitutions, and VH-CDR3 comprising SEQ ID NO:39 with up to four amino acid substitutions (e.g., 0, 1, 2, 3, or 4); or

[0219] (f) VH-CDR1 comprising SEQ ID NO:45 with up to two (e.g., 0, 1, or 2) amino acid substitutions, VH-CDR2 comprising SEQ ID NO:46 with up to four (e.g., 0, 1, 2, 3, or 4) amino acid substitutions, and VH-CDR3 comprising SEQ ID NO:47 with up to four (e.g., 0, 1, 2, 3, or 4) amino acid substitutions; and

[0220] (ii) the amino acid sequence of the VL comprises:

[0221] (a) VL-CDR1 comprising SEQ ID NO:8 with up to three (e.g., 0, 1, 2, or

[0222] 3) amino acid substitutions, VL-CDR2 comprising SEQ ID NO:9 with up to two (e.g., 0, 1, or 2) amino acid substitutions, and VL-CDR3 comprising SEQ ID NO: 10 with up to three (e.g., 0, 1, 2, or 3) amino acid substitutions;

[0223] (b) VL-CDR1 comprising SEQ ID NO:16 with up to three (e.g., 0, 1, 2, or 3) amino acid substitutions, VL-CDR2 comprising SEQ ID NO: 17 with up to two (e.g., 0, 1, or 2) amino acid substitutions, and VL-CDR3 comprising SEQ ID NO: 18 with up to three (e.g., 0, 1, 2, or 3) amino acid substitutions;

[0224] (c) VL-CDR1 comprising SEQ ID NO:24 with up to three (e.g., 0, 1, 2, or 3) amino acid substitutions, VL-CDR2 comprising SEQ ID NO:25 with up to two (e.g., 0, 1, or 2) amino acid substitutions, and VL-CDR3 comprising SEQ ID NO:26 with up to three (e.g., 0, 1, 2, or 3) amino acid substitutions; (d) VL-CDR1 comprising SEQ ID NO:32 with up to three (e.g., 0, 1, 2, or 3) amino acid substitutions, VL-CDR2 comprising SEQ ID NO:33 with up to two (e.g., 0, 1, or 2) amino acid substitutions, and VL-CDR3 comprising SEQ ID NO:34 with up to two (e.g., 0, 1, or 2) amino acid substitutions;

[0225] (e) VL-CDR1 comprising SEQ ID NO:40 with up to three (e.g., 0, 1, 2, or 3) amino acid substitutions, VL-CDR2 comprising SEQ ID NO:41 with up to two (e.g., 0, 1, or 2) amino acid substitutions, and VL-CDR3 comprising SEQ ID NO:42 with up to two (e.g., 0, 1, or 2) amino acid substitutions; or

[0226] (f) VL-CDR1 comprising SEQ ID NO:48 with up to three (e.g., 0, 1, 2, or 3) amino acid substitutions, VL-CDR2 comprising SEQ ID NO:49 with up to two (e.g., 0, 1, or 2) amino acid substitutions, and VL-CDR3 comprising SEQ ID NO:50 with up to two (e.g., 0, 1, or 2) amino acid substitutions.

[0227] In some embodiments a MIF-binding protein for use in the methods disclosed herein comprises (i) in the VH, a VH-CDR1 comprising SEQ ID NO:5 with up to two (e.g., 0, 1, or 2) amino acid substitutions, VH-CDR2 comprising SEQ ID NO:6 with up to four (e.g., 0, 1, 2, 3, or 4) amino acid substitutions, and VH-CDR3 comprising SEQ ID NO:7 with up to three (e.g., 0, 1, 2, or 3) amino acid substitutions; and (ii) in the VL, a VL-CDR1 comprising SEQ ID NO:8 with up to three (e.g., 0, 1, 2, or 3) amino acid substitutions, VL-CDR2 comprising SEQ ID NO:9 with up to two (e.g., 0, 1, or 2) amino acid substitutions, and VL-CDR3 comprising SEQ ID NO: 10 with up to three (e.g., 0, 1, 2, or 3) amino acid substitutions.

[0228] In some embodiments the VH comprises the amino acid sequence corresponding to SEQ ID NO: 11 and the VL comprises the amino acid sequence corresponding to SEQ ID NO: 12; wherein the VH comprises up to six (e.g., 0, 1, 2, 3, 4, 5, or 6) amino acid substitutions and the VL comprises up to six amino acid substitutions.

[0229] In some embodiments a MIF-binding protein comprises at least one out of the above-mentioned maximum number of substitutions in: (i) one CDR; (ii) two CDRs; (iii) three CDRs; (iv) four CDRs; (v) five CDRs; or (vi) six CDRs.

[0230] In some embodiments of a MIF-binding protein, the number of substitutions in any one of the sequence-specified heavy chain or light chain CDRs does not exceed the above-specified maximums (e.g., 4 substitutions for VH CDR2), the overall amino acid sequence identity of the VH is at least 80% identical to a VH amino acid sequence disclosed herein, and the overall amino acid sequence identity of the VL is at least 80% identical to a VL amino acid sequence disclosed herein, e.g., 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, or another percent identity from at least 80% to 100% identical to a VH amino acid sequence disclosed herein (for comparison to a VH amino acid sequence) and to a VL amino acid sequence disclosed herein (for comparison to a VL amino acid sequence), wherein the MIF-binding protein specifically binds to MIF protein as disclosed herein. For clarity, the amino acid sequence of a VH and VL do not need to have the same percent amino acid sequence identities to their respective reference sequences, e.g., the amino acid sequence of a MIF-binding protein VH can be 90% identical to a VH reference sequence disclosed herein; and the VL of the same MIF-binding protein can be 97% identical to a VL reference sequence disclosed herein.

[0231] In some embodiments, for a MIF-binding protein disclosed herein the amino acid sequence of the VH is at least 80% identical to the amino acid sequence of SEQ ID NO: 11 and the amino acid sequence of the VL is at least 80% identical to the amino acid sequence of SEQ ID NO: 12, e.g., 82%, 85%, 87%, 90%, 92%, 93%, 95%, 96%, 97%, 98%, 99%, or another percent amino acid sequence identity from at least 80% to 100% for the respective VH and VL amino acid sequences.

[0232] In some embodiments a MIF-binding protein for use in the methods disclosed herein comprises (i) in the VH, a VH-CDR1 comprising SEQ ID NO:5, VH-CDR2 comprising SEQ ID NO:6, and VH-CDR3 comprising SEQ ID NO:7; and (ii) in the VL, a VL-CDR1 comprising SEQ ID NO:8, VL-CDR2 comprising SEQ ID NO:9, and VL-CDR3 comprising SEQ ID NO: 10.

[0233] In some preferred embodiments the MIF-binding protein comprises a VH comprising the amino acid sequence corresponding to SEQ ID NO: 11 and a VL comprising the amino acid sequence corresponding to SEQ ID NO: 12.

[0234] In the just-mentioned MIF-binding protein, the residue numbering of the VH and VL regions according to the Kabat numbering scheme is as follows:

[0235] For VH: Framework region 1 (FR1): 1-30; CDR1: 31-35; FR2: 36-49; CDR2: 50-65; FR3: 66-94; CDR3: 95-102; FR4: 103-113.

[0236] For VL: Framework region 1 (FR1): 1-23; CDR1 : 24-34; FR2: 35-49; CDR2: 50-56; FR3: 57-88; CDR3: 89-97; FR4: 98-107.

[0237] In one preferred embodiment the MIF-binding protein to be used in the methods disclosed herein is the fully human divalent antibody described as BAX69 described in U.S. Patent No. 8,668,909 and known commercially as Imalumab (Baxalta / Takeda). Sequence Variation

[0238] The skilled artisan is aware that amino acid substitutions in one or more CDRs of an antibody, e.g., in a MIF-binding protein, in principle have the potential to alter binding characteristics of an antigen-binding domain comprising the altered CDR (e.g., affinity, specificity, or immunogenicity), and further that the number of theoretically possible amino acid substitutions is very large. However, the skilled artisan is likewise aware that the likelihood of substantial functional alterations an antigen-binding domain can be minimized, in practice, by a combination of (i) general and individual antibody-specific heuristics for selecting substitutions and / or (ii) sequence and structure-based computational tools to avoid substitutions likely to compromise key MIF-binding protein characteristics.

[0239] Accordingly, in some embodiments, where any CDR substitutions are to be made, CDR substitutions are made based on the generally expected average binding free energy (A A G) contribution of each CDR, where CDRs with a lower binding energy contribution should generally be the first to have amino acid substitutions introduced, i.e., the preferred order of CDR selection for substitutions from lower to higher A A G is: (1) VL CDR2 < (2) VL CDR1 < (3) VL CDR3 = VH CDR1 < (4) VH CDR2 < (5) VH CDR3. See, e.g., Robin et al., (2014), Journal of Molecular Biology, 426:3729-3743. Thus, for example, an amino acid substitution in VL CDR2, which generally contributes very little free energy to paratope-antigen binding, is less likely to have a consequence on the affinity of an antibody for its antigen than a substitution in VH CDR2 generally found to contribute substantially to the free binding energy. In addition, it has been observed that certain amino acids and amino acid types are over- represented in CDRs and, conversely, some amino acids are under-represented. Further, most of the amino acids that are over-represented in CDRs are also found to make a disproportionate contribution to the free energy of binding to a cognate antigen. This suggests that in making substitutions in CDRs, substitutions at positions occupied by amino acids known to be among the most represented / overrepresented in CDRs and contributing the highest free energy of binding should generally be avoided. Accordingly in some embodiments, substitutions of the following group of amino acids, though not forbidden, should be avoided when possible:

[0240] Tyr, Trp, Phe, His, Asn, Asp, and Pro If not possible or desired to avoid substitution of one of the above amino acids, then preferred suggested substitutions are as follows based on free energy of binding contribution (with ordered preference) and structural / functional conservation in ordered preference from left to right:

[0241] Tyr — > Trp, Phe, Leu, He, Vai, or Met

[0242] Trp -> Tyr, Phe, Leu, He, Met, or Vai

[0243] Phe — > Tyr, Trp, Leu, He, Met, or Vai

[0244] His -> Arg, Asn, Lys, Thr

[0245] Asn -> Asp, His, Ser, Thr

[0246] Asp -> Asn, His, Ser, Thr

[0247] Pro -^Val, Thr, Gly, His, Ala

[0248] In some embodiments, where the amino acid to be substituted is not from among the above-privileged group of amino acids (z.e., Y, T, F, H, N, D, and P), then a substitution should be the most conservative one possible based on similarity of physicochemical properties, e.g., based on “Grantham’s distance” (Grantham, 1974, Science, doi: 10.1126 / science.l85.4154.862) relating to the properties of composition, polarity, and molecular volume, where the shorter the distance the more conservative the substitution.

[0249] Alternative methods for determining conservative amino acid substitutions are known in the art as described in, e.g., Yampolsky et al., (2005), Genetics, 170(4):1459- 1472.

[0250] It is also recognized in the art that certain variable domain positions (mostly CDR positions) are found more frequently to contribute to the free energy of binding to the antigen. In fact, it has been shown that the 30 highest contribution residue positions alone contribute about 80% of the total binding free energy (Robin et al., supra) and are therefore likely to be the most sensitive to substitution, particularly non-conservative substitutions or substitutions that diverge from the above-mentioned preferred substitutions. In the Kabat numbering scheme the more sensitive CDR (or CDR- proximal) positions are frequently found as follows:

[0251] VHpositions (Kabat): (CDR1) 27, 32, 33, and 35; (CDR2) 52, 52A, 53, 54, 56, and 58; and (CDR3) 95, 96, 97, 98, and 99; and

[0252] VLpositions (Kabat): (CDR1) 32; (CDR2) 49 and 50; (CDR3) 91, 92, 94, and

[0253] 96 With reference to a MIF -binding protein disclosed herein (e.g., amino acid sequence of VH SEQ ID NO:11 and VL SEQ ID NO:12) the corresponding amino acids for the above-mentioned “sensitive” CDR positions are:

[0254] VHpositions (Kabat): (CDR1) F27, Y32, S33, and N35; (CDR2) G52, S52A, S53, G54, T56, and Y58; and (CDR3) S95, Q96, W97, L98, and Y99; and

[0255] VLpositions (Kabat): (CDR1) Y32; (CDR2) F49 and V50; (CDR3) S91, F92, T94, and L96.

[0256] In some preferred embodiments, where CDR substitutions are to be made, the order of CDR selection for substitution follows the above-mentioned order (based on CDR free binding energy contribution), i.e., (1) VL CDR2 < (2) VL CDR1 < (3) VL CDR3 = VH CDR1 < (4) VH CDR2 < (5) VH CDR3.

[0257] Thus, based on at least the above-mentioned criteria, the skilled artisan can without undue effort provide a large number of sequence variants of MIF-binding protein VH or VL sequences without a substantial loss in affinity or specificity. As referred to herein, a “substantial loss,” in reference to affinity or specificity as used herein, is about a 10 % to about a 95% reduction in affinity or specificity relative to a reference MIF-binding protein, e.g., 15%, 18%, 20%, 25%, 30%, 35%, 37%, 40%, 42%, 45%, 50%, 55%, 60% 65%, 70%, 75%, 80%, 85%, 90%, or another percent reduction in affinity or specificity of a sequence variant MIF-binding protein relative to a reference MIF-binding protein disclosed herein, e.g., a MIF-binding protein comprising SEQ ID NOs:ll and 12 as VH and VL amino acid sequences, respectively.

[0258] Exemplary methods for producing sequence-varied forms of a MIF-binding protein include:

[0259] • construction of mutagenesis libraries (Xu et al., J Biotechnol., 194:27-36, 2015);

[0260] • introducing a nucleic acid encoding the polypeptide into a mutator cell, e.g.,

[0261] XL- 1 Red, XL-mutS and XL-mutS-Kanr bacterial cells (Stratagene);

[0262] • DNA shuffling, e.g., as disclosed in Stemmer, Nature 370: 389-91, 1994; and

[0263] • site directed mutagenesis, e.g., as described in Dieffenbach (ed) and Dveksler

[0264] (ed) (In: PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratories, NY, 1995).

[0265] Exemplary methods for determining biological activity of sequence-variant MIF- binding proteins of the disclosure will be apparent to the skilled artisan and / or described herein, e.g., antigen binding. For example, methods for determining antigen binding, competitive inhibition of binding, affinity, association, dissociation and therapeutic efficacy are described herein.

[0266] The skilled person in the art is aware that a number of antibody variable domain and CDR numbering schemes have been developed in the art as reviewed in, e.g., Dondelinger et al., (2018), Frontiers in Immunology, 9:2278. These include the Kabat, Chothia, IMGT, Martin, North-AHO. Herein, unless otherwise specified, CDRs are defined according to the Kabat numbering scheme. However, in some cases SEQ ID NOs, as recited herein, include additional residues not included in Kabat-defined CDRs, which may however be considered to be part of a CDR according to other antibody numbering schemes, e.g., North-AHO.

[0267] In some embodiments a MIF-binding protein comprises no more than one amino acid substitution in each CDR. In other embodiments the MIF-binding protein comprises no more than one substitution in any of the VH CDRS and one amino acid substitution in any of the VL CDRS.

[0268] The skilled person in the art is aware that a MIF-binding protein, e.g., a MIF antibody can be provided in various antibody formats or structures comprising any of the variable domain amino acid sequences disclosed herein. Accordingly, in some embodiments a MIF protein comprises both a VH and a VL domain in a single polypeptide chain. Suitable examples of such single polypeptide MIF binding proteins include, but are not limited to, (i) a single chain Fvfragment (scFv), (ii) a dimeric scFv (di-scFv), or (iii) at least one of (i) and / or (ii) linked to a Fc or a heavy chain constant domain (CH)2 and / or CH3.

[0269] In some embodiments one or more CDRs VH domain from a MIF-binding protein is used to generate a single domain antibody AKA a “nanobody.” Methods for generating a nanobody by CDR grafting from a mammalian antibody onto frameworks derived from variable domains of heavy-chain-only antibodies (VHH) to generate nanobodies are known in the art as exemplified in, e.g., Wagner et al., (2018), International Journal of Molecular Sciences, 19(11), 3444. See also, Vincke et al., (2009), Journal of Biological Chemistry, 284(5):3273-3284.

[0270] In other embodiments a MIF-binding protein is: (i) a divalent antibody, (ii) a Fab’; (iii) a diabody; (iv); a triabody; or (v) a tetrabody.

[0271] In some embodiments any of the MIF-binding proteins disclosed herein further include a functional moiety selected from among: (i) a detection moiety; (ii) a stabilization moiety; (iii) a delivery moiety; and (iv) any combination thereof. In some embodiments, a MIF-binding protein of the present disclosure is provided as a conjugate with a functional moiety. For example, the functional moiety is selected from the group consisting of a radioisotope, a detectable label, a therapeutic compound, a peptide, a protein, a compound that increases the half-life of the MIF- binding protein in a subject, and combinations thereof.

[0272] The functional moiety can be directly or indirectly bound to the MIF-binding protein (e.g., can comprise a linker in the case of indirect binding). Examples of detectable moieties include, a radioisotope (e.g., Carbon- 11 or Fluorine- 18), a detectable label (e.g., near-infrared (NIR) fluorophore dyes such as, BTC1070, BTC980, BTC982), or NIR-quantum dots. See, e.g., Gil et al., (2021), (Science, 24(3): 102189. In some embodiments a MIF-binding protein is detectably labelled, e.g., by conjugation of a detectable moiety.

[0273] In some embodiments a functional moiety is encoded as a fusion protein with a MIF-binding protein disclosed herein (e.g., as an N-terminal or C-terminal fusion protein). Accordingly, in some embodiments a detectable moiety is a reporter protein fused to the MIF-binding protein. In some embodiments the reporter protein is a fluorescent protein or an enzyme. In some embodiments, where the reporter protein is a fluorescent protein, the fluorescent protein is selected from the group consisting of: a green fluorescent protein (GFP), a yellow fluorescent protein (YFP), mCerulean3, and mScarlet3. In other embodiments, where the reporter protein is an enzyme, the enzyme is selected from the group consisting of: 0-Galactosidase (LacZ), a luciferase, 0- lactamase, and HaloTag.

[0274] Examples of stabilizing moieties include, but are not limited to, polyethylene glycol (PEG), (Q311R / M428L-substituted) Fc domains (Ko et al., 2022, Experimental Molecular Medicine, 54:1850-1861), and unstructured Pro-Ala-Ser (PAS) polypeptide chains (Schlapschy et al., 2013, Protein Engineering, Design and Selection, 26(8):489- 501). Examples of delivery moieties include, but are not limited to cell-penetrating peptides, particularly those facilitating transport across the blood-brain barrier, (reviewed in, e.g., Blades et al., 2023, Labelled Compounds and Radiopharmaceuticals, DOI 10.1002 / jlcr.4023), polypeptides such as scFvs or nanobodies (fused to the MIF-binding protein) that target receptors on the blood brain barrier (e.g., transferrin receptor TfR or insulin receptor), to facilitate receptor- mediated transcytosis-see, e.g., Kouhi et al., (2021), International Journal of Molecular Sciences, 22(12): 6331. In other embodiments, the targeting moiety is a polypeptide comprising a transcytosis-enabling module (TEM) for example in the form of an anti- scFv (e.g., a TfR-scFv or a CD98-scFv), as described in Edavettal et al., (2022), Med, 3(12):860-882 and in International Patent Application PCT / IB2021 / 052889.

[0275] Constant Regions

[0276] In some embodiments MIF -binding proteins and / or antibodies disclosed herein comprise a constant region of an antibody. This includes antigen-binding fragments of an antibody fused to a Fc.

[0277] Sequences of constant regions useful for producing the proteins of the present disclosure may be obtained from a number of sources. In some preferred embodiments, the constant region or portion thereof of the protein is derived from a human antibody. The constant region or portion thereof may be derived from any antibody class, including IgM, IgG, IgD, IgA and IgE, and any antibody isotype, including IgGl, IgG2, IgG3 and IgG4. In some embodiments, the constant region is human isotype IgG4 or a stabilized IgG4 constant region. In other embodiments the constant region is an IgGl constant region, which can facilitate uptake of a MIF or MIF-aggregate-bound antibody complexes into microglia thereby facilitating clearance of extracellular aggregated forms of MIF in the brain. In other embodiments the constant region in a MIF -binding protein (e.g., a MIF antibody) is an IgG2b constant region.

[0278] In some embodiments, the Fc region of the constant region has a reduced ability to induce effector function, e.g., compared to a native or wild-type human IgGl or IgG3 Fc region. In the context of the present disclosure, “effector functions” refer to those biological activities mediated by cells or proteins that bind to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody that result in activation of an Fc receptor. Examples of effector functions induced by antibodies include in relevant part: antibody-dependent-cell-phagocytosis (ADCP). Methods for assessing the level of effector function of an Fc region-containing protein are known in the art.

[0279] In some embodiments, where a MIF-binding protein includes an Fc region, the Fc region is an IgG4 Fc or an IgGl region (i.e., from an IgG4 or IgGl constant region), e.g., a human IgG4 Fc or IgGl Fc region. Sequences for IgG Fc regions are available in publicly accessible databases (e.g., available from National Center for Biotechnology Information).

[0280] In some embodiments, where an IgG4 Fc region is utilized, the constant region is a stabilized IgG4 constant region. The term “stabilized IgG4 constant region” will be understood to mean an IgG4 constant region that has been modified to reduce Fab arm exchange or the propensity to undergo Fab arm exchange or formation of a halfantibody or a propensity to form a half antibody. “Fab arm exchange” refers to a type of protein modification for human IgG4, in which an IgG4 heavy chain and attached light chain (half-molecule) is swapped for a heavy-light chain pair from another IgG4 molecule. Thus, IgG4 molecules may acquire two distinct Fab arms recognizing two distinct antigens (resulting in bispecific molecules). Fab arm exchange occurs naturally in vivo and can be induced in vitro by purified blood cells or reducing agents such as reduced glutathione. A “half antibody” forms when an IgG4 antibody dissociates to form two molecules each containing a single heavy chain and a single light chain.

[0281] In some embodiments, a stabilized IgG4 constant region comprises a proline at position 241 of the hinge region according to the system of Kabat (Kabat et al., Sequences of Proteins of Immunological Interest Washington DC United States Department of Health and Human Services, 1987 and / or 1991). This position corresponds to position 228 of the hinge region according to the EU numbering system (Kabat et al., Sequences of Proteins of Immunological Interest Washington DC United States Department of Health and Human Services, 2001 and Edelman et al., Proc. Natl. Acad. USA, 63, 78-85, 1969). In human IgG4, this residue is generally a serine. Following substitution of the serine for proline, the IgG4 hinge region comprises a sequence CPPC. In this regard, the skilled person will be aware that the “hinge region” is a proline-rich portion of an antibody heavy chain constant region that links the Fc and Fab regions that confers mobility on the two Fab arms of an antibody. The hinge region includes cysteine residues which are involved in inter-heavy chain disulfide bonds. It is generally defined as stretching from Glu226 to Pro243 of human IgGl according to the numbering system of Kabat. Hinge regions of other IgG isotypes may be aligned with the IgGl sequence by placing the first and last cysteine residues forming inter-heavy chain disulfide (S-S) bonds in the same positions (see for example WG2010 / 080538).

[0282] Additional examples of stabilized IgG4 antibodies include antibodies in which arginine at position 409 in a heavy chain constant region of human IgG4 (according to the EU numbering system) is substituted with lysine, threonine, methionine, or leucine (e.g., as described in W02006 / 033386). The Fc region of the constant region may additionally or alternatively comprise a residue selected from the group consisting of: alanine, valine, glycine, isoleucine and leucine at the position corresponding to 405 (according to the EU numbering system). Optionally, the hinge region comprises a proline at position 241 (z.e., a CPPC sequence) (as described above).

[0283] In another example, the Fc region is a region modified to have reduced effector function, i.e., a “non-immunostimulatory Fc region”. For example, the Fc region is an IgGl Fc region comprising a substitution at one or more positions selected from the group consisting of 268, 309, 330 and 331. In another example, the Fc region is an IgGl Fc region comprising one or more of the following changes E233P, L234V, L235A and deletion of G236 and / or one or more of the following changes A327G, A330S and P331S (Armour et al., Eur J Immunol. 29:2613-2624, 1999; Shields et al., J Biol Chem. 276(9) :6591-604, 2001). Additional examples of non-immunostimulatory Fc regions are described, for example, in Dall’Acqua et al., J Immunol. 177: 1129-1138 2006; and / or Hezareh J Virol ; 75: 12161-12168, 2001).

[0284] In some embodiments, the Fc region is a chimeric Fc region, e.g., comprising at least one CH2 domain from an IgG4 antibody and at least one CH3 domain from an IgGl antibody, wherein the Fc region comprises a substitution at one or more amino acid positions selected from the group consisting of 240, 262, 264, 266, 297, 299, 307, 309, 323, 399, 409 and 427 (EU numbering) (e.g., as described in W02010 / 085682). Exemplary substitutions include 240F, 262L, 264T, 266F, 297Q, 299 A, 299K, 307P, 309K, 309M, 309P, 323F, 399S, and 427F.

[0285] Targeting Polynucleotides

[0286] Also disclosed herein are targeting polynucleotides as agents that inhibit MIF- CD74 signaling by reducing the level of MIF mRNA or CD74 mRNA and / or their level of translation.

[0287] Programmable Nucleases

[0288] In some embodiments of the methods disclosed herein, the agent that inhibits MIF-CD74 signaling comprises encodes a programmable nuclease that reduces the level of MIF mRNA or CD74 mRNA. As used herein, the term "programmable nuclease" relates to nucleases that are "targeted" ("programmed") to recognize and edit a pre-determined genomic location. In some embodiments the encoded polypeptide is a programmable nuclease "targeted" or "programmed" to introduce, within a population of target cells (e.g., microglia) a genetic modification into the MIF gene and / or the CD74 gene or a regulatory region thereof. In some embodiments, the genetic modification is a deletion or substitution in the gene or in a regulatory region thereof. In other embodiments, the nuclease is a mutated nuclease that is catalytically inactive, but is fused to a transcriptional repressor to reversibly inhibit expression from the targeted gene locus, e.g., the MIF ox CD74 gene.

[0289] Programmable nucleases that can be used in accordance with the present disclosure include, but are not limited to, RNA-guided engineered nuclease (RGEN) derived from the bacterial clustered regularly interspaced short palindromic repeat (CRISPR)-cas (CRISPR-associated) system, zinc-finger nuclease (ZFN), transcription activator-like nuclease (TALEN), and argonaute. In some embodiments, the nuclease is an RNA-guided engineered nuclease (RGEN). In some embodiments the RGEN is from an archaeal genome or is a recombinant version thereof. In some embodiments the RGEN is from a bacterial genome or is a recombinant version thereof. In some embodiments the RGEN is from a Type I (CRISPR)-cas (CRISPR-associated) system. In some embodiments the RGEN is from a Type II (CRISPR)-cas (CRISPR-associated) system. In some embodiments the RGEN is from a Type III (CRISPR)-cas (CRISPR-associated) system. In some embodiments the nuclease is a class I RGEN. In some embodiments the nuclease is a class II RGEN. In some embodiments the RGEN is a multi-component enzyme. In some embodiments the RGEN is a single component enzyme. In some embodiments the RGEN is CAS3. In some embodiments the RGEN is CASIO. In some embodiments the RGEN is CAS9. In some embodiments the RGEN is Cpfl (Zetsche et al., 2015). In some embodiments the RGEN is targeted by a single RNA or DNA. In some embodiments the RGEN is targeted by more than one RNA and / or DNA. In some embodiments the programmable nuclease may be a DNA programmed argonaute (WO 14 / 189628).

[0290] In some embodiments the targeting polynucleotide, e.g., a guide RNA (gRNA) for CRISPR-based inhibition of MIF and / or CD74 expression is provided in an expression vector to be delivered in vivo using any of a number of non-viral transfection methods or viral transduction methods known in the art, e.g., recombinant virus transduction, liposome-based transfection, electroporation, or nanoparticle based transfection. Suitable examples of gRNA sequences are available commercially. For example, a vector for expression of both CD74 sgRNA and Cas9-2A-GFP is available from Applied Biological Materials (Catalog No. 15566136). An exemplary CD74 gRNA comprises the sequence GCGCGCTGGTCATCCATGAC (SEQ ID NO:53). sgRNA vectors are likewise commercially available for MIF from, e.g. , OriGene (Cat No. RC205106)

[0291] As used herein, an "expression vector" is a DNA or RNA vector that is capable of effecting expression of one or more polynucleotides in host cells (e.g., microglia). The vector is typically a plasmid or recombinant virus. Any suitable expression vector can be used, examples of which include, but are not limited to, a plasmid or viral vector. In some embodiments, the viral vector is a lentivirus, an adenovirus, a herpes, adenovirus, or an adeno-associated virus (AAV).

[0292] Such vectors and recombinant viruses will include one or more promoters for expressing the polynucleotide such as a gRNA for gene knockdown. Suitable promoters include, but are not limited to, the human cytomegalovirus (CMV) promoter. Cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, RNA polymerase III (in the case of shRNA or miRNA expression), and P- actin promoters, can also be used. In some embodiments the promoter is microglial-selective promoter such as the human HEXB promoter (see, e.g., Shah et al., 2022, Frontiers in Cellular Neuroscience, doi 10.3389 / fhcel.2022.808598). The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.

[0293] In other embodiments, the programmable nuclease may be programmed to recognize a genomic location by a combination of DNA-binding zinc-finger protein (ZFP) domains. ZFPs recognize a specific 3 -bp in a DNA sequence, a combination of ZFPs can be used to recognize a specific a specific genomic location. In some embodiments, the programmable nuclease may be programmed to recognize a genomic location by transcription activator-like effectors (TALEs) DNA binding domains. In an alternate embodiment, the programmable nuclease may be programmed to recognize a genomic location by one or more RNA sequences. In an alternate embodiment, the programmable nuclease may be programmed by one or more DNA sequences. In an alternate embodiment, the programmable nuclease may be programmed by one or more hybrid DNA / RNA sequences. In an alternate embodiment, the programmable nuclease may be programmed by one or more of an RNA sequence, a DNA sequences and a hybrid DNA / RNA sequence.

[0294] RNA Interference

[0295] The terms "RNA interference", "RNAi" or "gene silencing" refer generally to a process in which a double-stranded RNA molecule reduces the expression of a nucleic acid sequence with which the double-stranded RNA molecule shares substantial or total homology. However, it has been shown that RNA interference can also be achieved using non-RNA double stranded molecules (see, for example, US 20070004667).

[0296] In some embodiments, a targeting polynucleotide for use in the methods disclosed herein comprises nucleic acid molecules comprising and / or encoding double-stranded regions for RNA interference against the CD74 mRNA (human CD74 mRNA: GenBank Accession No. NM_001025159.3). In other embodiments the RNA interference is against MIF mRNA (GenBank Accession No. NM_002415.2). The nucleic acid molecules are typically RNA but may comprise chemically-modified nucleotides and non-nucleotides.

[0297] The double-stranded regions should be at least 19 contiguous nucleotides, for example about 19 to 23 nucleotides, or may be longer, for example 30 or 50 nucleotides, or 100 nucleotides or more. The full-length sequence corresponding to the entire gene transcript may be used. Preferably, they are about 19 to about 23 nucleotides in length.

[0298] The degree of identity of a double-stranded region of a nucleic acid molecule to the targeted transcript should be at least 90% and more preferably 95-100%. The nucleic acid molecule may of course comprise unrelated sequences which may function to stabilize the molecule.

[0299] The term "short interfering RNA" or "siRNA" as used herein refers to a nucleic acid molecule which comprises ribonucleotides capable of inhibiting or down regulating gene expression, for example by mediating RNAi in a sequence-specific manner, wherein the double stranded portion is less than 50 nucleotides in length, preferably about 19 to about 23 nucleotides in length. For example, the siRNA can be a nucleic acid molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The siRNA can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary.

[0300] As used herein, the term siRNA is meant to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi, for example micro-RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid (siRNA), short interfering modified oligonucleotide, chemically-modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), and others. In addition, as used herein, the term RNAi is meant to be equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, translational inhibition, or epigenetics. For example, siRNA molecules can be used to epigenetically silence genes at both the post- transcriptional level or the pre-transcriptional level. In a non-limiting example, epigenetic regulation of gene expression by siRNA molecules can result from siRNA mediated modification of chromatin structure to alter gene expression.

[0301] By "shRNA" or "short-hairpin RNA" is meant an RNA molecule where less than about 50 nucleotides, preferably about 19 to about 23 nucleotides, is base paired with a complementary sequence located on the same RNA molecule, and where said sequence and complementary sequence are separated by an unpaired region of at least about 4 to about 15 nucleotides which forms a single-stranded loop above the stem structure created by the two regions of base complementarity. Included shRNAs are dual or bi-finger and multi-finger hairpin dsRNAs, in which the RNA molecule comprises two or more of such stem-loop structures separated by single-stranded spacer regions.

[0302] Once designed, the nucleic acid molecules comprising a double-stranded region can be generated by any method known in the art, for example, by in vitro transcription, recombinantly, or by synthetic means.

[0303] Modifications or analogs of nucleotides can be introduced to improve the properties of the nucleic acid molecules. Improved properties include increased nuclease resistance and / or increased ability to permeate cell membranes. Accordingly, the terms "nucleic acid molecule" and "double-stranded RNA molecule" includes synthetically modified bases such as, but not limited to, inosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl-, 2-propyl- and other alkyl- adenines, 5-halo uracil, 5-halo cytosine, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiuracil, 8- halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines, 8-amino guanine, 8-thiol guanine, 8-thioalkyl guanines, 8-hydroxyl guanine and other substituted guanines, other aza and deaza adenines, other aza and deaza guanines, 5 -trifluoromethyl uracil and 5- trifluoro cytosine.

[0304] Chemically modified siRNAs particularly suited for in vivo delivery are described in the art in, e.g., W02014201306, W02007051303. Exemplary siRNAs that can be used to target CD74 mRNA are commercially available, e.g., from ThermoFisher (Cat Nos: s2715, S2716, and s225179) and Origene (Cat. No. SR319632). Likewise, MIF-targeting siRNAs are also commercially available, e.g., from ThermoFisher (Cat Nos: S194614, s8780, and S194615).

[0305] Genetically Modified Cells and Recombinant Expression of MIF-Binding Proteins

[0306] Also disclosed herein is a genetically modified cell comprising any of the isolated nucleic acids or expression vectors encoding a MIF-binding protein as disclosed herein. In some embodiments the genetically modified cells are mammalian cells, yeast cells, bacterial cells, insect cells, plant cells, or filamentous fungal cells. In some preferred embodiments the modified mammalian cells are rodent cells or human cells. Thus, also disclosed herein is a method for producing a MIF-binding protein disclosed herein by culturing a genetically modified cells disclosed herein comprising expression vectors for recombinant expression of the MIF-binding proteins for recombinant expression of such proteins, and subsequently purifying / isolating the expressed protein. In some embodiments, a MIF-binding protein disclosed herein is produced by culturing a hybridoma under conditions sufficient to produce the protein, e.g., as described herein and / or as is known in the art.

[0307] For example, genetically modified host cells, such as E. coli cells, yeast cells, insect cells, or mammalian cells, such as simian COS cells, Chinese Hamster Ovary (CHO) cells, human embryonic kidney (HEK) cells, or myeloma cells that do not otherwise produce the protein can be used for expression and producing a MIF-protein disclosed herein. Exemplary cells used for expressing a MIF-binding protein are CHO cells, myeloma cells or HEK cells. In other embodiments, the genetically modified host cells are filamentous fungi cells, e.g., from Aspergillus oryzae, which can be particularly useful for recombinant expression of antibodies at large scale and lowered cost as described in, e.g., Huyn et al., (2020), Fungal Biology and. Biotechnology, doi.org / 10.1186 / s40694-020-00098-w.

[0308] Means for introducing the isolated nucleic acid or expression construct comprising same into a cell for expression are known to those skilled in the art. The technique used for a given cell depends on the known successful techniques. Means for introducing recombinant DNA into cells include microinjection, transfection mediated by DEAE-dextran, transfection mediated by liposomes such as by using lipofectamine (Gibco, MD, USA) and / or cellfectin (Gibco, MD, USA), PEG-mediated DNA uptake, electroporation and microparticle bombardment such as by using DNA-coated tungsten or gold particles (Agracetus Inc., WI, USA) amongst others.

[0309] Mammalian host cells used to produce the protein may be cultured in a variety of media, depending on the cell type used. Commercially available media such as Ham’s F10 (Sigma), Minimal Essential Medium (MEM), (Sigma), RPM1-1640 (Sigma), and Dulbecco’s Modified Eagle’s Medium (DMEM), Sigma) are suitable for culturing mammalian cells. Media for culturing other cell types discussed herein, e.g., yeast or filamentous fungi, are known in the art.

[0310] Purification of MIF-Binding Proteins

[0311] Methods for purifying binding proteins, e.g., antibodies are known in the art and / or described herein.

[0312] In some embodiments where a MIF-binding protein is secreted into culture medium, supernatants from such expression systems can be first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants. Alternatively, or additionally, supernatants can be filtered and / or separated from cells expressing the protein, e.g. , using continuous centrifugation.

[0313] A MIF -binding protein prepared from cells can be purified using, for example, ion exchange, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, affinity chromatography (e.g., protein A affinity chromatography or protein G chromatography), or any combination of the foregoing. These methods are known in the art and described, for example in WO99 / 57134 or Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988). See also Fishman and Berg, (2019), Cold Spring Harbor Protocols, doi: 10.110 l / pdb.top099101.

[0314] The skilled artisan will also be aware that a protein can be modified to include a tag to facilitate purification or detection, e.g., a poly-histidine tag, e.g., a hexa-histidine tag, or an influenza virus hemagglutinin (HA) tag, or a FLAG tag, or a glutathione S- transferase (GST) tag. The resulting protein is then purified using methods known in the art, such as, affinity purification. For example, a protein comprising a hexa-his tag is purified by contacting a sample comprising the protein with nickel-nitrilotriacetic acid (Ni-NTA) that specifically binds a hexa-his tag immobilized on a solid or semisolid support, washing the sample to remove unbound protein, and subsequently eluting the bound protein. Alternatively, or in addition a ligand or antibody that binds to a tag is used in an affinity purification method.

[0315] Compositions

[0316] Also disclosed herein are compositions, including pharmaceutical compositions, comprising a MIF-binding protein a non- viral expression vector or a recombinant virus for expression of a targeting polynucleotide against CD74 or MIF; and a pharmaceutically acceptable carrier. In some preferred embodiments a pharmaceutical composition for use in a treatment method disclosed herein contains a MIF-binding protein disclosed herein. A “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” herein refers to any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Such carriers are well known to those of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable carriers / excipients can be found in Remington’s Pharmaceutical Sciences, Gennaro, AR, ed., 20th edition, 2000: Williams and Wilkins PA, USA. Exemplary pharmaceutically acceptable carriers can include salts, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. For example, compositions described herein may be provided in liquid form, and formulated in saline based aqueous solution of physiological pH, with or without detergents such polysorbate-80 at 0.01-1%, or carbohydrate additives, such mannitol, sorbitol, or trehalose. Commonly used buffers include histidine, acetate, phosphate, or citrate. The infusion solution may include 0 to 10% dextrose.

[0317] In some embodiments the pharmaceutical compositions disclosed herein also include one or more agents to facilitate delivery of the active agent, e.g., a MIF-binding protein or targeting polynucleotide against MIF or CD74, across the blood-brain barrier (BBB). Accordingly, in some embodiments the pharmaceutical composition includes lipid-shelled microbubbles or lipid shelled nanobubbles. In some embodiments the lipid-shelled microbubbles comprise l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-

[0318] [carboxy(polyethylene glycol)-2000 (DSPE-PEG2000). The generation, therapeutic formulation, and use of lipid-shelled microbubbles or lipid-shelled nanobubbles, particularly in relation to biotherapeutic delivery across the BBB is known in the art as exemplified in, e.g., Omata et al., (2020), Advanced Drug Delivery Reviews, 154- 155:236-244; Yang et al., (2022), Interdisciplinary Materials, doi.org / 10.1002 / idm2.12050; and in international patent publications WO 2021 / 108861 and WO 2020 / 077380.

[0319] In some embodiments, a pharmaceutical composition disclosed herein is formulated for an intravenous, intra-arterial, intrathecal, intraventricular, intracistemal, intracerebral, intranasal, intraperitoneal, intramuscular, or subcutaneous route of administration.

[0320] Formulation of (i) a MIF-binding protein or another of the foregoing active agents (ii)-(v) into a suitable form for administration to a subject (e.g., a pharmaceutical composition) are known in the art and include, for example, methods as described in Remington’s Pharmaceutical Sciences, Academic Press; 23rdedition (2020); and U.S. Pharmacopeia: The National Formulary (2023). Rockville, MD: United States Pharmacopeial Convention, Inc.

[0321] The pharmaceutical compositions of this disclosure are particularly useful for parenteral administration, such as intravenous, intra-arterial administration or administration into the CNS (e.g., intrathecal route) or into the brain (e.g., intraventricular route). The compositions for administration will commonly comprise a solution of a MIF-binding protein dissolved in a pharmaceutically acceptable carrier, for example an aqueous carrier. A variety of aqueous carriers can be used, e.g., buffered saline and the like. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of a MIF-binding protein of the present disclosure in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight, level of uptake in the brain, and the like in accordance with the particular mode of administration selected, and a specific patient’s needs (e.g., disease stage). Exemplary carriers include water, physiological saline, Ringer's solution, dextrose solution, and 5% human serum albumin. In some cases, no- aqueous vehicles such as mixed oils and ethyl oleate may also be used. The vehicles may contain minor amounts of additives that enhance isotonicity and chemical stability, e.g., buffers and preservatives. The skilled person will appreciate that carriers for pharmaceutical compositions disclosed herein will be selected based upon the active agent being formulated and may differ from each other based accordingly to the extent particular active agents may require specific stabilizing agents. Notwithstanding any specific differences in such formulations, each formulation is physiologically compatible for in vivo administration in view of the selected route of administration.

[0322] Methods of Use

[0323] Also disclosed herein is a method for preventing or treating a CNS condition associated with neuroinflammation, the method comprising administering to the subject a therapeutically effective amount of an agent that inhibits macrophage migration inhibitory factor (MIF)-CD74 signaling. In some embodiments the CNS condition associated with neuroinflammation is a condition selected from the group consisting of: Alzheimer’s disease, mild cognitive impairment (MCI), schizophrenia, autism spectrum disorder (ASD), major depression disorder, anxiety disorder, traumatic brain injury (TBI), amyotrophic lateral sclerosis (ALS), Parkinson’s Disease (PD). In some preferred embodiments disclosed herein is a method for preventing, treating, or delaying progression of Alzheimer’s disease (AD) or mild cognitive impairment (MCI) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an agent that inhibits macrophage migration inhibitory factor (MIF)-CD74 signaling. In some embodiments the method is for treatment of the condition.

[0324] Similarly disclosed is the use of an agent that inhibits MIF-CD74 signalling in the manufacture of a medicament for preventing, treating, or delaying progression of AD or MCI. In some embodiments the agent for administration that inhibits MIF-CD74 signalling comprises a MIF-binding protein, e.g., a MIF-binding protein as disclosed herein. In some preferred embodiments the MIF-binding protein to be administered is a MIF-binding protein containing at least one immunoglobulin variable region that binds specifically to MIF. In some embodiments the MIF-binding protein to be administered includes a nanobody. In other embodiments the MIF-binding protein comprises an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL), wherein the MIF-binding protein binds specifically, through an antigen binding site formed by the VH and VL, to an epitope of human MIF protein. In some embodiments MIF-binding protein to be administered comprises any one of the MIF-binding antibodies described herein. In some embodiments the MIF antibody to be administered is a MIF antibody, wherein the VH comprises the amino acid sequence corresponding to SEQ ID NO: 11 and the VL comprises the amino sequence corresponding to SEQ ID NO: 12. In some embodiments the MIF-binding antibody is the antibody known as Imalumab (BAX69). In other embodiments the MIF-binding protein is a soluble protein comprising the soluble ectodomain of CD74 corresponding to SEQ ID NO:4.

[0325] In other embodiments the agent that inhibits MIF-CD74 signaling comprises a CD74-targeting polynucleotide that reduces an expression level of CD74 in a cell population in the subject, whereby MIF-CD74 signaling is inhibited in the cell population. In other embodiments the agent that inhibits MIF-CD74 signaling comprises a MIF-targeting polynucleotide that reduces an expression level of MIF in a cell population in the subject, whereby MIF-CD74 signaling is inhibited in the cell population. In some embodiments a treatment method, as disclosed herein, includes administering a targeting polynucleotide selected from the group consisting of: guide RNA (gRNA), an siRNA, a shRNA, and an antisense oligonucleotide (ASO), as disclosed herein. In some preferred embodiments, where a targeting polynucleotide is to be administered, the targeting polynucleotide is a gRNA against CD74.

[0326] In some preferred embodiments an agent that inhibits MIF-CD74 signaling is provided for administration according to the methods disclosed herein as a pharmaceutical composition further comprising a pharmaceutically acceptable excipient and / or an agent to facilitate crossing of the blood brain barrier as disclosed herein.

[0327] In some embodiments the subject is a rodent, e.g., a mouse such as a genetically modified mouse or rat model of AD. In other embodiments the subject to be treated is a non-human primate. In some preferred embodiments the subject to be treated is a human subject.

[0328] In some embodiments of the above methods, where the route of administration of the pharmaceutical composition is a systemic route of administration (e.g., a parenteral route of administration) particularly intravenous or intra-arterial administration., the above methods also include a step of enhancing BBB permeability transiently before, during, or after administration of the pharmaceutical composition or the active agent. In some embodiments transient BBB permeabilization includes a step of scanning ultrasound transient permeabilization. In some preferred embodiments scanning ultrasound transient permeabilization is used in combination with lipid- shelled microbubbles or nanobubbles, e.g., DSPE-PEG2000 microbubbles that are either pre-formulated in the pharmaceutical composition to be administered or administered separately. Suitable methods for scanning ultrasound transient BBB permeabilization are described in, e.g., international patent application publication WO 2021 / 108861.

[0329] In embodiments combining scanning ultrasound permeabilization with the use of lipid-shelled microbubbles or lipid shelled-nanobubbles, permeabilization is achieved by mechanical interactions between the microbubbles or nanobubbles and the blood vessel wall as pulsed focused ultrasound is applied, resulting in cycles of compression and rarefaction of the microbubbles. This leads to a transient disruption of tight junctions and the uptake of blood-borne factors by the brain. Microbubbles within the target volume become “acoustically activated” by what is known as acoustic cavitation. In this process, the microbubbles expand and contract with acoustic pressure rarefaction and compression over several cycles.

[0330] In some embodiments microbubbles or nanobubbles are provided as a continuous infusion. In other embodiments they provided as a single bolus dose. In some preferred embodiments continuous infusion of microbubbles separately or preformulated with the active agent such a MIF-binding protein disclosed herein is provided over the duration of the acoustic energy application. In some embodiments, a microbubble agent is administered intravenously through the systemic circulation. Delivery of acoustic energy may comprise the delivery of acoustic energy from an acoustic energy source such as a focused ultrasound transducer to deliver transcranial ultrasound. See, e.g., Kee et al., (2023), Scientific Reports, 6963; doi.org / 10.1038 / s41598-023-33671-5. Various ultrasound parameters can be manipulated to influence the permeability increase in the blood-brain barrier and these include pressure amplitude, ultrasound frequency, burst length, pulse repetition frequency, focal spot size and focal depth.

[0331] Increased permeability of the blood-brain barrier may be determined by any suitable imaging method. Preferably, the imaging method is MRI, an optical imaging method, positron emission tomography (PET), computerized tomography (CT) or computerized axial tomography (CAT) or ultrasound.

[0332] Neuroinflammatory Conditions

[0333] The above-mentioned methods are useful in prophylaxis and / or treatment of a wide range of neuroinflammatory conditions based on their common etiology underpinned by pathological MIF-CD74 signaling and aberrant and / or impaired microglial function. Accordingly, in some embodiments, a subject to be treated suffers from a neuroinflammatory condition selected from the group consisting of: Alzheimer’s disease, frontotemporal dementia, schizophrenia, autism spectrum disorder, Pick’s disease, chronic traumatic encephalopathy, progressive supranuclear palsy, Parkinson’s disease, and Parkinsonian disorders. In some embodiments the subject to be treated is suffering from Alzheimer’s disease or frontotemporal dementia.

[0334] Diagnostic criteria and prognostic approaches for a variety of conditions associated with neuroinflammation are known in the art, as reviewed in, e.g., for Alzheimer’s Disease (van der Schaar et al., 2022, Alzheimer’s Research & Therapy, doi.org / 10.1186 / sl3195-022-00971-3), Frontotemporal Dementia (Antonioni et al., 2023, International Journal of Molecular Sciences, doi: 10.3390 / ijms241411732), schizophrenia (Orsolini et al., Journal of Clinical Medicine, doi: 10.3390 / jcml 1175040), autism spectrum disorder (Okoye et al., 2023, Cureus, doi: 10.7759 / cureus.43226), and Parkinson’s disease (Armstrong et al., 2020, JAMA, doi: 10.1001 / jama.2019.22360). Generally, diagnosis of such conditions will utilize a combination of behavioral / psychometric, neuroimaging, and molecular biomarker assays. In particular, brain MIF-PET imaging using ligands that specifically target MIF protein are particularly useful in diagnosis and prognosis of neurodegenerative conditions. See, e.g., Petersen et al., (2022), Current Opinion in Neurology, 35(2):230- 239. Neuroimaging methods such as Tau-PET and amyloid-PET imaging are particularly useful for evaluating a subject’s ongoing response and progress during and following any of the prophylactic and therapeutic method regimens disclosed herein.

[0335] Disease models (including ex vivo human cellular models) for various conditions associated with neuroinflammation are known in the art, e.g. , for AD, dual transgenic knock-in mice expressing human MIF in combination with 5xFAD mice as a model of Alzheimer’s disease (Barendrecht et al., 2023, Alzheimer’s Research & Therapy, 15(16); doi.org / 10.1186 / sl3195-022-01144-y); for ASD, see Hughes et al. (2023), Brain Behavior and Immunity, 108:245-254. For Parkinson’s disease, see Dovonou et al. (2023), Translational Neurodegeneration, 12:36. Useful assays for assessing efficacy include spatial learning and memory tests (e.g. , Morris water maze), motor behavior tests (e.g, using a Rotarod test) included learning and memory, biochemical and immunocytochemical assessments of Tau hyperphosphorylation and / or aggregation.

[0336] Combination Therapies

[0337] In some embodiments, an agent that inhibits MIF-CDF signaling, as disclosed in the present disclosure is administered in combination with another therapeutic agent useful for treating a condition described herein, either as combined or additional treatment steps or as additional components of a therapeutic formulation.

[0338] For example, the other therapeutic is an antibody against amyloid-0 protofibrils, e.g., lecanemab “Leqembi” (Eisai), a humanized IgGl (CAS 1260393-98-3), approved in the U.S. for the treatment of Alzheimer’s disease. Alternatively, or additionally, the other compound is an immunosuppressant. In other embodiments another therapeutic agent for use in combination with a composition disclosed herein is a cholinesterase inhibitor, e.g, is donepezil (Aricept), galantamine (Razadyne), or rivastagmine (Exelon). Alternatively, or additionally, a further therapeutic agent is a serotonin receptor 7 (5-HT7R) inverse agonist, e.g., Amisulpride, found to reduce Tau hyperphosphorylation as described in Jahreis et al., (2023), Alzheimer’s & Dementia, doi.org / 10.1002 / alz.13090. Alternatively, or additionally, the other therapeutic agent is an inhibitor of RIPK3, recently shown to play a role in necroptosis of neurons in Alzheimer’s disease (Balusu et al., 2023, Science, 381(6663):1176-1182). Alternatively, or additionally, another therapeutic agent is 5-(l,3-benzodioxol-5-yl)-3- (3 -bromophenyl)- IH-pyrazole (anlel38b), a Tau aggregation inhibitor.

[0339] In other embodiments, an additional therapeutic agent is antisense oligonucleotide (ASO) against Tau mRNA, e.g., BIIB080 (IONIS Pharmaceuticals), as described in, Mummery et al., (2023), Nature Medicine, 29:1437-1447).

[0340] Dosages and Timing of Administration

[0341] Suitable dosages of an agent that inhibits MIF-CD74 signaling, e.g., a MIF- binding protein or CD74-targeting polynucleotide, as disclosed herein, will vary depending on the specific MIF-binding protein, the condition to be treated, and / or the subject being treated. It is within the ability of a skilled physician to determine a suitable dosage, e.g., by commencing with a sub-optimal dosage and incrementally modifying the dosage to determine an optimal or useful dosage. Alternatively, to determine an appropriate dosage for treatment / prophylaxis, data from the cell culture assays or animal studies are used, wherein a suitable dose is within a range of circulating concentrations that include the EDso of the active compound with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. A therapeutically / prophylactically effective dose can be estimated initially from cell culture assays, particularly ones making use of human cell 2D or 3D culture models (e.g., patient hiPSC-derived models). A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (z.e., the concentration or amount of the compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma maybe measured, for example, by high performance liquid chromatography .

[0342] In some preferred embodiments, a method of the present disclosure comprises administering a prophylactically or therapeutically effective amount of a protein described herein.

[0343] In the context of the disclosed methods herein, the term “therapeutically effective amount” refers to a quantity of a therapeutic agent (e.g., a MIF-binding protein or expression vector or recombinant virus encoding the MIF-binding protein) which, when administered to a subject in need of treatment, improves the prognosis and / or state of the subject and / or that reduces or inhibits one or more symptoms of a clinical condition described herein to a level that is below that observed and accepted as clinically diagnostic or clinically characteristic of that condition. The amount to be administered to a subject will depend on the particular characteristics of the condition to be treated, the type and stage of condition being treated, the mode of administration, and the characteristics of the subject, such as general health, other diseases, age, sex, genotype, and body weight. A person skilled in the art will be able to determine appropriate dosages depending on these and other factors. Accordingly, this term is not to be construed to limit the present disclosure to a specific quantity, e.g., amount of protein(s), nucleic acids, rather the present disclosure encompasses any amount of a MIF-binding protein, targeting polynucleotide, or other agent that inhibits MIF-CD74 signalling, sufficient to achieve the stated result in a subject.

[0344] As used herein, the term “prophylactically effective amount” shall be taken to mean a sufficient quantity of a protein to prevent or inhibit or delay the onset of one or more detectable symptoms of a clinical condition. The skilled artisan will be aware that such an amount will vary depending on, for example, the specific MIF-binding protein(s) administered and / or the particular subject and / or the type or severity or level of condition and / or predisposition (genetic or otherwise) to the condition. Accordingly, this term is not to be construed to limit the present disclosure to a specific quantity, of the disclosed MIF-binding proteins and related compositions (e.g. , encoding targeting polynucleotides), rather the present disclosure encompasses any amount of such agents to achieve a clinically meaningful prophylactic effect in a subject.

[0345] In some embodiments a route of administration used in the therapeutic, prophylactic, diagnostic, or prognostic methods disclosed herein is intravenous, intraarterial, intrathecal, intracerebral, intracerebroventricular, intranasal, intramuscular, or subcutaneous.

[0346] In some embodiments, where a MIF-binding protein disclosed herein is to be administered systemically, suitable dosage amounts range from about 2 mg / kg up to about 100 mg / kg of an individual’s body weight per day, e.g., 2.5 mg / kg, 5.0 mg / kg, 10 mg / kg, 15 mg / kg, 22 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 37 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, or another dose from about 2 mg / kg to about 100 mg / kg. In some embodiments, where the route of administration is intrathecal, intracerebral, or intracerebrovascular, the dosing is in a lower range of about 0.1 mg / kg to about 20 mg / kg to reflect the higher relative dose reaching the brain due to the more direct route of administration. For repeated administrations over several days or longer, depending on the severity of the neuroinflammation-associated condition to be treated, the treatment can be sustained until a desired suppression of symptoms is achieved.

[0347] In some examples, the MIF-binding protein is administered systemically at an initial (or loading) dose of between about 2 mg / kg to about 40 mg / kg, such as from about 5 mg / kg to about 30 mg / kg, e.g., 7 mg / kg, 10 mg / kg, 12 mg / kg, 15 mg / kg, 18 mg / kg, 20 mg / kg, 22 mg / kg, 25 mg / kg, 27 mg / kg, or another dose from about 5 mg / kg to about 30 mg / kg. The skilled person in the art will appreciate that depending on the particular MIF-binding protein to be administered and the route of administration, dosing can vary. See, e.g., Mahalingam et al., 2020, British Journal of Clinical Pharmacology, doi.org / 10.1111 / bcp.14289, for a dosing example for the MIF antibody BAX69 (imalumab). The MIF-binding protein can then be administered at a lower maintenance dose of 10% to about 50% of the initial (“loading”) dose, e.g., 12%, 15%, 20%, 25%, 30%, 35%, 40%, or another lower maintenance dose from about 10% to about 50% of the initial dose. The maintenance doses may be administered every 7-30 days, such as, every 10-15 days, for example, every 10 or 11 or 12 or 13 or 14 or 15 days. In some embodiments, doses are administered at periodic intervals, e.g., every week, every 2 weeks, every 3 weeks, once a month, once every 2 months, or another interval from about every week to about every 2 months. In some embodiments a treatment period encompassing one of the foregoing dosing intervals last from about 2 months to about 24 months, e.g. , 3 months, 4 months, 5 months, 6 months, 8 months, 9 months, 10 months, 12 months, 16 months, months, 20 months, 22 months, or another treatment period from about 2 months to about 24 months.

[0348] In the case of a subject that is not adequately responding to treatment following a treatment period, a second treatment period may be initiated and / or an increased dosing regimen may be used.

[0349] In some embodiments, where a subject experiences an adverse reaction, the initial (or loading) dose may be split over numerous days in one week or over numerous consecutive days.

[0350] Administration of a pharmaceutical composition to provide a therapeutic agent disclosed herein according to the methods of the present disclosure can be continuous or intermittent, depending, for example, on the recipient’s physiological condition, whether the purpose of the administration is prophylactic or therapeutic, and other factors known to skilled practitioners. In some embodiments the administration of a an agent that inhibits MIF-CD74 signaling (e.g., a MIF antibody) is essentially continuous over a preselected period of time. In other embodiments administration is provided in a series of spaced doses, e.g., either during or after development of a neuroinflammation- related condition to be treated, e.g., AD.

[0351] ADDITIONAL EMBODIMENTS I

[0352] Methods for preventing, treating or delaying progression of neurodegenerative disease

[0353] In some aspects, the techniques described herein relate to a method for preventing, treating, or delaying progression of Alzheimer's disease (AD) or mild cognitive impairment (MCI) in a subject in need thereof, the method including administering to the subject a therapeutically effective amount of an agent that inhibits and / or abrogates macrophage migration inhibitory factor (MIF)-CD74 signalling.

[0354] In some embodiments, the techniques described herein relate to a method, wherein the subject has AD.

[0355] In some embodiments, the techniques described herein relate to a method, wherein the subject has MCI. In certain aspects, the techniques described herein relate to a method for preventing, treating, or delaying progression of schizophrenia in a subject in need thereof, the method including administering to the subject a therapeutically effective amount of an agent that inhibits and / or abrogates macrophage migration inhibitory factor (MIF)-CD74 signalling.

[0356] In some embodiments, the techniques described herein relate to a method, wherein the subject has schizophrenia.

[0357] In certain aspects, the techniques described herein relate to a method for preventing, treating, or delaying progression of anxiety associated with brain disease in a subject in need thereof, the method including administering to the subject a therapeutically effective amount of an agent that inhibits and / or abrogates macrophage migration inhibitory factor (MIF)-CD74 signalling.

[0358] In some embodiments, the techniques described herein relate to a method, wherein the subject has anxiety associated with brain disease.

[0359] In some embodiments, the techniques described herein relate to a method or claim 43, wherein the brain disease is neurodegenerative or non-neurodegenerative. In some embodiments, the brain disease is selected from the list: AD, Parkinson's disease, multiple sclerosis, and amyotrophic lateral sclerosis (ALS), frontotemporal dementia, Huntington's disease, and Lewy body dementia.

[0360] In some embodiments, the techniques described herein relate to a method, wherein the agent that inhibits MIF-CD74 signalling includes a MIF-binding protein.

[0361] Antibodies and antigen-binding fragments thereof

[0362] In some embodiments, the techniques described herein relate to a method, wherein the agent that inhibits MIF-CD74 signalling includes an MIF-binding protein, and that MIF-bniding protein includes at least one immunoglobulin variable region that binds specifically to MIF.

[0363] In some embodiments, the techniques described herein relate to a method, wherein the MIF-binding protein includes a nanobody.

[0364] In some embodiments, the techniques described herein relate to a method, wherein the nanobody is humanized.

[0365] In some embodiments, the techniques described herein relate to a method, wherein the MIF-binding protein includes an antibody or antigen-binding fragment thereof. In some embodiments, the techniques described herein relate to a method, wherein the antibody is an IgGl, IgG2, or IgG4 antibody or antigen binding fragment thereof.

[0366] In some embodiments, the techniques described herein relate to a method, wherein the antibody is an IgGl antibody or antigen binding fragment thereof. In some embodiments, the antibody is an IgG2 antibody. In some embodiments, the antibody is an IgG4 antibody.

[0367] In some embodiments, the techniques described herein relate to a method, wherein the antibody is a humanized antibody or antigen binding fragment thereof.

[0368] In some embodiments, the techniques described herein relate to a method, wherein the humanized antibody is a human chimeric antibody or antigen binding fragment thereof.

[0369] In some embodiments, the antibody or antigen binding fragment thereof is a human or humanized IgG2 or IgG4 monoclonal antibody. In various embodiments, the antibody or antigen binding fragment thereof is selected from the group consisting of an IgG2 kappa antibody, an IgG2 lambda antibody, an IgG4 kappa antibody or an IgG4 lambda antibody.

[0370] In some embodiments, the techniques described herein relate to a method, wherein the antibody or antigen-binding fragment thereof includes three heavy chain variable regions (VH) CDRs and three light chain variable region (VL) CDRs, wherein: (i) the three VH CDRs respectively have the sequences of: (a) VH-CDR1 including SEQ ID NO: 5 with up to two amino acid substitutions, VH-CDR2 including SEQ ID NO: 6 with up to four amino acid substitutions, and VH-CDR3 including SEQ ID NO: 7 with up to three amino acid substitutions; (b) VH-CDR1 including SEQ ID NO: 13 with up to two amino acid substitutions, VH-CDR2 including SEQ ID NO:14 with up to four amino acid substitutions, and VH-CDR3 including SEQ ID NO: 15 with up to four amino acid substitutions; (c) VH-CDR1 including SEQ ID NO:21 with up to two amino acid substitutions, VH-CDR2 including SEQ ID NO:22 with up to four amino acid substitutions, and VH-CDR3 including SEQ ID NO:23 with up to three amino acid substitutions; (d) VH-CDR1 including SEQ ID NO:29 with up to two amino acid substitutions, VH-CDR2 including SEQ ID NO:30 with up to four amino acid substitutions, and VH-CDR3 including SEQ ID NO:31 with up to four amino acid substitutions; (e) VH-CDR1 including SEQ ID NO:37 with up to two amino acid substitutions, VH-CDR2 including SEQ ID NO:38 with up to four amino acid substitutions, and VH-CDR3 including SEQ ID NO:39 with up to four amino acid substitutions; or (f) VH-CDR1 including SEQ ID NO:45 with up to two amino acid substitutions, VH-CDR2 including SEQ ID NO:46 with up to four amino acid substitutions, and VH-CDR3 including SEQ ID NO:47 with up to four amino acid substitutions; and (ii) the three VL CDRs respectively have the sequences of: (a) VL- CDR1 including SEQ ID NO:8 with up to three amino acid substitutions, VL-CDR2 including SEQ ID NO:9 with up to two amino acid substitutions, and VL-CDR3 including SEQ ID NO: 10 with up to three amino acid substitutions; (b) VL-CDR1 including SEQ ID NO: 16 with up to three amino acid substitutions, VL-CDR2 including SEQ ID NO: 17 with up to two amino acid substitutions, and VL-CDR3 including SEQ ID NO: 18 with up to three amino acid substitutions; (c) VL-CDR1 including SEQ ID NO:24 with up to three amino acid substitutions, VL-CDR2 including SEQ ID NO:25 with up to two amino acid substitutions, and VL-CDR3 including SEQ ID NO:26 with up to three amino acid substitutions; (d) VL-CDR1 including SEQ ID NO:32 with up to three amino acid substitutions, VL-CDR2 including SEQ ID NO:33 with up to two amino acid substitutions, and VL-CDR3 including SEQ ID NO:34 with up to two amino acid substitutions; (e) VL-CDR1 including SEQ ID NO:40 with up to three amino acid substitutions, VL-CDR2 including SEQ ID NO:41 with up to two amino acid substitutions, and VL-CDR3 including SEQ ID NO:42 with up to two amino acid substitutions; or (f) VL-CDR1 including SEQ ID NO:48 with up to three amino acid substitutions, VL-CDR2 including SEQ ID NO:49 with up to two amino acid substitutions, and VL-CDR3 including SEQ ID NO:50 with up to two amino acid substitutions.

[0371] In some embodiments, the techniques described herein relate to a method, wherein the antibody or antigen-binding fragment thereof includes three heavy chain variable regions (VH) CDRs and three light chain variable region (VL) CDRs, wherein: (i) the three VH CDRs respectively have the sequences of: (a) VH-CDR1 including SEQ ID NO:5, VH-CDR2 including SEQ ID NO:6, and VH-CDR3 including SEQ ID NO:7; (b) VH-CDR1 including SEQ ID NO:13, VH-CDR2 including SEQ ID NO:14, and VH-CDR3 including SEQ ID NO: 15; (c) VH-CDR1 including SEQ ID NO:21, VH-CDR2 including SEQ ID NO:22, and VH-CDR3 including SEQ ID NO:23; (d) VH-CDR1 including SEQ ID NO:29, VH-CDR2 including SEQ ID NO:30, and VH- CDR3 including SEQ ID NO:31; (e) VH-CDR1 including SEQ ID NO:37, VH-CDR2 including SEQ ID NO:38, and VH-CDR3 including SEQ ID NO:39; or (f) VH-CDR1 including SEQ ID NO:45, VH-CDR2 including SEQ ID NO:46, and VH-CDR3 including SEQ ID NO:47; and (ii) the three VL CDRs respectively have the sequences of: (a) VL-CDR1 including SEQ ID NO:8, VL-CDR2 including SEQ ID NO:9, and VL-CDR3 including SEQ ID NO: 10; (b) VL-CDR1 including SEQ ID NO: 16, VL- CDR2 including SEQ ID NO: 17, and VL-CDR3 including SEQ ID NO: 18; (c) VL- CDR1 including SEQ ID NO:24, VL-CDR2 including SEQ ID NO:25, and VL-CDR3 including SEQ ID NO:26; (d) VL-CDR1 including SEQ ID NO:32, VL-CDR2 including SEQ ID NO:33, and VL-CDR3 including SEQ ID NO:34; (e) VL-CDR1 including SEQ ID NO:40, VL-CDR2 including SEQ ID NO:41, and VL-CDR3 including SEQ ID NO:42; or (f) VL-CDR1 including SEQ ID NO:48, VL-CDR2 including SEQ ID NO:49, and VL-CDR3 including SEQ ID NO:50.

[0372] In some embodiments, the techniques described herein relate to a method, wherein the antibody or antigen-binding fragment thereof includes three VH CDRs and three VL CDRs: (i) the three VH CDRs respectively have the sequences of: VH-CDR1 including SEQ ID NO:5 with up to two amino acid substitutions, VH-CDR2 including SEQ ID NO:6 with up to four amino acid substitutions, and VH-CDR3 including SEQ ID NO: 7 with up to three amino acid substitutions; and (ii) the three VL CDRs respectively have the sequences of: VL-CDR1 including SEQ ID NO:8 with up to three amino acid substitutions, VL-CDR2 including SEQ ID NO:9 with up to two amino acid substitutions, and VL-CDR3 including SEQ ID NO: 10 with up to three amino acid substitutions.

[0373] In some embodiments, the techniques described herein relate to a method, wherein the antibody or antigen-binding fragment thereof includes three VH CDRs and three VL CDRs: (i) the three VH CDRs respectively have the sequences of: VH-CDR1 including SEQ ID NO:5, VH-CDR2 including SEQ ID NO:6, and VH-CDR3 including SEQ ID NO:7; and (ii) the three VL CDRs respectively have the sequences of: VL- CDR1 including SEQ ID NO:8, VL-CDR2 including SEQ ID NO:9, and VL-CDR3 including SEQ ID NO: 10.

[0374] In some embodiments, the techniques described herein relate to a method, wherein the VH of the antibody or antigen-binding fragment thereof includes the amino acid sequence corresponding to any one of SEQ ID NOs:l l, 19, 27, 35, 43, or 51; and the VL includes the amino acid sequence of any one of SEQ ID NOs:12, 20, 28, 36, 44, or 52; and optionally wherein the VH includes up to six amino acid substitutions and the VL includes up to six amino acid substitutions.

[0375] In some embodiments, the techniques described herein relate to a method, wherein the antibody or the antigen-binding fragment thereof is a divalent antibody, bispecific antibody, a F(ab), a Fab', a F(ab)'2, a diabody, a triabody, a tetrabody, a single chain Fv fragment (scFv), or a dimeric scFv (di-scFv).

[0376] In some embodiments, the techniques described herein relate to a method, wherein the antibody or antigen binding fragment thereof is a Fab, optionally wherein the Fab is PEGylated. In some embodiments, the techniques described herein relate to a method, wherein the antibody or antigen binding fragment thereof is a Fv, optionally wherein the variable chains have been cross-linked, optionally wherein the cross-linkage is performed through glutaraldehyde, intermolecular disulfides or a peptide linker.

[0377] In some embodiments, the techniques described herein relate to a method, wherein the antibody or antigen binding fragment thereof is a full-length monoclonal antibody.

[0378] In some embodiments, the techniques described herein relate to a method, wherein the antibody or antigen binding fragment there, wherein the antibody or antigen binding fragment thereof is bispecific.

[0379] In some embodiments, the techniques described herein relate to a method, wherein the antibody or antigen binding fragment thereof includes an Fc region.

[0380] In some embodiments, the techniques described herein relate to a method, wherein the Fc has engineered mutations that reduce antibody binding to FcyR and / or reduce complement fixation.

[0381] Pharmaceutical compositions

[0382] In some embodiments, the agent that inhibits and / or abrogates MIF-CD74 signalling is formulated as a pharmaceutical composition including the antibody or antigen binding fragment thereof and a pharmaceutically acceptable carrier.

[0383] In some embodiments, the techniques described herein relate to a method, wherein the antibody or antigen-binding fragment thereof is formulated as a pharmaceutical composition including the antibody or antigen binding fragment thereof and a pharmaceutically acceptable carrier.

[0384] In some embodiments, the antibody or antigen-binding fragment thereof is administered as a pharmaceutical composition including the antibody or antigen binding fragment thereof and a pharmaceutically acceptable carrier.

[0385] In some embodiments, the pharmaceutical composition is formulated for parenteral administration. In some embodiments, the techniques described herein relate to a method, wherein the pharmaceutical composition is formulated for intravenous administration. In some embodiments, the techniques described herein relate to a method, wherein the pharmaceutical composition is formulated for subcutaneous administration.

[0386] In some embodiments, the pharmaceutical composition is administered parenterally. In some embodiments, the pharmaceutical composition is administered by intravenous administration. In some embodiments, the pharmaceutical composition is administered by subcutaneous administration.

[0387] Dose Regimen

[0388] In some embodiments, the techniques described herein relate to a method, wherein the antibody or antigen binding fragment thereof is administered for at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months. In some embodiments, the techniques described herein relate to a method, wherein the antibody or antigen binding fragment thereof is administered for at least 3 months. In some embodiments, the techniques described herein relate to a method, wherein the antibody or antigen binding fragment thereof is administered for at least 6 months. In some embodiments, the techniques described herein relate to a method, wherein the antibody or antigen binding fragment thereof is administered for at least 12 months.

[0389] Therapeutic combinations

[0390] In some embodiments, the techniques described herein relate to a method, wherein the agent that inhibits MIF-CD74 signalling is formulated for coadministration with a glucocorticoid or glucocorticoid-like drug.

[0391] In some embodiments, the glucocorticoid is a natural glucocorticoid. In some embodiments, the natural glucocorticoid is cortisol or corticosterone.

[0392] In some embodiments, the glucocorticoid is a synthetic glucocorticoid. In some embodiments, the synthetic glucocorticoid is hydrocortisone, prednisone, methylprednisolone, dexamethasone, or fluticasone.

[0393] In some embodiments, the glucocorticoid-like drug is a progestin. In some embodiments, the progestin is medroxyprogesterone acetate.

[0394] In some embodiments, the techniques described herein relate to a method, wherein the glucocorticoid is formulated as a pharmaceutical composition comprising the glucocorticoid and a pharmaceutically acceptable carrier.

[0395] In some embodiments, the techniques described herein relate to a method, wherein the agent that inhibits MIF-CD74 signalling, and the glucocorticoid are coadministered as a fixed dose combination. In some embodiments, the techniques described herein relate to a method, wherein the agent that inhibits MIF-CD74 signalling, and the glucocorticoid are co-administered as a free dose combination.

[0396] In some embodiments, the fixed dose combination is administered parenterally. In some embodiments, the fixed dose combination is administered by intravenous administration. In some embodiments, the fixed dose combination is administered by subcutaneous administration. In some embodiments, glucocorticoid or glucocorticoid-like drug is administered as a free dose. In some embodiments, free dose is formulated as an oral free dose. In some embodiments, the oral free dose combination is formulated as a solid dosage form, such as a tablet or capsule.

[0397] In some embodiments, the free dose glucocorticoid or glucocorticoid-like drug and the anti-MIF signalling agent are administered concomitantly.

[0398] CD74 targeting

[0399] In some embodiments, the techniques described herein relate to a method, wherein the agent that inhibits MIF-CD74 signalling includes a CD74-targeting polynucleotide that reduces an expression level of CD74 in a cell population in the subject, whereby MIF-CD74 signalling is inhibited in the cell population.

[0400] In some embodiments, the techniques described herein relate to a method, wherein the CD74-targeting polynucleotide is selected from the group consisting of: guide RNA (gRNA), an siRNA, a shRNA, and an antisense oligonucleotide (ASO).

[0401] In some embodiments, the techniques described herein relate to a method, wherein the CD74-targeting polynucleotide is a gRNA.

[0402] Other embodiments

[0403] In some embodiments, the techniques described herein relate to a method, wherein the subject is a human subject.

[0404] In some embodiments, the techniques described herein relate to a method, wherein MIF is oxidized-MIF (ox-MIF).

[0405] In some embodiments, the agent that inhibits and / or abrogates MIF-CD74 signalling is formulated as a pharmaceutical composition including the antibody or antigen binding fragment thereof or a pharmaceutically acceptable carrier.

[0406] In some embodiments, the techniques described herein relate to a method, wherein an agent inhibits MIF-CD74 signalling, and that agent is capable of includes a CD74- targeting polynucleotide that reduces an expression level of CD74 in a cell population in the subject, whereby MIF-CD74 signalling is inhibited in the cell population.

[0407] In some embodiments, the agent that inhibits and / or abrogates MIF-CD74 signalling is capable of increasing microglial amyloid-0 phagocytosis. In some embodiments, the agent is capable of increasing microglial amyloid-0 phagocytosis in a dose dependent manner. ADDITIONAL EMBODIMENTS II

[0408] 1. A method for preventing, treating, or delaying progression of Alzheimer’ s disease (AD) or mild cognitive impairment (MCI) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an agent that inhibits macrophage migration inhibitory factor (MIF)-CD74 signalling.

[0409] 2. The method according to claim 1 for treating AD or MCI.

[0410] 3. The use of an agent that inhibits macrophage migration inhibitory factor (MIF)- CD74 signalling in the manufacture of a medicament for preventing, treating, or delaying progression of Alzheimer’s disease (AD) or mild cognitive impairment (MCI).

[0411] 4. The use according to claim Error! Reference source not found, for treating AD or MCI.

[0412] 5. The method according to claim Error! Reference source not found, or claim Error! Reference source not found., or the use according to claim Error! Reference source not found, or claim Error! Reference source not found., wherein the agent that inhibits MIF-CD74 signalling comprises a MIF-binding protein.

[0413] 6. The method or use according to claim Error! Reference source not found., wherein the MIF-binding protein comprises at least one immunoglobulin variable region that binds specifically to MIF.

[0414] 7. The method or use according to claim Error! Reference source not found, or claim Error! Reference source not found., wherein the MIF-binding protein comprises a nanobody.

[0415] 8. The method or use according to claim Error! Reference source not found, or claim Error! Reference source not found., wherein the MIF-binding protein comprises an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL), wherein the MIF-binding protein binds specifically, through an antigen binding site formed by the VH and VL, to an epitope of human MIF protein.

[0416] 9. The method or use according to claim Error! Reference source not found., wherein:

[0417] (i) the amino acid sequence of the VH comprises:

[0418] (a) VH-CDR1 comprising SEQ ID NO:5 with up to two amino acid substitutions, VH-CDR2 comprising SEQ ID NO:6 with up to four amino acid substitutions, and VH-CDR3 comprising SEQ ID NO:7 with up to three amino acid substitutions;

[0419] (b) VH-CDR1 comprising SEQ ID NO: 13 with up to two amino acid substitutions, VH-CDR2 comprising SEQ ID NO: 14 with up to four amino acid substitutions, and VH-CDR3 comprising SEQ ID NO: 15 with up to four amino acid substitutions;

[0420] (c) VH-CDR1 comprising SEQ ID NO:21 with up to two amino acid substitutions, VH-CDR2 comprising SEQ ID NO:22 with up to four amino acid substitutions, and VH-CDR3 comprising SEQ ID NO:23 with up to three amino acid substitutions;

[0421] (d) VH-CDR1 comprising SEQ ID NO:29 with up to two amino acid substitutions, VH-CDR2 comprising SEQ ID NO:30 with up to four amino acid substitutions, and VH-CDR3 comprising SEQ ID NO:31 with up to four amino acid substitutions;

[0422] (e) VH-CDR1 comprising SEQ ID NO:37 with up to two amino acid substitutions, VH-CDR2 comprising SEQ ID NO:38 with up to four amino acid substitutions, and VH-CDR3 comprising SEQ ID NO:39 with up to four amino acid substitutions; or

[0423] (f) VH-CDR1 comprising SEQ ID NO:45 with up to two amino acid substitutions, VH-CDR2 comprising SEQ ID NO:46 with up to four amino acid substitutions, and VH-CDR3 comprising SEQ ID NO:47 with up to four amino acid substitutions; and

[0424] (ii) the amino acid sequence of the VL comprises:

[0425] (a) VL-CDR1 comprising SEQ ID NO:8 with up to three amino acid substitutions, VL-CDR2 comprising SEQ ID NO:9 with up to two amino acid substitutions, and VL-CDR3 comprising SEQ ID NO: 10 with up to three amino acid substitutions;

[0426] (b) VL-CDR1 comprising SEQ ID NO: 16 with up to three amino acid substitutions, VL-CDR2 comprising SEQ ID NO: 17 with up to two amino acid substitutions, and VL-CDR3 comprising SEQ ID NO: 18 with up to three amino acid substitutions;

[0427] (c) VL-CDR1 comprising SEQ ID NO:24 with up to three amino acid substitutions, VL-CDR2 comprising SEQ ID NO:25 with up to two amino acid substitutions, and VL-CDR3 comprising SEQ ID NO:26 with up to three amino acid substitutions;

[0428] (d) VL-CDR1 comprising SEQ ID NO:32 with up to three amino acid substitutions, VL-CDR2 comprising SEQ ID NO:33 with up to two amino acid substitutions, and VL-CDR3 comprising SEQ ID NO:34 with up to two amino acid substitutions;

[0429] (e) VL-CDR1 comprising SEQ ID NO:40 with up to three amino acid substitutions, VL-CDR2 comprising SEQ ID NO:41 with up to two amino acid substitutions, and VL-CDR3 comprising SEQ ID NO:42 with up to two amino acid substitutions; or

[0430] (f) VL-CDR1 comprising SEQ ID NO:48 with up to three amino acid substitutions, VL-CDR2 comprising SEQ ID NO:49 with up to two amino acid substitutions, and VL-CDR3 comprising SEQ ID NO:50 with up to two amino acid substitutions.

[0431] 10. The method or use according to claim Error! Reference source not found., wherein:

[0432] (i) the amino acid sequence of the VH comprises: VH-CDR1 comprising SEQ ID NO:5 with up to two amino acid substitutions, VH-CDR2 comprising SEQ ID NO:6 with up to four amino acid substitutions, and VH-CDR3 comprising SEQ ID NO:7 with up to three amino acid substitutions; and

[0433] (ii) the amino acid sequence of the VL comprises:VL-CDRl comprising SEQ ID NO:8 with up to three amino acid substitutions, VL-CDR2 comprising SEQ ID NO:9 with up to two amino acid substitutions, and VL-CDR3 comprising SEQ ID NO: 10 with up to three amino acid substitutions.

[0434] 11. The method or use according to claim Error! Reference source not found, or claim Error! Reference source not found., wherein the total number of substitutions in the VH CDRS comprises up to six amino acid substitutions, and the total number of substitutions in the VL CDRS comprises up to six amino acid substitutions. 12. The method or use according to any one of claims Error! Reference source not found, to Error! Reference source not found., wherein the VH comprises the amino acid sequence corresponding to any one of SEQ ID NOs:ll, 19, 27, 35, 43, or 51; and the VL comprises the amino acid sequence of any one of SEQ ID NOs:12, 20, 28, 36, 44, or 52; and wherein the VH comprises up to six amino acid substitutions and the VL comprises up to six amino acid substitutions.

[0435] 13. The method or use according to claim Error! Reference source not found., wherein the VH comprises the amino acid sequence corresponding to SEQ ID NO: 11 and the VL comprises the amino acid sequence corresponding to SEQ ID NO: 12; wherein the VH comprises up to six amino acid substitutions and the VL comprises up to six amino acid substitutions.

[0436] 14. The method or use according to claim Error! Reference source not found., wherein:

[0437] (i) the amino acid sequence of: VH-CDR1 corresponds to SEQ ID NO:5, VH- CDR2 corresponds to SEQ ID NO:6, and VH-CDR3 corresponds to SEQ ID NO:7; and

[0438] (ii) the amino acid sequence of: VL-CDR1 corresponds to SEQ ID NO:8, VL- CDR2 corresponds to SEQ ID NO:9, and VL-CDR3 corresponds to SEQ ID NO:10.

[0439] 15. The method or use according to claim Error! Reference source not found., wherein the VH comprises the amino acid sequence corresponding to SEQ ID NO: 11 and the VL comprises the amino acid sequence corresponding to SEQ ID NO: 12.

[0440] 16. The method or use according to any one of claims Error! Reference source not found, to Error! Reference source not found., wherein the VH and the VL are in a single polypeptide chain.

[0441] 17. The method or use according to claim Error! Reference source not found., wherein the MIF-binding protein is:

[0442] (i) a single chain Fv fragment (scFv);

[0443] (ii) a dimeric scFv (di-scFv); or (iii) at least one of (i) and / or (ii) linked to a Fc or a heavy chain constant domain (CH)2 and / or CH3.

[0444] 18. The method or use according to any one of claims Error! Reference source not found, to Error! Reference source not found., wherein the VH and the VL are in separate polypeptide chains.

[0445] 19. The method or use according to claim Error! Reference source not found., wherein MIF-binding protein is:

[0446] (i) a divalent antibody

[0447] (ii) a Fab’;

[0448] (iii) a diabody;

[0449] (iv) a triabody; or

[0450] (v) a tetrabody.

[0451] 20. The method according to claim 1 or the use according to claim Error! Reference source not found., wherein the agent that inhibits MIF-CD74 signaling comprises a CD74-targeting polynucleotide that reduces an expression level of CD74 in a cell population in the subject, whereby MIF-CD74 signaling is inhibited in the cell population.

[0452] 21. The method or use according to claim Error! Reference source not found., wherein the CD74-targeting polynucleotide is selected from the group consisting of: guide RNA (gRNA), an siRNA, a shRNA, and an antisense oligonucleotide (ASO).

[0453] 22. The method or use according to claim Error! Reference source not found., wherein the CD74-targeting polynucleotide is a gRNA.

[0454] 23. The method or use according to any one of claim 1 to Error! Reference source not found., wherein the route of administration or formulation of the medicament is intravenous, intrathecal, intra-arterial, intra-cerebral, intracerebroventricular, intranasal, or subcutaneous.

[0455] 24. The method according to any one of claims 1 to 23, wherein the subject is a human subject.

[0456] 25. The method or use according to any one of claims 1 to Error! Reference source not found., wherein the subject is a human subject. The present disclosure includes the following non-limiting Examples.

[0457] EXAMPLES

[0458] Materials and Methods

[0459] Culture of cortical organoids (COs)

[0460] COs were grown in individual wells using a novel dorsal forebrain protocol derived from Velasco et al. to improve cortical fate, reliability, and embryoid body survival in round bottom plates. Briefly, iPSCs were thawed concurrently and passaged twice before seeding in Stemflex Media (ThermoFisher, A3349401). Prior to seeding, iPSCs were dissociated when 50-80% confluent and plated in U-bottom 96-well plates at 9x103 cells per well with ROCK inhibitor (Peprotech, 1293823). Organoids were cultured in neural induction media containing inhibitory molecules added to direct cortical fate via single-SMAD, Wnt signaling inhibition, as well as Shh inhibition. Organoids were fed on automated Biotek feeders and washers. On the fifth week of culture, organoids were switched to a DMEM-based maturation media with the addition of Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix (VWR, 47743-718). On the eighth week of culture, COs were fed maturation medium containing BDNF (Qkine, QK050) and GDNF (Peprotech, 450-10) with GFR Matrigel. Matrigel was omitted during the ninth week of culture. COs were maintained with automated feeding and imaging.

[0461] Live Microglial CRISPRi Amyloid Beta Phagocytosis Screen iPSC-derived Microglia containing dCas9-KRAB integrated at the CLYBL locus were thawed and plated in 96 well format at 50,000 cells / well. Microglia were transduced the following day with the appropriate lentiviral gRNA (Cellecta, n=30 total, listed below) at MOI of 2. After 4 days, microglia were replated into 384 well format at 8,000 cells / well. The following day cells were treated with Amyloid Beta fibrils conjugated to pHrodo STP Ester (Thermo), and promptly imaged every 30 minutes for 24 hours.

[0462] Guide RNA sequence selection

[0463] Guide RNA sequences were selected based on Horlbeck et. al., 2016, and purchased from Cellecta in the pRSGERPCS2h2-U6-2Bpi-HeatCS2h2-EFl-TagRFP-2A-Puro background. Sequences are listed below. Live fluorescence imaging of NICOs

[0464] NICOs or microglia imaged using ImageXpress Confocal (Molecular Devices) at 4x or lOx magnification under 37 °C and 5% CO2. Videos had images acquired every 30 minutes. Microglia were labeled either virally (Cellecta) or via CMTMR dye (Cayman).

[0465] Phagocytosis Score Quantification

[0466] To compare across treatment and dosage, the curve obtained for each individual well was transformed into a single score: AUC, area under the curve. We is the sum at each time point of the phagocytosis score PS = Abeta / Microglia, where, ABeta is the total area of detected ABeta engulfed by microglia, and microglia the total area covered by microglia in the image (either CMTMR dye or RFP. Then we compute SSMD of the AUC at each time interval, and use t-tests to derive statistical significance accounting for multiple comparisons across groups. scRNAseq Preparation

[0467] NICOs for each experimental parameter were transferred from their culture plates, pooled in a single tube, and washed once with PBS. PBS was removed and NICOs were incubated in TrypLE Express (Gibco, 12604-013) containing DNAse (1:20, Worthington Biochemical, LK003163) and Actinomycin D (1:1000, Sigma, A1410). NICOs were incubated for 20 minutes at 37 °C at 100 rpm on an orbital shaker followed by gentle manual dissociation to a single cell solution. 10% v / v FBS in ice cold PBS was added to the cell solution (1 :2). The cell solution was centrifuged at 300 g for 5 minutes followed by aspiration of enzyme solution. Cells were resuspended in ice cold PBS with 0.5% w / v bovine serum albumin (BSA) followed by filtration through a 40 pm mesh cell strainer. Removal of dead cells was done by centrifuging cell solution at 200 g for 3 minutes, aspirating solution, and resuspending cell clump in ice cold 0.5% w / v BSA in PBS solution. Centrifugation and resuspension was repeated five times. The cell pellets were resuspended in 100 pL solution of PBS with 0.5% w / v BSA and CDl lb microbeads (Miltenyi Biotec, 130-093-636) and incubated for 15 minutes at 4 °C. The cell solutions were further diluted in 0.5% w / v BSA in PBS and filtered through Miltenyi MS Columns (130-042-201) on a Miltenyi OctoMacs Magnetic Separation Unit (130-042-109). The collected negative (organoid cells) and positive (microglia cells) fractions were counted and stored on ice until loading onto a Chromium Controller. For microglia cultured in 2D with neurons, the same enzymatic dissociation and microglia isolation process was used for microglia collection for scRNAseq. Libraries were prepared according to the Next GEM Single Cell 3' Reagent Kits v3.1 protocol, and submitted to Novogene for sequencing. scRNAseq Analysis

[0468] Quality control filtering on the scRNAseq data was conducted using standard cell ranger pipelines. All downstream analysis was conducted using Python scRNAseq analysis package, scanpy. UMAP projections were projected into 2 dimensions using a 20-nearest neighbor distance metric and an effective minimum distance of 0.25. Dot plots and heat maps for gene expression represent mean log-transformed counts normalized by gene. Low expression genes, genes in which no cells exceeded normalized expression of 1.5 in at least one condition, were excluded in heatmaps comparing individual gene expression across groups. Geneset scores for heatmaps and violin plots were computed using the scanpy .tl.score_genes tool. The score is the average expression of a set of genes subtracted with the average expression of a reference set of genes. The reference set is randomly sampled from the gene pool for each binned expression value. Differential gene expression was conducted using the scanpy .tl.rank genes groups function, using the “t-test” method with an adjusted p- value cutoff of 0.05 and log2 fold change minimum of 0.2.

[0469] Example 1: CD74 Transcript Levels Are Knocked Down in Microglia Using gRNA Microglia were infected with lentiviral guide RNAs for CD74, ABB, INPP5D and PTK2B at various MOIs, and incubated for 3-5 days. Microglia were lysed and transcripts were quantified via qPCR. The results indicated that guide RNAs resulted in gene-specific inhibition of RNA levels as shown in heat map (FIG. 1). Notably, CD74 gRNA lentiviruses knock down specifically CD74 transcript levels.

[0470] Example 2: MIF-CD74 Interaction is a Critical Pathway in Microglial Function and Dysfunction

[0471] In one approach, Applicants performed heterozygous and homozygous knock-outs in neuroimmune cortical organoids (NICO). Gene expression was analyzed using scRNAseq. Results indicated that relative to control, CD74 was elevated relative to wild type (FIG. 2, left side). In another approach, a microglial live CRISPRi screen of 30 genes in induced pluripotent stem cells (iPSCs), identified CD74 transcriptional inhibition as the top target increasing Amyloid-P phagocytosis. Amyloid-P phagocytosis, was measured via pHrodo signal. Total overlap of pHrodo (Green) and Microglia (Red) (“phagocytosis score”) is divided by total microglial signal to account for differences in transduction efficiency. Phagocytosis scores are shown as line graphs (FIG. 2, right side bottom panel). Both approaches identified CD74 as a potential therapeutic target for neuromodulation.

[0472] Example 3: TREM2 Expression Levels in Microglia Correlate With Microglia Disease-Related Phenotype

[0473] TREM2-deficient microglia are deficient for disease associated microglia (DAM) markers and TREM2-deficient microglia express increased levels of homeostatic markers relative to wild-type control microglia (FIG. 3B). ISO represents isogenic expression, TREM2 HET represents heterozygous TREM2-KO, and TREM2 HOM represents homozygous TREM2-KO in microglia.

[0474] Using fluorescence imaging of labelled wildtype control microglia and TREM2 homozygous knockouts, we further demonstrated that TREM2-deficient microglia exhibited decreased migration in neuroimmune cortical organoids (NICOs) relative to wild-type control microglia in NICOs (FIG. 4A). Microglial density within cortical organoids where there is loss of one or both TREM2 alleles is consistent with TREM2 playing a role in microglial migration within the brain (FIG. 4B).

[0475] Example 4: Identification of MIF-CD74 Signalling as a Key Modulator of Microglial Response in Neuroinflammation-Driven CNS conditions

[0476] To show target validation of CD74, we developed a catalytically inactive Cas9 protein (dCas9) fused to the Krtippel-associated box (KRAB) repressor in neuroimmune organoids (dCAS9-KRAB NICOs) to study the effect of CD74 knockdown on homeostatic and DAM markers as well as microglial motility within organoids (FIG. 5A).

[0477] The results showed that CD74 knockdown modulates homeostatic markers, specifically lowering expression of P2RY12 receptor (FIG. 5B, upper panel), and increasing expression of DAM inflammatory marker, secreted phosphoprotein 1 (SPP1) (FIG. 5B, lower panel). Results also showed that the dCAS9-KRAB mediated knockdown of CD74 in microglia induced increased microglial motility within the cortical organoids (FIG. 5C, upper and lower panels).

[0478] Example 5: Imalumab Increases Amyloid-0 phagocytosis in Microglia Having identified the role of CD74 on DAM inflammatory marker expression and microglial motility within cortical organoids, we decided to explore the role of MIF- CD74 signalling on microglia activity in neuroimmune cortical organoids (NICOs).

[0479] The Applicants examined the effects anti-MIF antibodies on Amyloid-P phagocytosis. - A Donanemab biosimilar was compared to Imalumab, and an IgGl isotype antibody was used as a control. The results showed that Imalumab is much more effective in increasing Amyloid-P phagocytosis than was Donanemab (FIG. 6A). Additionally, it was noted that Imalumab increased Amyloid-P phagocytosis in a dose-dependent manner. Doses of 0.4, 2, 10 and 50pg / mL were provided. (FIG. 6B, p values are displayed).

[0480] Following experiments on the MIF component of the MIF-CD74 complex, Applicants examined the effects of anti-CD74 antibody Milatuzumab and MIF-targeting small molecule BTZO-1 on Amyloid-P phagocytosis. Surprisingly, neither anti-CD74 antibody Milatuzumab nor MIF-targeting small molecule BTZO-1 increased Amyloid- P phagocytosis in a dose dependent study, Milatuzumab: 0.4 - 50 pg / mL, BTZO-1: .05- 5 pM (FIG.7A and FIG. 7B, respectively).

[0481] Additionally, various anti-CD74, anti-MIF, CD74-targeting, and MIF targeting small molecules were assessed for effect on Amyloid-P phagocytosis at varying doses. See FIG. 8A; the full list of agents listed on the x-axis from left to right is provided in Appendix 2. The results showed that Imalumab is highly effective in increasing Amyloid-P phagocytosis as compared to the other agents tested. FIG. 8B confirms that Imalumab is much more effective than Donanemab in increasing Amyloid-P phagocytosis.

[0482] It will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0483] All publications cited herein are hereby incorporated by reference in their entirety. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

[0484] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

[0485] WIGPSGGFTFYADSVKG

[0486] SEQ ID NO:23

[0487] BAX74 VH-CDR3

[0488] GTPDYGGNSLDH

[0489] SEQ ID NO:24

[0490] BAX74 VL-CDR1

[0491] RASQSIGTYLS

[0492] SEQ ID NO:25

[0493] BAX74 VL-CDR2

[0494] ATSRLQS

[0495] SEQ ID NO:26

[0496] BAX74 VL-CDR3

[0497] QQTYSTPLT

[0498] SEQ ID NO:27

[0499] BAX74 VH full sequence

[0500] EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYYMIWVRQAPGKGLEWVSWIG

[0501] PSGGFTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGTPDYGG

[0502] NSLDHWGQTLVTVSS

[0503] SEQ ID NO:28

[0504] BAX74 VL full sequence

[0505] DIQMTQSPSSLPASVGDRVTITCRASQSIGTYLSWYQHKPGNAPKLLIYATSRL

[0506] QSGVPSRFSGGGSGTRFTLAISSLQPDDFATYFCQQTYSTPLTFGGGTKVDIK

[0507] SEQ ID NO:29

[0508] BAX94 VH-CDR1

[0509] IYAMD

[0510] SEQ ID NO:30

[0511] BAX94 VH-CDR2

[0512] GIVPSGGFTKYADSVKG

[0513] SEQ ID NO:31

[0514] BAX94 VH-CDR3

[0515] VNVIAVAGTGYYYYGMDV SEQ ID NO:32

[0516] BAX94 VL-CDR1 RASQGVSSSSLA

[0517] SEQ ID NO:33

[0518] BAX94 VL-CDR2

[0519] GTSSRAT

[0520] SEQ ID NO:34

[0521] BAX94 VL-CDR3

[0522] QQYGRSLT

[0523] SEQ ID NO:35

[0524] BAX94 VH full sequence

[0525] EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYAMDWVRQAPGKGLEWVSGIVP

[0526] SGGFTKYADSVKGRFTISRDNSKNTLYQMNSLRAEDTAVYYCARVNVIAVAG

[0527] TGYYYYGMDWGQGTTVTVSS

[0528] SEQ ID NO:36

[0529] BAX94 VL full sequence

[0530] DIQMTQSPGTLSLSPGERATLSCRASQGVSSSSLAWYQQKPGQAPRLLIYGTSS

[0531] RATGIPDRFSGSASGTDFTLTISRLQPEDFAVYYCQQYGRSLTFGGGTVEIK

[0532] SEQ ID NO:37

[0533] BAX152 VH-CDR1

[0534] IYAMD

[0535] SEQ ID NO:38

[0536] BAX152 VH-CDR2

[0537] GIVPSGGFTKYADSVKG

[0538] SEQ ID NO:39

[0539] BAX152 VH-CDR3

[0540] VNVIAVAGTGYYYYGMDV

[0541] SEQ ID NO:40

[0542] BAX152 VL-CDR1

[0543] RASQSVRSSYLA SEQ ID NO:41

[0544] BAX152 VL-CDR2 GASNRAT

[0545] SEQ ID NO:42

[0546] BAX152 VL-CDR3

[0547] QQYGNSLT

[0548] SEQ ID NO:43

[0549] BAX 152 VH full sequence

[0550] EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYAMDWVRQAPGKGLEWVSGIVP

[0551] SGGFTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVNVIAVA

[0552] GTGYYYYGMDVWGQTTVTVSS

[0553] SEQ ID NO:44

[0554] BAX 152 VL full sequence

[0555] DIQMTQSPVTLSLSPGERATLSCRASQSVRSSYLAWYQQKPGQTPRLLIYGASN

[0556] RATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGNSLTFGGGTKVEIK

[0557] SEQ ID NO:45

[0558] BAXA10 VH-CDR1

[0559] WYAMD

[0560] SEQ ID NO:46

[0561] BAXA10 VH-CDR2

[0562] GIYPSGGRTKYADSVKG

[0563] SEQ ID NO:47

[0564] BAXA10 VH-CDR3

[0565] VNVIAVAGTGYYYYGMDV

[0566] SEQ ID NO:48

[0567] BAXA10 VL-CDR1

[0568] RASQGVSSSSLA

[0569] SEQ ID NO:49

[0570] BAXA10 VL-CDR2

[0571] GTSSRAT

[0572] Appendix 2: Agents tested |ig / ml Milatuzumab lOpig / ml Milatuzumab 5μM BTZO-1

[0573] EQUIVALENTS AND INCORPORATION BY REFERENCE

[0574] All references cited herein are incorporated by reference to the same extent as if each individual publication, database entry (e.g., Genbank sequences or GenelD entries), patent application, or patent, was specifically and individually indicated incorporated by reference in its entirety, for all purposes. This statement of incorporation by reference is intended by Applicants, pursuant to 37 C.F.R. § 1.57(b)(1), to relate to each and every individual publication, database entry (e.g., Genbank sequences or GenelD entries), patent application, or patent, each of which is clearly identified in compliance with 37 C.F.R. § 1.57(b)(2), even if such citation is not immediately adjacent to a dedicated statement of incorporation by reference. The inclusion of dedicated statements of incorporation by reference, if any, within the specification does not in any way weaken this general statement of incorporation by reference. Citation of the references herein is not intended as an admission that the reference is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents.

[0575] While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it is understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.

Claims

WHAT IS CLAIMED1. A method for preventing, treating, or delaying progression of Alzheimer’s disease (AD) or mild cognitive impairment (MCI) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an agent that inhibits and / or abrogates macrophage migration inhibitory factor (MIF)- CD74 signalling.

2. The method of claim 1, wherein the agent that inhibits MIF-CD74 signalling comprises a MIF-binding protein.

3. The method of claim 2, wherein the MIF-binding protein comprises at least one immunoglobulin variable region that binds specifically to MIF.

4. The method of claim 3, wherein the MIF-binding protein comprises an antibody or antigen-binding fragment thereof.

5. The method of claim 4, wherein the MIF-binding protein comprises a nanobody.

6. The method of claim 5, wherein the nanobody is humanized.

7. The method of claim 4, wherein the antibody is an IgGl, IgG2, or IgG4 antibody or antigen binding fragment thereof.

8. The method of claim 7, wherein the antibody is an IgGl antibody or antigen binding fragment thereof.

9. The method of claim 7 or 8, wherein the antibody is a humanized antibody or antigen binding fragment thereof.

10. The method of claim 9, wherein the humanized antibody is a human chimeric antibody or antigen binding fragment thereof.

11. The method of any one of claims 4-10, wherein the antibody or antigen-binding fragment thereof comprises three heavy chain variable regions (VH) CDRs and three light chain variable region (VL) CDRs, wherein:(i) the three VH CDRs respectively have the sequences of:(a) VH-CDR1 comprising SEQ ID NO:5 with up to two amino acid substitutions, VH-CDR2 comprising SEQ ID NO:6 with up to four amino acid substitutions, and VH-CDR3 comprising SEQ ID NO:7 with up to three amino acid substitutions;(b) VH-CDR1 comprising SEQ ID NO: 13 with up to two amino acid substitutions, VH-CDR2 comprising SEQ ID NO: 14 with up to four amino acid substitutions, and VH-CDR3 comprising SEQ ID NO: 15 with up to four amino acid substitutions;(c) VH-CDR1 comprising SEQ ID NO:21 with up to two amino acid substitutions, VH-CDR2 comprising SEQ ID NO:22 with up to four amino acid substitutions, and VH-CDR3 comprising SEQ ID NO:23 with up to three amino acid substitutions;(d) VH-CDR1 comprising SEQ ID NO:29 with up to two amino acid substitutions, VH-CDR2 comprising SEQ ID NO:30 with up to four amino acid substitutions, and VH-CDR3 comprising SEQ ID NO:31 with up to four amino acid substitutions;(e) VH-CDR1 comprising SEQ ID NO:37 with up to two amino acid substitutions, VH-CDR2 comprising SEQ ID NO:38 with up to four amino acid substitutions, and VH-CDR3 comprising SEQ ID NO:39 with up to four amino acid substitutions; or(f) VH-CDR1 comprising SEQ ID NO:45 with up to two amino acid substitutions, VH-CDR2 comprising SEQ ID NO:46 with up to four amino acid substitutions, and VH-CDR3 comprising SEQ ID NO:47 with up to four amino acid substitutions; and(ii) the three VL CDRs respectively have the sequences of:(a) VL-CDR1 comprising SEQ ID NO:8 with up to three amino acid substitutions, VL-CDR2 comprising SEQ ID NO:9 with up to two amino acid substitutions, and VL-CDR3 comprising SEQ ID NO: 10 with up to three amino acid substitutions;(b) VL-CDR1 comprising SEQ ID NO: 16 with up to three amino acid substitutions, VL-CDR2 comprising SEQ ID NO:17 with up to two amino acidsubstitutions, and VL-CDR3 comprising SEQ ID NO: 18 with up to three amino acid substitutions;(c) VL-CDR1 comprising SEQ ID NO:24 with up to three amino acid substitutions, VL-CDR2 comprising SEQ ID NO:25 with up to two amino acid substitutions, and VL-CDR3 comprising SEQ ID NO:26 with up to three amino acid substitutions;(d) VL-CDR1 comprising SEQ ID NO:32 with up to three amino acid substitutions, VL-CDR2 comprising SEQ ID NO:33 with up to two amino acid substitutions, and VL-CDR3 comprising SEQ ID NO:34 with up to two amino acid substitutions;(e) VL-CDR1 comprising SEQ ID NO:40 with up to three amino acid substitutions, VL-CDR2 comprising SEQ ID NO:41 with up to two amino acid substitutions, and VL-CDR3 comprising SEQ ID NO:42 with up to two amino acid substitutions; or(f) VL-CDR1 comprising SEQ ID NO:48 with up to three amino acid substitutions, VL-CDR2 comprising SEQ ID NO:49 with up to two amino acid substitutions, and VL-CDR3 comprising SEQ ID NO:50 with up to two amino acid substitutions.

12. The method of claim 11, wherein the antibody or antigen-binding fragment thereof comprises three heavy chain variable regions (VH) CDRs and three light chain variable region (VL) CDRs, wherein:(i) the three VH CDRs respectively have the sequences of:(a) VH-CDR1 comprising SEQ ID NO:5, VH-CDR2 comprising SEQ ID NO:6, and VH-CDR3 comprising SEQ ID NO:7;(b) VH-CDR1 comprising SEQ ID NO: 13, VH-CDR2 comprising SEQ ID NO: 14, and VH-CDR3 comprising SEQ ID NO: 15;(c) VH-CDR1 comprising SEQ ID NO:21, VH-CDR2 comprising SEQ ID NO:22, and VH-CDR3 comprising SEQ ID NO:23;(d) VH-CDR1 comprising SEQ ID NO:29, VH-CDR2 comprising SEQ ID NO:30, and VH-CDR3 comprising SEQ ID NO:31;(e) VH-CDR1 comprising SEQ ID NO:37, VH-CDR2 comprising SEQ ID NO:38, and VH-CDR3 comprising SEQ ID NO:39; or(f) VH-CDR1 comprising SEQ ID NO:45, VH-CDR2 comprising SEQ ID NO:46, and VH-CDR3 comprising SEQ ID NO:47; and(ii) the three VL CDRs respectively have the sequences of:(a) VL-CDR1 comprising SEQ ID NO:8, VL-CDR2 comprising SEQ ID NO:9, and VL-CDR3 comprising SEQ ID NO: 10;(b) VL-CDR1 comprising SEQ ID NO: 16, VL-CDR2 comprising SEQ ID NO:17, and VL-CDR3 comprising SEQ ID NO:18;(c) VL-CDR1 comprising SEQ ID NO:24, VL-CDR2 comprising SEQ ID NO:25, and VL-CDR3 comprising SEQ ID NO:26;(d) VL-CDR1 comprising SEQ ID NO:32, VL-CDR2 comprising SEQ ID NO:33, and VL-CDR3 comprising SEQ ID NO:34;(e) VL-CDR1 comprising SEQ ID NO:40, VL-CDR2 comprising SEQ ID NO:41, and VL-CDR3 comprising SEQ ID NO:42; or(f) VL-CDR1 comprising SEQ ID NO:48, VL-CDR2 comprising SEQ ID NO:49, and VL-CDR3 comprising SEQ ID NO:50.

13. The method of claim 12, wherein the antibody or antigen-binding fragment thereof comprises three VH CDRs and three VL CDRs:(i) the three VH CDRs respectively have the sequences of: VH-CDR1 comprising SEQ ID NO:5 with up to two amino acid substitutions, VH-CDR2 comprising SEQ ID NO:6 with up to four amino acid substitutions, and VH-CDR3 comprising SEQ ID NO: 7 with up to three amino acid substitutions; and(ii) the three VL CDRs respectively have the sequences of: VL-CDR1 comprising SEQ ID NO:8 with up to three amino acid substitutions, VL-CDR2 comprising SEQ ID NO:9 with up to two amino acid substitutions, and VL-CDR3 comprising SEQ ID NO: 10 with up to three amino acid substitutions.

14. The method of claim 13, wherein the antibody or antigen-binding fragment thereof comprises three VH CDRs and three VL CDRs:(i) the three VH CDRs respectively have the sequences of: VH-CDR1 comprising SEQ ID NO:5, VH-CDR2 comprising SEQ ID NO:6, and VH-CDR3 comprising SEQ ID NO: 7; and(ii) the three VL CDRs respectively have the sequences of: VL-CDR1 comprising SEQ ID NO:8, VL-CDR2 comprising SEQ ID NO:9, and VL-CDR3 comprising SEQ ID NO: 10.

15. The method of any one of claims 11-14, wherein the VH of the antibody or antigen-binding fragment thereof comprises the amino acid sequence corresponding to any one of SEQ ID NOs:ll, 19, 27, 35, 43, or 51; and the VL comprises the amino acid sequence of any one of SEQ ID NOs:12, 20, 28, 36, 44, or 52; and optionally wherein the VH comprises up to six amino acid substitutions and the VL comprises up to six amino acid substitutions.

16. The method of any one of claims 11-15, wherein the antibody or the antigenbinding fragment thereof is a divalent antibody, bispecific antibody, a F(ab), a Fab’, a F(ab)’2, a diabody, a triabody, a tetrabody, a single chain Fv fragment (scFv), or a dimeric scFv (di-scFv).

17. The method of any one of claims 11-16, wherein the antibody or antigen binding fragment thereof is a Fab, optionally wherein the Fab is PEGylated.

18. The method of any one of claims 11-17, wherein the antibody or antigen binding fragment thereof is a Fv, optionally wherein the variable chains have been cross-linked, optionally wherein the cross-linkage is performed through glutaraldehyde, intermolecular disulfides or a peptide linker.

19. The method of any one of claims 11-18, wherein the antibody or antigen binding fragment thereof is a full-length monoclonal antibody.

20. The method of any one of claims 11-19, wherein the antibody or antigen binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen binding fragment thereof is bispecific.

21. The method of any one of claims 11 -20, wherein the antibody or antigen binding fragment thereof comprises an Fc region.

22. The method of claim 21, wherein the Fc has engineered mutations that reduce antibody binding to FcyR and / or reduce complement fixation.

23. The method of any one of claims 4-22, wherein the antibody or antigen-binding fragment thereof is formulated as a pharmaceutical composition comprising the antibody or antigen binding fragment thereof and a pharmaceutically acceptable carrier.

24. The method of claim 23, wherein the pharmaceutical composition is formulated for parenteral administration.

25. The method of claim 24, wherein the pharmaceutical composition is formulated for intravenous administration.

26. The method of claim 25, wherein the pharmaceutical composition is formulated for subcutaneous administration.

27. The method of any one of claims 4-26, wherein the antibody or antigen binding fragment thereof is administered for at least 3 months.

28. The method of claim 27, wherein the antibody or antigen binding fragment thereof is administered for at least 6 months.

29. The method of claim 28, wherein the antibody or antigen binding fragment thereof is administered for at least 12 months.

30. The method of any one of claims 1-29, wherein the agent that inhibits MIF- CD74 signalling is formulated for co-administration with a glucocorticoid.

31. The method of claim 30, wherein the glucocorticoid is formulated as a pharmaceutical composition comprising the glucocorticoid and a pharmaceutically acceptable carrier.

32. The method of claim 30 or 31, wherein the agent that inhibits MIF-CD74 signalling and the glucocorticoid are co-administered as a fixed dose combination.

33. The method of any one of claims 1-32, wherein MIF is oxidized-MIF (ox-MIF).

34. The method of claim 1, wherein the agent that inhibits MIF-CD74 signalling comprises a CD74-targeting polynucleotide that reduces an expression level of CD74 in a cell population in the subject, whereby MIF-CD74 signalling is inhibited in the cell population.

35. The method of claim 34, wherein the CD74-targeting polynucleotide is selected from the group consisting of: guide RNA (gRNA), an siRNA, a shRNA, and an antisense oligonucleotide (ASO).

36. The method of claim 35, wherein the CD74-targeting polynucleotide is a gRNA.

37. The method of any one of claims 1-36, wherein the subject is a human subject.

38. The method of any one of claims 1-37, wherein the subject has AD.

39. The method of any one of claims 1-38, wherein the subject has MCI.

40. A method for preventing, treating, or delaying progression of schizophrenia in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an agent that inhibits and / or abrogates macrophage migration inhibitory factor (MIF)-CD74 signalling.

41. The method of claim 40, wherein the subject has schizophrenia.

42. The method of claim 40 or 41, wherein the agent is any one of the agents from claims 1-the agent of any one of claims 1-35.

43. A method for preventing, treating, or delaying progression of anxiety associated with brain disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an agent that inhibits and / or abrogates macrophage migration inhibitory factor (MIF)-CD74 signalling.

44. The method of claim 43, wherein the subject has anxiety associated with brain disease.

45. The method of claim 43 or claim 44, wherein the brain disease is selected from AD, Parkinson’s disease, multiple sclerosis, and amyotrophic lateral sclerosis (ALS), frontotemporal dementia, Huntington’s disease, and Lewy body dementia.

46. The method of any one of claims 43-45, wherein the agent is any one of the agents from claims 1-the agent of any one of claims 1-35.

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