Immunoglobulin single variable domains directed against CD74 and uses derived thereof

FIELD OF THE INVENTION

The invention relates to immunoglobulin single variable domains directed against CD74. Furthermore, the invention relates to nucleic acids encoding such domains and to host cells expressing or capable of expressing such domains. Also encompassed are compositions, in particular pharmaceutical compositions, comprising such domains. The immunoglobulin single variable domains and compositions of the invention can be used for therapeutic, prophylactic or diagnostic purposes. Specific applications include the use of immunoglobulin single variable domains directed against CD74 for the prevention and/or treatment of inflammatory diseases, including cancer, as well as for detecting, monitoring and/or diagnosing a particular disease in the field of inflammation.

BACKGROUND

Cytokines play a critical role in the immune system as they are responsible for initiating the host inflammatory immune response (i.e. the recruitment and activation of leukocytes and plasma proteins to the site of infection) and coordinating the cellular and humoral responses against invading organisms or molecules (Abbas and Lichtman, 2003). While they are essential for the control of an invader, and are usually self-limiting, persistence and/or deregulation of pro-inflammatory cytokines can culminate into major problems for the host, leading to a wide variety of chronic inflammatory diseases, tissue damage and in some instances even death. Many inflammation-associated diseases occur commonly in developed countries and treatment of these diseases is usually non-curative and is aimed at suppressing inflammatory end-organ damage. Hereby, multiple clinical studies have indicated that macrophage migration inhibitory factor (MIF) is a key culprit in initiating and prolonging the inflammatory status (reviewed in Calandra and Roger 2003).

MIF is a pro-inflammatory cytokine that has gained substantial attention as a protein that is able to sustain inflammatory responses, thus playing a key role in inflammation-associated disease processes including autoimmune diseases (rheumatoid arthritis, atherosclerosis, asthma, inflammatory bowel and Crohn's disease and Alzheimer's disease), metabolic disorders (diabetes and obesity), systemic infections (inflammation-associated anemia (i.e. ACD)) as well as sepsis and cancer, despite the presence of anti-inflammatory agents (Calandra and Roger 2003; Flaster et al., 2007). It is produced by a variety of cells and tissues in the body. Monocytes, macrophages, dendritic cells, neutrophils, mast cells, basophils, eosinophils, and epithelial surfaces are all documented to be able to produce MI F upon stimulation of these cells by an antigen (Calandra and Roger, 2003; Daryadel et al., 2006).

As reviewed in Morand et al. 2006, the molecular mode of action of MI F involves extracellular, receptor-mediated signaling pathways as well as intracellular interactions. Extracellular MI F interacts with the cell surface receptor CD74. Notably, CD74 not only is a cell surface receptor for MI F, it is known as a trans-membrane protein which, on antigen presenting cells, is the invariant chain that plays a role in the assembly and trafficking of M HC class I I molecules from the endoplasmic reticulum to the cell surface (Borghese and Clanchy, 2011). It is found on antigen presenting cells, is upregulated in several cancers and is also expressed by non-immune cells during inflammation. The complete signaling mechanism is not well understood, however, it is known that upon interaction with CD74, MI F invokes the release of the cytoplasmic portion of CD74 hereby activating N FKB (Borghese and Clanchy, 2011; Leng and Bucala, 2006). Furthermore, the (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) pathways are also activated. This ultimately leads to the production of Cyclin Dl (plays a role in proliferation and the cell cycle) and ETS/AP1 (involved in the gene expression of TLR4, CAMs (cell surface adhesion molecules), and inflammatory molecules including but not limited to: TN F, IFN-γ, I L-2, I L-6, I L-8 and MI F itself) (Morand et al., 2006; Calandra and Roger, 2003). In addition to this, the MI F/CD74 interaction leads to the activation of cytosolic phospholipase A ₂ , production of arachidonic acid and activation of cyclooxygenase 2. This combined leads to the down regulation of p53, which in combination with M IF's anti-oxidative properties leads to inhibition of apoptosis (Morand et al., 2006). Recently it was found that another ligand D-dopachrome tautomerase (D-DT) binds CD74. D-DT has very close genetic, functional and structural homology with MI F and is now also referred to as MI F-2 (Merck et al. 2011). Like M I F, it is present in most tissues and exists in pre-formed pools, it is released upon stimulation and circulates in serum and also binds to the receptor complex CD74/CD44, leading to a similar signal transduction cascade as MI F. Given the great facet of disease process in which M I F has been found to play a key role, MI F is an attractive therapeutic target to alleviate pathogenesis of several of these processes. Strategies for blocking MIF-mediated effects include specific anti-MI F antibodies, as well as molecules that disrupt the MI F/CD74 interaction by blocking access to the receptor either by modification of the receptor or MI F.

One of the most successful MI F inhibitors demonstrated in literature to date is the synthetic molecule ISO-1 (also known as (S, ?)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester). As described by Al-Abed et al., 2005, ISO-1 was found to prevent the interaction of MI F with its CD74 receptor by binding to the catalytically active site within the MIF molecule. In addition, ISO-1 was shown to be a potent inhibitor of MIF tautomerase activity (which was also shown to be of importance for MIF's biological activity). As a result, blocking the MIF-CD74 interaction by ISO-1 was found to be beneficial in several inflammatory diseases including but not limited to chronic asthma, systemic lupus erythematosus, colon carcinoma and sepsis (Chen et al., 2010; Leng et al., 2010; Zhang et al., 2009; Conroy et al., 2010).

To date, there exists only one immunotherapeutic agent targeting CD74 that is in clinical trial, known as Milatuzumab. It is a humanized form of a mouse monoclonal antibody that shows selective binding and rapid internalization on CD74 positive cancer cells. Ongoing clinical trials suggest that Milatuzumab has no adverse effects on patients, and when used in association with other factors such as chemotherapeutic agents it is beneficial for treatment of several lymphomas (Berkova et al., 2010; Borghese and Clanchy 2011; Govindan et al. 2013).

While there has been some success in the development of MIF blocking agents, the development of additional and/or novel molecules able to block the MIF/CD74 interaction or block the CD74 molecule will be of added value for treatment of several inflammatory diseases.

SUMMARY OF THE INVENTION

The present invention provides novel molecules that block the MIF/CD74 interaction by targeting the CD74 receptor. This strategy has several advantages since CD74 has also been identified as receptor not only for MIF, but also for a newly discovered member of the MIF family, D-DT, as well as an important receptor in the establishment of H. pylori infections. Targeting CD74 not only adds additional value to inhibiting classical MIF and circumvents the effects mediated through D-DT, it also allows to interfere with pathologies associated with CD74 that are not directly related to MIF. It might thus be beneficial for treatment of other CD74-mediated processes.

Thus, in a first aspect, the invention relates to an immunoglobulin single variable domain that is directed against and/or that specifically binds to human CD74 (SEQ ID NO: 1) and/or mouse CD74 (SEQ ID NO: 4). According to a particular embodiment, the immunoglobulin single variable domain of the invention inhibits MIF and D-DT from binding to CD74.

Generally, the immunoglobulin single variable domain of the invention comprises an amino acid sequence that comprises 4 framework regions (F ) and 3 complementarity determining regions (CDR) according to the following formula (1): F 1-CD 1-F 2-CD 2-F 3-CD 3-F 4 (1);

or any suitable fragment thereof.

In particular, the immunoglobulin single variable domain of the invention comprises an amino acid sequence that comprises 4 framework regions (FR1 to FR4) and 3 complementarity determining regions (CDR1 to CDR3), according to the following formula (1):

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1) and wherein CDR1 is chosen from the group consisting of: a) SEQ ID NOs: 37-45,

b) A polypeptide that has at least 80% amino acid identity with SEQ ID NOs: 37-45, c) A polypeptide that has 3, 2 or 1 amino acid difference with SEQ ID NOs: 37-45, and wherein CDR2 is chosen from the group consisting of: a) SEQ ID NOs: 55-63,

b) A polypeptide that has at least 80% amino acid identity with SEQ ID NOs: 55-63, c) A polypeptide that has 3, 2 or 1 amino acid difference with SEQ ID NOs: 55-63, and wherein CDR3 is chosen from the group consisting of: a) SEQ ID NOs: 73-81,

b) A polypeptide that has at least 80% amino acid identity with SEQ ID NOs: 73-81, c) A polypeptide that has 3, 2 or 1 amino acid difference with SEQ ID NOs: 73-81.

Preferably, the immunoglobulin single variable domain of the invention is a nanobody (V _H H). In particular, the nanobody has an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-21 or variants thereof.

According to further embodiments, the immunoglobulin single variable domain of the invention is comprised in a polypeptide or may be fused to a moiety, either directly or through a linker. The moiety may be a detectable label or a therapeutically active agent. The immunoglobulin single variable domain of the invention may also be immobilized on a solid support.

A further aspect of the invention relates to a complex comprising an immunoglobulin single variable domain of the invention. In particular, the complex may be crystalline. In other aspects, a nucleic acid sequence encoding an amino acid sequence of an immunoglobulin single variable domain of the invention is provided. Also envisaged is a recombinant vector comprising the nucleic acid sequence or a cell comprising the vector or the nucleic acid sequence.

The invention also encompasses a pharmaceutical composition comprising the immunoglobulin single variable domain of the invention and optionally, at least one of a pharmaceutically acceptable carrier, adjuvant or diluent.

In general, the immunoglobulin single variable domains as described herein can be used as a medicine. In particular, the immunoglobulin single variable domains as described herein are provided for use in the prevention and/or treatment of an inflammatory disease, including cancer. Alternatively or additionally, the immunoglobulin single variable domain as described herein may be useful for detecting a protein. According to one particular embodiment, the immunoglobulin single variable domains as described herein are provided for use in modulating CD74 receptor signaling.

Also envisaged is a method for producing an immunoglobulin single variable domain of the invention, said method comprising the steps of: - expressing, in a suitable expression system, a nucleic acid sequence encoding an immunoglobulin single variable domain of the invention, and optionally

- isolating and/or purifying said immunoglobulin single variable domain.

Further, the invention provides a kit comprising an immunoglobulin single variable domain as described herein and a buffer. Also envisaged is a solid support comprising an immunoglobulin single variable domain of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1. FACS profile of Nb_49 binding to CD74 on the surface of the THP-1 cell line. A represents the live gate on the total THP-1 cell population. B represents an overlay histogram comparing isotype control IgG (red) with a monoclonal antibody against CD74 (green). C represents an overlay comparing no staining (red) with an Alexa488 labeled irrelevant Nanobody (green) and Alexa488 labeled Nb_49 (blue). FIGURE 2. FACS profile of Nb_49 binding to CD74 on naive wild type (WT) C57Black/6 mouse PECs. A represents the gating on CDllb-positive cells (Myeloid cells) within the total PEC population. B represents an overlay comparing isotype control IgG (red) with a monoclonal antibody against CD74 (green). C represents an overlay comparing Alexa488 labeled irrelevant nanobody (red) with Alexa488 labeled Nb_49 (blue). This is a representative figure for 1 out of 3 independent experiments.

FIGURE 3. FACS profile of Nb_49 binding to CD74 on T. brucei brucei infected WT C57Black/6 mouse Pecs. A represents the gating on CDllb-positive cells (Myeloid cells) within the total PEC population. B represents an overlay comparing isotype control IgG (red) with a monoclonal antibody against CD74 (green). C represents an overlay comparing Alexa488 labeled irrelevant nanobody (red) with Alexa488 labeled Nb_49 (blue). This is a representative figure for 1 out of 3 independent experiments.

FIGURE 4. Representative FACS profile of MIF binding inhibition on PECs from naive WT C57Black/6 mice. A represents PECs without addition of anything. B represents PECs with the additon of APC- labeled MIF (200ng).

FIGURE 5. TNF-production by THP-1 cells following LPS stimulation under various treatment conditions. FIGURE 6. TNF-production by PECs from naive wild type (WT) (A), CD74 ^7" (B) and MIF ^7" C57Black/6 mice (C) following LPS stimulation under various treatment conditions.

FIGURE 7. Parasitemia (A), anemia (B), and survival (C) profiles for MIF ^7" , CD74 ^7" , and wild type (WT) C57black/6 mice infected with T. brucei brucei (AnTatl.lE).

FIGURE 8. Nb binding titration on recombinant human CD74 ^73"232 (first panel) and mouse CD74 ^56"215 (second panel)

FIGURE 9. Nb_49/MIF competition experiment via ELISA. A fixed concentration of MIF (200ng/well) was used. All OD values are minus the blank.

FIGURE 10. Nb_49 competition study for D-DT (left panel) and MIF (right panel) on Pecs from MIF ^7" mice via FACS. FIGURE 11. Expression of CD74 on MC-38 cells. Representative FACS profile showing binding of Nb_49 on MC-38 cells (first panel). Red line are cells alone, blue line are cells in presence of Alexa488 labeled Nb-BCIIlO and orange line represents cells in presence of Alexa488 labeled Nb_49. The median fluorescence intensity of the signal obtained with either cells alone (black bars), isotype control (Nb- BCII10, light grey bars) or Nb_49 (dark grey bars) (second panel). For each sample duplicates were used.

FIGURE 12. Nb internalization assessment via FACS using the Raji cell line. DETAILED DESCRIPTION OF THE INVENTION

The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

Unless otherwise defined herein, scientific and technical terms and phrases used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclatures used in connection with, and techniques of molecular and cellular biology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002).

As used herein, the terms "polypeptide", "protein", "peptide" are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. As used herein, the terms "nucleic acid molecule", "polynucleotide", "polynucleic acid", "nucleic acid" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three- dimensional structure, and may perform any function, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger NA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. The nucleic acid molecule may be linear or circular.

As used herein, the terms "determining," "measuring," "assessing," and "assaying" are used interchangeably and include both quantitative and qualitative determinations.

As used herein, the term "specifically recognizing" or "specifically binding to" or simply "specific for" refers to the ability of an immunoglobulin or an immunoglobulin fragment, such as an immunoglobulin single variable domain, to preferentially bind to a desirable antigen, in particular CD74 as defined herein, that is present in a homogeneous mixture of different antigens and does not necessarily imply high affinity (as defined further herein). In certain embodiments, a specific binding interaction will discriminate between desirable and undesirable antigens in a sample, in some embodiments more than about 10 to 100-fold or more (e.g., more than about 1000- or 10,000-fold). The terms "specifically bind", "selectively bind", "preferentially bind", and grammatical equivalents thereof, are used interchangeably herein. The term "affinity", as used herein, refers to the degree to which an immunoglobulin single variable domain, binds to an antigen so as to shift the equilibrium of antigen and immunoglobulin single variable domain toward the presence of a complex formed by their binding. Thus, for example, where an antigen and antibody (fragment) are combined in relatively equal concentration, an antibody (fragment) of high affinity will bind to the available antigen so as to shift the equilibrium toward high concentration of the resulting complex. The dissociation constant is commonly used to describe the affinity between the antibody (fragment) and the antigenic target. Typically, the dissociation constant is lower than 10 ^s M. Preferably, the dissociation constant is lower than 10 ^"6 M, more preferably, lower than 10 ^"7 M. Most preferably, the dissociation constant is lower than 10 ^s M.

An immunoglobulin single variable domain that can specifically bind to and/or that has affinity for a specific antigen or antigenic determinant (e.g. epitope) is said to be "against" or "directed against" said antigen or antigenic determinant. An immunoglobulin single variable domain according to the invention is said to be "cross-reactive" for two different antigens or antigenic determinants (such as CD74 from two different species of mammal, such as human CD74 and mouse CD74) if it is specific for both these different antigens or antigenic determinants.

A "deletion" is defined here as a change in either amino acid or nucleotide sequence in which one or more amino acid or nucleotide residues, respectively, are absent as compared to an amino acid sequence or nucleotide sequence of a parental polypeptide or nucleic acid. Within the context of a protein or a fragment thereof, a deletion can involve deletion of about 2, about 5, about 10, up to about 20, up to about 30 or up to about 50 or more amino acids. A protein or a fragment thereof may contain more than one deletion.

An "insertion" or "addition" is that change in an amino acid or nucleotide sequence which has resulted in the addition of one or more amino acid or nucleotide residues, respectively, as compared to an amino acid sequence or nucleotide sequence of a parental protein. "Insertion" generally refers to addition to one or more amino acid residues within an amino acid sequence of a polypeptide, while "addition" can be an insertion or refer to amino acid residues added at an N- or C-terminus, or both termini. Within the context of a protein or a fragment thereof, an insertion or addition is usually of about 1 , about 3, about 5, about 10, up to about 20, up to about 30 or up to about 50 or more amino acids. A protein or fragment thereof may contain more than one insertion.

A "substitution", as used herein, results from the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively as compared to an amino acid sequence or nucleotide sequence of a parental protein or a fragment thereof. It is understood that a protein or a fragment thereof may have conservative amino acid substitutions which have substantially no effect on the protein's activity. By conservative substitutions is intended combinations such as gly, ala; val, ile, leu, met; asp, glu; asn, gin; ser, thr; lys, arg; cys, met; and phe, tyr, trp.

The term "sequence identity" as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Determining the percentage of sequence identity can be done manually, or by making use of computer programs that are available in the art. The term "biologically active", with respect to CD74, refers to a CD74 having a biochemical function (e.g. a binding function, a signal transduction function, or an ability to change conformation as a result of ligand binding) of a naturally occurring CD74.

The terms "therapeutically effective amount", "therapeutically effective dose" and "effective amount", as used herein, mean the amount needed to achieve the desired result or results.

The term "pharmaceutically acceptable", as used herein, means a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. A first aspect of the present invention relates to an immunoglobulin single variable domain that is directed against and/or that specifically binds to CD74.

"CD74" (Cluster of Differentiation 74), as used herein, has been described in the art and is the cell surface form of a trans-membrane protein most commonly known as the invariant chain of the MHC-II complex or li (Leng et al., 2003) that plays a role in the assembly and trafficking of MHC class II molecules from the endoplasmic reticulum to the cell surface (Borghese and Clanchy, 2011). In addition, it also plays a key role in B-cell differentiation, DC motility, thymic selection and most importantly as a receptor for MIF (as defined further herein). While the crystal structure of CD74 has not been resolved, features of CD74 are well known in the art. Taking human CD74 (SEQ ID NO: 1) as a reference and non-limiting example, the CD74 molecule consists of a cytoplasmic region (AA 1-46), a transmembrane region (AA 47-72) while the remainder of the molecule is luminal/extracellular (AA 73- 296). Considering more closely the luminal/extracellular region, some key features include not only the MIF binding region corresponding to amino acid residues 109-149 but also a trimerization domain (AA 133-207). With respect to the intracellular form, this region is thought to be situated in the endosomal lumen allowing for interaction with three MHC-II heterodimers. On the cell surface however, due to the overlapping of the MIF binding region and the trimerization domain, it is thought to influence MIF binding (Borghese and Clanchy, 2011). CD74 is highly conserved amongst mammals. As a non-limiting example, mouse (UniProt P04441) and human (UniProt P04233) CD74 exhibit a sequence identity of about 72.3%, as can be easily measured in a BLASTp alignment.

Generally, the present invention provides for immunoglobulin single variable domains directed against and/or specifically binding to any CD74, in particular to a mammalian CD74. Thus, in particular, CD74 as referred to herein is of mammalian origin, particularly from mouse, rat, human, and the like. These cross-species variants of the CD74 protein are referred to herein as "orthologues" of CD74. Thus, CD74 as referred to in the present invention includes such orthologues. Non-limiting examples of orthologues of CD74 include mouse CD74 (Uniprot P04441, HG2A_MOUSE, and as in SEQ ID NO: 3) or human CD74 (Uniprot P04233, HG2A_HUMAN, and as in SEQ ID NO: 1). The present invention is in its broadest sense not particularly limited to or defined by a specific antigenic determinant, epitope, part, domain, subunit or conformation of CD74, and in particular of human CD74 (SEQ ID NO: 1) and/or mouse CD74 (SEQ ID NO: 4) against which the immunoglobulin single variable domains are directed. According to a specific embodiment, the present invention provides for immunoglobulin single variable domains directed against and/or specifically binding to a fragment of the full length CD74 protein, more specifically the extracellular domain of CD74, and in particular the extracellular domain of human CD74 (AA 73-296; SEQ ID NO: 6) and/or the extracellular domain of mouse CD74 (AA 56-279; SEQ ID NO: 7).

Generally, the immunoglobulin single variable domains of the invention will at least bind to those forms of CD74 proteins that are most relevant from a biological and/or therapeutic point of view, as will be clear to the skilled person. It will thus be understood that the immunoglobulin single variable domains of the invention are capable of binding CD74 in either a naturally occurring or non-naturally occurring (i.e., altered by man) form. The term "naturally-occurring", as used herein, means a CD74 protein that is naturally produced. In particular, wild type polymorphic variants and isoforms of CD74, as well as orthologues across different species are examples of naturally occurring proteins, and are found for example, and without limitation, in a mammal, more specifically in a human, or in a virus, or in a plant, or in an insect, amongst others). Thus, such CD74 proteins are found in nature. The term "non-naturally occurring", as used herein, means a CD74 protein that is not naturally-occurring. In certain circumstances, it may be advantageous that the CD74 protein is a non-naturally occurring protein. For example, to increase cellular expression levels of a recombinant CD74, one might consider introducing certain mutations in the CD74 protein of interest. Non-limiting examples of non-naturally occurring CD74 include, without limitation, CD74 with an N- and/or C-terminal deletion, CD74 with a substitution, an insertion or addition, or any combination thereof, in relation to its amino acid or nucleotide sequence, or other variants of naturally-occurring CD74. According to specific embodiments within the scope of the present invention, a non-naturally occurring CD74 protein may have an amino acid sequence that is at least 80% identical to, at least 90% identical to, at least 95% identical to, at least 97% identical to, or at least 99% identical to, a naturally-occurring CD74. Generally, it is expected that the immunoglobulin single variable domains according to the invention will generally bind to all naturally occurring or synthetic analogs, variants, mutants, alleles, parts, fragments, and isoforms of a particular CD74; or at least to those analogs, variants, mutants, alleles, parts, fragments, and isoforms of a particular CD74 that contain one or more antigenic determinants or epitopes that are essentially the same as the antigenic determinant(s) or epitope(s) to which the immunoglobulin single variable domains of the invention bind to a particular CD74.

It will be appreciated that, according to the invention, immunoglobulin single variable domains that are directed against CD74 from one species may or may not show cross-reactivity with CD74 from another species. For example, immunoglobulin single variable domains directed against human CD74, in particular human CD74 (SEQ ID NO: 1) may or may not show cross-reactivity with CD74 from one or more other species of animals that are often used in animal models for diseases (for example, mouse, rat, rabbit, pig or dog). It will be clear to the skilled person that such cross-reactivity, when present, may have advantages for diagnostic and/or therapeutic development, since it allows the immunoglobulin single variable domains to be tested in such disease models. Various methods may be used to determine specific binding (as defined hereinbefore) between the immunoglobulin single variable domain and CD74, including for example, enzyme linked immunosorbent assays (ELISA), surface Plasmon resonance assays, phage display, and the like, which are common practice in the art, for example, in discussed in Sambrook et al. (2001), Molecular Cloning, A Laboratory Manual. Third Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, and are further illustrated in the Example section. It will be appreciated that for this purpose often a unique label or tag will be used, such as a peptide label, a nucleic acid label, a chemical label, a fluorescent label, or a radio frequency tag, as described further herein.

According to a particular embodiment, the immunoglobulin single variable domains of the present invention can inhibit MIF from binding to CD74. More specifically, the immunoglobulin single variable domains of the present invention can specifically displace MIF from CD74, in particular from human CD74 (SEQ ID NO: 1) or mouse CD74 (SEQ ID NO: 4). As used herein, "MIF" or "macrophage migration inhibitory factor" is a pro-inflammatory cytokine and is well-known in the art. It is a natural ligand of the CD74 receptor. As an example, the amino acid sequence of human MIF is defined by UniProt P14174 (MIF_HUMAN; SEQ ID NO: 8). It was surprisingly found that the immunoglobulin single variable domains of the present invention can also inhibit D-DT (MIF-2) from binding to CD74. Thus, according to other embodiments, the immunoglobulin single variable domains of the present invention can inhibit D-DT (MIF-2) from binding to CD74. More specifically, the immunoglobulin single variable domains of the present invention can specifically displace D-DT (MIF-2) from CD74, in particular from human CD74 (SEQ ID NO: 1) or mouse CD74 (SEQ ID NO: 4). As used herein, "D-DT" or "D-dopachrome tautomerase", refers to another natural ligand (besides MIF) that binds the CD74 receptor. D-DT has very close genetic, functional and structural homology with MIF and is now also referred to as MIF-2 (Merck et al. 2011). As an example, the amino acid sequence of human MIF-2 (D-DT) is defined by UniProt Q53Y51 (Q53Y51_HUMAN; SEQ ID NO: 10).

According to preferred embodiments, the immunoglobulin single variable domains of the present invention can specifically displace MIF and D-DT (MIF-2) on CD74, with an average displacement of MIF or D-DT binding signal of at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or more. Thus, the average MIF or D-DT (MIF-2) displacement on CD74 is 40% or more, 50% or more, 60% or more, 70% or more, even more preferably, 80% or more, 90% or more. Percentages of average displacement can be determined in several ways, e.g. by a ligand displacement assay known in the art. A preferred way of measuring average displacement is by using flow cytometry, e.g. according to the FACS based competition assay as in Examples 7 and 8.

In general, immunoglobulin single variable domains as referred to in the present invention are meant to comprise a single amino acid chain that comprises 4 "framework sequences or regions" or F 's (termed FR1, FR2, FR3, FR4) and 3 "complementarity determining regions" or CDR's (termed CDR1, CDR2, CDR3), each non-contiguous with the others. The delineation of the CDR sequences (and thus also of the FR sequences) is based on the IMGT unique numbering system for V-domains and V-like domains (Lefranc et al. 2003). More specifically, immunoglobulin single variable domains are envisaged that comprise an amino acid sequence comprising 4 framework regions (FR1 to FR4) and 3 complementarity determining regions (CDR1 to CDR3), preferably according to the following formula (1): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1)

,or any suitable fragment thereof (which will then usually contain at least some of the amino acid residues that form at least one of the complementarity determining regions).

Immunoglobulin single variable domains comprising 4 FRs and 3 CDRs are known to the person skilled in the art and have been described, as a non-limiting example, in Wesolowski et al. (2009). Typical, but non-limiting, examples of immunoglobulin single variable domains include light chain variable domain sequences (e.g. a V _L domain sequence), or heavy chain variable domain sequences (e.g. a V _H domain sequence) which are usually derived from conventional four-chain antibodies. Preferably, the immunoglobulin single variable domains are derived from camelid antibodies, preferably from heavy chain camelid antibodies, devoid of light chains, and are known as V _H H domain sequences or Nanobodies (as described further herein).

The term "Nanobody" (Nb), as used herein, refers to the smallest antigen binding fragment or single variable domain (V _H H) derived from naturally occurring heavy chain antibody and is known to the person skilled in the art. They are derived from heavy chain only antibodies, seen in camelids (Hamers- Casterman et al. 1993; Desmyter et al. 1996). In the family of "camelids" immunoglobulins devoid of light polypeptide chains are found. "Camelids" comprise old world camelids (Camelus bactrianus and Camelus dromedarius) and new world camelids (for example Lama paccos, Lama glama, Lama guanicoe and Lama vicugna). Said single variable domain heavy chain antibody is herein designated as a Nanobody or a V _H H antibody. Nanobody™ and Nanobodies™ are trademarks of Ablynx NV (Belgium). The small size and unique biophysical properties of Nbs excel conventional antibody fragments for the recognition of uncommon or hidden epitopes and for binding into cavities or active sites of protein targets. Further, Nbs can be designed as multispecific and/or multivalent antibodies or attached to reporter molecules (Conrath et al. 2001). Nbs are stable and rigid single domain proteins that can easily be manufactured and survive the gastro-intestinal system. Therefore, Nbs can be used in many applications including drug discovery and therapy (Saerens et al. 2008) but also as a versatile and valuable tool for purification, functional study and crystallization of proteins (Conrath et al. 2009).

Thus, the immunoglobulin single variable domains of the invention, in particular the Nanobodies of the invention, generally comprise a single amino acid chain that typically comprises 4 "framework sequences" or F 's and 3 "complementarity determining regions" or CDR's according to formula (1)

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1).

The term "complementarity determining region" or "CDR" refers to variable regions in immunoglobulin single variable domains and contains the amino acid sequences capable of specifically binding to antigenic targets. These CDR regions account for the basic specificity of the nanobody for a particular antigenic determinant structure. Such regions are also referred to as "hypervariable regions." The immunoglobulin single variable domains have 3 CDR regions, each non-contiguous with the others (termed CDR1, CDR2, CDR3). It should be clear that framework regions of immunoglobulin single variable domains may also contribute to the binding of their antigens (Desmyter et al 2002; Korotkov et al. 2009). Non-limiting examples of such immunoglobulin single variable domains according to the present invention as well as particular combinations of FR's and CDR's are as described herein (see Tables 7-8). The delineation of the CDR sequences (and thus also the FR sequences) is based on the IMGT unique numbering system for V-domains and V-like domains (Lefranc et al. 2003). Alternatively, the delineation of the FR and CDR sequences can be done by using the Kabat numbering system as applied to V _H H domains from Camelids in the article of Riechmann and Muyldermans (2000). As will be known by the person skilled in the art, the immunoglobulin single variable domains, in particular the Nanobodies, can in particular be characterized by the presence of one or more Camelidae hallmark residues in one or more of the framework sequences (according to Kabat numbering), as described for example in WO 08/020079, on page 75, Table A-3, incorporated herein by reference).

In a preferred embodiment, the invention provides immunoglobulin single variable domains against CD74 with an amino acid sequence selected from the group consisting of amino acid sequences that essentially consist of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), in which the CDR sequences of said amino acid sequences have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% amino acid identity with the CDR sequences (see Table 8) of at least one of the immunoglobulin single variable domains of SEQ ID NOS: 12-21. It will be understood that for determining the degree of amino acid identity of the amino acid sequences of the CDRs of one or more sequences of the immunoglobulin single variable domains, the amino acid residues that from the framework regions are disregarded. Some preferred but non-limiting examples of immunoglobulin single variable domains of the invention are given in Table 7, and are defined by SEQ ID NOs: 12-21.

It should be noted that the immunoglobulin single variable domains, in particular the Nanobodies, of the invention in their broadest sense are not limited to a specific biological source or to a specific method of preparation. For example, the immunoglobulin single variable domains of the invention, in particular the Nanobodies, can generally be obtained: (1) by isolating the V _H H domain of a naturally occurring heavy chain antibody; (2) by expression of a nucleotide sequence encoding a naturally occurring V _H H domain; (3) by "humanization" of a naturally occurring V _H H domain or by expression of a nucleic acid encoding a such humanized V _H H domain; (4) by "camelization" of a naturally occurring VH domain from any animal species, and in particular from a mammalian species, such as from a human being, or by expression of a nucleic acid encoding such a camelized VH domain; (5) by "camelization" of a "domain antibody" or "Dab" as described in the art, or by expression of a nucleic acid encoding such a camelized VH domain; (6) by using synthetic or semi-synthetic techniques for preparing proteins, polypeptides or other amino acid sequences known per se; (7) by preparing a nucleic acid encoding a nanobody using techniques for nucleic acid synthesis known per se, followed by expression of the nucleic acid thus obtained; and/or (8) by any combination of one or more of the foregoing.

One preferred class of immunoglobulin single variable domains corresponds to the V _H H domains of naturally occurring heavy chain antibodies directed against CD74. Although naive or synthetic libraries of immunoglobulin single variable domains may contain binders against the target, a preferred embodiment of this invention includes the immunization of a Camelidae with the target to expose the immune system of the animal to the target.

Thus, such V _H H sequences can generally be generated or obtained by suitably immunizing a species of Camelid with a target CD74 protein, by obtaining a suitable biological sample from said Camelid (such as a blood sample, or any sample of B-cells), and by generating V _H H sequences directed against the target, starting from said sample, using any suitable technique known per se. Such techniques will be clear to the skilled person. Alternatively, such naturally occurring V _H H domains can be obtained from naive libraries of Camelid V _H H sequences, for example by screening such a library using the target or at least one part, fragment, antigenic determinant or epitope thereof using one or more screening techniques known per se. Such libraries and techniques are for example described in W09937681, WO0190190, WO03025020 and WO03035694. Alternatively, improved synthetic or semi-synthetic libraries derived from naive V _H H libraries may be used, such as V _H H libraries obtained from naive V _H H libraries by techniques such as random mutagenesis and/or CD shuffling, as for example described in WO0043507. Yet another technique for obtaining V _H H sequences directed against the target involves suitably immunizing a transgenic mammal that is capable of expressing heavy chain antibodies (i.e. so as to raise an immune response and/or heavy chain antibodies directed against a target), obtaining a suitable biological sample from said transgenic mammal (such as a blood sample, or any sample of B- cells), and then generating V _H H sequences directed against the target starting from said sample, using any suitable technique known per se. For example, for this purpose, the heavy chain antibody- expressing mice and the further methods and techniques described in WO02085945 and in WO04049794 can be used.

A particularly preferred class of immunoglobulin single variable domains of the invention, in particular Nanobodies of the invention, comprises immunoglobulin single variable domains with an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring V _H H domain, but that has been "humanized" , i.e. by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring V _H H sequence (and in particular in the framework sequences) by one or more of the amino acid residues that occur at the corresponding position(s) in a VH domain from a conventional 4-chain antibody from a human being. This can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the further description herein and the prior art on humanization referred to herein. Again, it should be noted that such humanized immunoglobulin single variable domains of the invention can be obtained in any suitable manner known per se (i.e. as indicated under points (1) - (8) above) and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring V _H H domain as a starting material. Humanized immunoglobulin single variable domains, in particular nanobodies, may have several advantages, such as a reduced immunogenicity, compared to the corresponding naturally occurring V _H H domains. Such humanization generally involves replacing one or more amino acid residues in the sequence of a naturally occurring V _H H with the amino acid residues that occur at the same position in a human VH domain, such as a human VH3 domain. The humanizing substitutions should be chosen such that the resulting humanized immunoglobulin single variable domains still retain the favourable properties of immunoglobulin single variable domains as defined herein. The skilled person will be able to select humanizing substitutions or suitable combinations of humanizing substitutions which optimize or achieve a desired or suitable balance between the favourable properties provided by the humanizing substitutions on the one hand and the favourable properties of naturally occurring V _H H domains on the other hand.

Another particularly preferred class of immunoglobulin single variable domains of the invention, in particular Nanobodies of the invention, comprises immunoglobulin single variable domains with an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VH domain, but that has been "camelized", i.e. by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain from a conventional 4-chain antibody by one or more of the amino acid residues that occur at the corresponding position(s) in a V _H H domain of a heavy chain antibody. Such "camelizing" substitutions are preferably inserted at amino acid positions that form and/or are present at the VH-VL interface, and/or at the so-called Camelidae hallmark residues, as defined herein (see for example WO9404678). Preferably, the VH sequence that is used as a starting material or starting point for generating or designing the camelized nanobody is preferably a VH sequence from a mammal, more preferably the VH sequence of a human being, such as a VH3 sequence. However, it should be noted that such camelized immunoglobulin single variable domains of the invention can be obtained in any suitable manner known per se (i.e. as indicated under points (1) - (8) above) and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring VH domain as a starting material.

For example both "humanization" and "camelization" can be performed by providing a nucleotide sequence that encodes a naturally occurring V _H H domain or VH domain, respectively, and then changing, in a manner known per se, one or more codons in said nucleotide sequence in such a way that the new nucleotide sequence encodes a "humanized" or "camelized" immunoglobulin single variable domains of the invention, respectively. This nucleic acid can then be expressed in a manner known per se, so as to provide the desired immunoglobulin single variable domains of the invention. Alternatively, based on the amino acid sequence of a naturally occurring V _H H domain or VH domain, respectively, the amino acid sequence of the desired humanized or camelized immunoglobulin single variable domains of the invention, respectively, can be designed and then synthesized de novo using techniques for peptide synthesis known per se. Also, based on the amino acid sequence or nucleotide sequence of a naturally occurring V _H H domain or VH domain, respectively, a nucleotide sequence encoding the desired humanized or camelized immunoglobulin single variable domains of the invention, respectively, can be designed and then synthesized de novo using techniques for nucleic acid synthesis known per se, after which the nucleic acid thus obtained can be expressed in a manner known per se, so as to provide the desired immunoglobulin single variable domains of the invention. Other suitable methods and techniques for obtaining the immunoglobulin single variable domains of the invention and/or nucleic acids encoding the same, starting from naturally occurring VH sequences or preferably V _H H sequences, will be clear from the skilled person, and may for example comprise combining one or more parts of one or more naturally occurring VH sequences (such as one or more FR sequences and/or CDR sequences), one or more parts of one or more naturally occurring V _H H sequences (such as one or more FR sequences or CDR sequences), and/or one or more synthetic or semi-synthetic sequences, in a suitable manner, so as to provide a nanobody of the invention or a nucleotide sequence or nucleic acid encoding the same.

Also within the scope of the invention are natural or synthetic analogs, mutants, variants, alleles, parts or fragments (herein collectively referred to as "variants") of the immunoglobulin single variable domains, in particular the Nanobodies, of the invention as defined herein, and in particular variants of the immunoglobulin single variable domains of SEQ ID NOs: 12-21 (see Tables 7-8). Thus, according to one embodiment of the invention, the term "immunoglobulin single variable domain of the invention" or "nanobody of the invention" in its broadest sense also covers such variants. Generally, in such variants, one or more amino acid residues may have been replaced, deleted and/or added, compared to the immunoglobulin single variable domains of the invention as defined herein. Such substitutions, insertions or deletions may be made in one or more of the FR's and/or in one or more of the CDR's, and in particular variants of the FR's and CDR's of the immunoglobulin single variable domains of SEQ ID NOs: 12-21 (see Tables 7-8). Variants, as used herein, are sequences wherein each or any framework region and each or any complementarity determining region shows at least 80% identity, preferably at least 85% identity, more preferably 90% identity, even more preferably 95% identity or, still even more preferably 99% identity with the corresponding region in the reference sequence (i.e. F l_variant versus FRl_reference, CDRl_variant versus CDRl_reference, FR2_variant versus FR2_reference, CDR2_variant versus CDR2_reference, FR3_variant versus FR3_reference, CDR3_variant versus CDR3_reference, FR4_variant versus FR4_reference), as can be measured electronically by making use of algorithms such as PILEUP and BLAST. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www/ncbi.nlm.nih.gov/). It will be understood that for determining the degree of amino acid identity of the amino acid sequences of the CDRs of one or more sequences of the immunoglobulin single variable domains, the amino acid residues that form the framework regions are disregarded. Similarly, for determining the degree of amino acid identity of the amino acid sequences of the FRs of one or more sequences of the immunoglobulin single variable domains of the invention, the amino acid residues that form the complementarity regions are disregarded. Such variants of immunoglobulin single variable domains may be of particular advantage since they may have improved potency/affinity.

By means of non-limiting examples, a substitution may for example be a conservative substitution (as described herein) and/or an amino acid residue may be replaced by another amino acid residue that naturally occurs at the same position in another V _H H domain. Thus, any one or more substitutions, deletions or insertions, or any combination thereof, that either improve the properties of the immunoglobulin single variable domains of the invention or that at least do not detract too much from the desired properties or from the balance or combination of desired properties of the Nanobody of the invention (i.e. to the extent that the immunoglobulin single variable domains is no longer suited for its intended use) are included within the scope of the invention. A skilled person will generally be able to determine and select suitable substitutions, deletions or insertions, or suitable combinations of thereof, based on the disclosure herein and optionally after a limited degree of routine experimentation, which may for example involve introducing a limited number of possible substitutions and determining their influence on the properties of the immunoglobulin single variable domains thus obtained.

Thus, according to a specific embodiment, the present invention encompasses immunoglobulin single variable domains comprising an amino acid sequence that comprises 4 framework regions (FR1 to FR4) and 3 complementarity determining regions (CDR1 to CDR3), according to the following formula (1):

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1) and wherein CDR1 is chosen from the group consisting of: a) SEQ ID NOs: 37-45, A polypeptide that has at least 80% amino acid identity with SEQ ID NOs: 37-45, c) A polypeptide that has 3, 2 or 1 amino acid difference with SEQ ID NOs: 37-45, and wherein CDR2 is chosen from the group consisting of: a) SEQ ID NOs: 55-63,

A polypeptide that has at least 80% amino acid identity with SEQ ID NOs: 55-63, c) A polypeptide that has 3, 2 or 1 amino acid difference with SEQ ID NOs: 55-63, and wherein CDR3 is chosen from the group consisting of: a) SEQ ID NOs: 73-81,

A polypeptide that has at least 80% amino acid identity with SEQ ID NOs: 73-81, c) A polypeptide that has 3, 2 or 1 amino acid difference with SEQ ID NOs: 73-81.

In a particular embodiment of the present invention, the immunoglobulin single variable domain directed against and/or specifically binding to CD74 is a Nanobody or V _H H, wherein the Nanobody has an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-21 or variants thereof. In a particularly preferred embodiment, the present invention provides for an immunoglobulin single variable domain comprising an amino acid sequence that comprises 4 framework regions (FR1 to FR4) and 3 complementarity determining regions (CDRl to CDR3), according to the following formula (1):

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1); wherein CDRl is SEQ ID NO: 37, and CDR2 is SEQ ID NO: 55, and CDR3 is SEQ ID NO: 73; or wherein CDRl is SEQ ID NO: 38, and CDR2 is SEQ ID NO: 56, and CDR3 is SEQ ID NO: 74; or wherein CDRl is SEQ ID NO: 39, and CDR2 is SEQ ID NO: 57, and CDR3 is SEQ ID NO: 75; or wherein CDRl is SEQ ID NO: 40, and CDR2 is SEQ ID NO: 58, and CDR3 is SEQ ID NO: 76; or wherein CDRl is SEQ ID NO: 41, and CDR2 is SEQ ID NO: 59, and CDR3 is SEQ ID NO: 77; or wherein CDRl is SEQ ID NO: 42, and CDR2 is SEQ ID NO: 60, and CDR3 is SEQ ID NO: 78; or wherein CDRl is SEQ ID NO: 43, and CDR2 is SEQ ID NO: 61, and CDR3 is SEQ ID NO: 79; or wherein CDRl is SEQ ID NO: 44, and CDR2 is SEQ ID NO: 62, and CDR3 is SEQ ID NO: 80; or wherein CDRl is SEQ ID NO: 45, and CDR2 is SEQ ID NO: 63, and CDR3 is SEQ ID NO: 81. More preferably, the immunoglobulin single variable domain directed against and/or specifically binding to CD74 has an amino acid sequence chosen from the group consisting of SEQ ID NOs: 12-21.

Further, and depending on the host organism used to express the immunoglobulin single variable domain of the invention, deletions and/or substitutions may be designed in such a way that one or more sites for post-translational modification (such as one or more glycosylation sites) are removed, as will be within the ability of the person skilled in the art. Alternatively, substitutions or insertions may be designed so as to introduce one or more sites for attachment of functional groups (as described herein), for example to allow site-specific pegylation. The immunoglobulin single variable domains within the scope of the invention may be further modified and/or may comprise (or can be further fused to) other moieties, as described further herein. Examples of modifications, as well as examples of amino acid residues within the immunoglobulin single variable domain, preferably the Nanobody sequence, that can be modified (i.e. either on the protein backbone but preferably on a side chain), methods and techniques that can be used to introduce such modifications and the potential uses and advantages of such modifications will be clear to the skilled person. For example, such a modification may involve the introduction (e.g. by covalent linking or in another suitable manner) of one or more functional groups, residues or moieties into or onto the immunoglobulin single variable domain of the invention, and in particular of one or more functional groups, residues or moieties that confer one or more desired properties or functionalities to the immunoglobulin single variable domain of the invention. Examples of such functional groups and of techniques for introducing them will be clear to the skilled person, and can generally comprise all functional groups and techniques mentioned in the general background art cited hereinabove as well as the functional groups and techniques known per se for the modification of pharmaceutical proteins, and in particular for the modification of antibodies or antibody fragments (including ScFv's and single domain antibodies), for which reference is for example made to Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, PA (1980). Such functional groups may for example be linked directly (for example covalently) to a immunoglobulin single variable domain of the invention, or optionally via a suitable linker or spacer, as will again be clear to the skilled person. One of the most widely used techniques for increasing the half-life and/or reducing immunogenicity of pharmaceutical proteins comprises attachment of a suitable pharmacologically acceptable polymer, such as poly(ethyleneglycol) (PEG) or derivatives thereof (such as methoxypoly(ethyleneglycol) or mPEG). Generally, any suitable form of pegylation can be used, such as the pegylation used in the art for antibodies and antibody fragments (including but not limited to (single) domain antibodies and ScFv's); reference is made to for example Chapman, Nat. Biotechnol., 54, 531-545 (2002); by Veronese and Harris, Adv. Drug Deliv. Rev. 54, 453-456 (2003), by Harris and Chess, Nat. Rev. Drug. Discov., 2, (2003) and in WO04060965. Various reagents for pegylation of proteins are also commercially available, for example from Nektar Therapeutics, USA. Preferably, site-directed pegylation is used, in particular via a cysteine-residue (see for example Yang et al., Protein Engineering, 16, 10, 761-770 (2003). For example, for this purpose, PEG may be attached to a cysteine residue that naturally occurs in an immunoglobulin single variable domain, or the immunoglobulin single variable domain may be modified so as to suitably introduce one or more cysteine residues for attachment of PEG, or an amino acid sequence comprising one or more cysteine residues for attachment of PEG may be fused to the N- and/or C-terminus of an immunoglobulin single variable domain, all using techniques of protein engineering known per se to the skilled person. Preferably, for the immunoglobulin single variable domains of the invention, a PEG is used with a molecular weight of more than 5000, such as more than 10,000 and less than 200,000, such as less than 100,000; for example in the range of 20,000-80,000. Another, usually less preferred modification comprises N-linked or O-linked glycosylation, usually as part of co-translational and/or post-translational modification, depending on the host cell used for expressing the immunoglobulin single variable domain or polypeptide of the invention. Another technique for increasing the half-life of an immunoglobulin single variable domain may comprise the engineering into bifunctional constructs (for example, one Nanobody against the target CD74 and one against a serum protein such as albumin) or into fusions of immunoglobulin single variable domains with peptides (for example, a peptide against a serum protein such as albumin). Yet another modification may comprise the introduction of one or more detectable labels or other signal-generating groups or moieties, depending on the intended use of the labeled immunoglobulin single variable domain. Thus, according to a preferred embodiment, the immunoglobulin single variable domain as used in the present invention is coupled or fused to a detectable label, either directly or through a linker. Suitable labels and techniques for attaching, using and detecting them will be clear to the skilled person, and for example include, but are not limited to, fluorescent labels, (such as I Dye800, VivoTag800, fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine and fluorescent metals such as Eu or others metals from the lanthanide series), phosphorescent labels, chemiluminescent labels or bioluminescent labels (such as luminal, isoluminol, theromatic acridinium ester, imidazole, acridinium salts, oxalate ester, dioxetane or GFP and its analogs), radio-isotopes (such as technetium 99m ( ⁹⁹ mTc), iodium 123 ( ¹²³ l), zirconium 89 ( ⁸⁹ Zr), iodium 125 ( ¹²⁵ l), indium 111 ( ^m ln), fluor 18 ( ¹⁸ F), copper 64 ( ⁶⁴ Cu), gallium 67 ( ⁶⁷ Ga), gallium 68 ( ⁶⁸ Ga)), metals, metals chelates or metallic cations or other metals or metallic cations that are particularly suited for use in in vivo, in vitro or in situ diagnosis and imaging, as well as chromophores and enzymes (such as malate dehydrogenase, staphylococcal nuclease, delta- V- steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, biotinavidin peroxidase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholine esterase). Other suitable labels will be clear to the skilled person, and for example include moieties that can be detected using NMR or ESR spectroscopy. Such labeled Nanobodies and polypeptides of the invention may for example be used for n vitro, in vivo or in situ assays (including immunoassays known per se such as ELISA, IA, EIA and other "sandwich assays", etc.) as well as in vivo diagnostic and imaging purposes, depending on the choice of the specific label. As will be clear to the skilled person, another modification may involve the introduction of a chelating group, for example to chelate one of the metals or metallic cations referred to above. Suitable chelating groups for example include, without limitation, 2,2',2"-(10-(2-((2,5- dioxopyrrolidin-l-yl)oxy)-2-oxoethyl)-l,4,7,10-tetraazacyclo dodecane-l,4,7-triyl)triacetic acid (DOTA), 2,2'-(7-(2-((2,5-dioxopyrrolidin-l-yl)oxy)-2-oxoethyl)-l,4,7 -triazonane-l,4-diyl)diacetic acid (NOTA), diethyl- enetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Yet another modification may comprise the introduction of a functional group that is one part of a specific binding pair, such as the biotin-(strept)avidin binding pair. Such a functional group may be used to link the immunoglobulin single variable domain to another protein, polypeptide or chemical compound that is bound to the other half of the binding pair, i.e. through formation of the binding pair. For example, a Nanobody of the invention may be conjugated to biotin, and linked to another protein, polypeptide, compound or carrier conjugated to avidin or streptavidin. For example, such a conjugated Nanobody may be used as a reporter, for example in a diagnostic system where a detectable signal-producing agent is conjugated to avidin or streptavidin. Such binding pairs may for example also be used to bind the Nanobody of the invention to a carrier, including carriers suitable for pharmaceutical purposes. One non-limiting example are the liposomal formulations described by Cao and Suresh, Journal of Drug Targetting, 8, 4, 257 (2000). Such binding pairs may also be used to link a therapeutically active agent (as defined further herein) to the immunoglobulin single variable domain of the invention.

In another preferred embodiment, the immunoglobulin single variable domain of the present invention is coupled to or fused to a moiety, in particular a therapeutically active agent, either directly or through a linker. As used herein, a "therapeutically active agent" means any molecule that has or may have a therapeutic effect (i.e. curative or stabilizing effect) in the context of treatment of an inflammatory disease (as described further herein). Preferably, a therapeutically active agent is a disease-modifying agent, which can be a cytotoxic agent, such as a toxin, or a cytotoxic drug, or an enzyme capable of converting a prodrug into a cytotoxic drug, or a radionuclide, or a cytotoxic cell, or which can be a non- cytotoxic agent. Even more preferably, a therapeutically active agent has a curative effect on the disease. Alternatively, a therapeutically active agent is a disease-stabilizing agent, in particular a molecule that has a stabilizing effect on the evolution of an inflammatory disease. Examples of stabilizing agents include anti-inflammatory agents, in particular non-steroid anti-inflammatory molecules. According to one specific embodiment, the therapeutically active agent is not a cytotoxic agent. Preferred "linker molecules" or "linkers" are peptides of 1 to 200 amino acids length, and are typically, but not necessarily, chosen or designed to be unstructured and flexible. For instance, one can choose amino acids that form no particular secondary structure. Or, amino acids can be chosen so that they do not form a stable tertiary structure. Or, the amino acid linkers may form a random coil. Such linkers include, but are not limited to, synthetic peptides rich in Gly, Ser, Thr, Gin, Glu or further amino acids that are frequently associated with unstructured regions in natural proteins (Dosztanyi, Z., Csizmok, V., Tompa, P., & Simon, I. (2005). lUPred: web server for the prediction of intrinsically unstructured regions of proteins based on estimated energy content. Bioinformatics (Oxford, England), 21(16), 3433- 4.). Non-limiting examples of suitable linker sequences include (GS)5 (GSGSGSGSGS; SEQ ID NO: 91), (GS)10 (GSGSGSGSGSGSGSGSGSGS; SEQ ID NO: 92), (G4S)3 (GGGGSGGGGSGGGGS; SEQ ID NO: 93), llama lgG2 hinge (AHHSEDPSSKAPKAPMA; SEQ ID NO: 94) or human IgA hinge (SPSTPPTPSPSTPPAS; SEQ ID NO: 95) linkers. Other non-limiting examples of suitable linker sequences are also described in the Example section.

Thus, according to specific embodiments, the amino acid (AA) linker sequence is a peptide of between 0 and 200 AA, between 0 and 150 AA, between 0 and 100 AA, between 0 and 90 AA, between 0 and 80 AA, between 0 and 70 AA, between 0 and 60 AA, between 0 and 50 AA, between 0 and 40 AA, between 0 and 30 amino acids, between 0 and 20 AA, between 0 and 10 amino acids, between 0 and 5 amino acids. Examples of sequences of short linkers include, but are not limited to, PPP, PP or GS.

For certain applications, it may be advantageous that the linker molecule comprises or consists of one or more particular sequence motifs. For example, a proteolytic cleavage site can be introduced into the linker molecule such that detectable label or moiety can be released. Useful cleavage sites are known in the art, and include a protease cleavage site such as Factor Xa cleavage site having the sequence IEG (SEQ ID NO: 96), the thrombin cleavage site having the sequence LVPR (SEQ ID NO: 97), the enterokinase cleaving site having the sequence DDDDK (SEQ ID NO: 98), or the PreScission cleavage site LEVLFQGP (SEQ ID NO: 99).

Alternatively, in case the immunoglobulin single variable domain is linked to a detectable label or moiety using chemoenzymatic methods for protein modification, the linker moiety may exist of different chemical entities, depending on the enzymes or the synthetic chemistry that is used to produce the covalently coupled molecule in vivo or in vitro (reviewed in: Rabuka 2010, Curr Opin Chem Biol 14: 790-796)

Also encompassed within the scope of the present invention are the immunoglobulin single variable domains of the invention that are in a "multivalent" form and are formed by bonding, chemically or by recombinant DNA techniques, together two or more monovalent immunoglobulin single variable domains. Non-limiting examples of multivalent constructs include "bivalent" constructs, "trivalent" constructs, "tetravalent" constructs, and so on. The immunoglobulin single variable domains comprised within a multivalent construct may be identical or different. In another particular embodiment, the immunoglobulin single variable domains of the invention are in a "multi-specific" form and are formed by bonding together two or more immunoglobulin single variable domains, of which at least one with a different specificity. Non-limiting examples of multi-specific constructs include "bi-specific" constructs, "tri-specific" constructs, "tetra-specific" constructs, and so on. To illustrate this further, any multivalent or multispecific (as defined herein) immunoglobulin single variable domain of the invention may be suitably directed against two or more different epitopes on the same antigen, for example against two or more different parts of CD74; or may be directed against two or more different antigens, for example against an epitope of CD74 and an epitope of an interacting partner. In particular, a monovalent immunoglobulin single variable domain of the invention is such that it will bind to the target CD74 protein with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM. Multivalent or multispecific immunoglobulin single variable domains of the invention may also have (or be engineered and/or selected for) increased avidity and/or improved selectivity for the desired CD74, and/or for any other desired property or combination of desired properties that may be obtained by the use of such multivalent or multispecific immunoglobulin single variable domains. Also, the amino acid sequences of the immunoglobulin single variable domains as described herein may be comprised in a polypeptide sequence.

A further aspect of the present invention relates to a complex comprising an immunoglobulin single variable domain of the invention. More specifically, a complex is provided comprising an immunoglobulin single variable domain of the invention, a CD74 target protein, and optionally at least one other interacting partner of CD74. As a non-limiting example, a complex may be purified by gel filtration. In a particular embodiment, the complex can be crystalline. Accordingly, a crystal of the complex is also provided, as well as methods of making said crystal, which are known to the person skilled in the art.

In another aspect, a nucleic acid sequence encoding an amino acid sequence of any of the immunoglobulin single variable domains of the invention is also part of the present invention and a non-limiting examples are provided in Table 7.. According to preferred embodiments, the invention relates to nucleic acid sequences of immunoglobulin single variable domains of the invention, in particular immunoglobulin single variable domains, in which the sequences have more than 80%, preferably more than 90%, more preferably more than 95%, such as 99% or more sequence identity (as defined herein) with the sequences of at least one of the nucleic acid sequences of the immunoglobulin single variable domains defined by SEQ ID NOs: 25-27 (see Table 7). For the calculation of the percentage sequence identity, the nucleic acid sequences of tags (e.g. His tag or EPEA tag) should be disregarded. Also, the nucleic acid sequences as described herein may be comprised in a nucleic acid sequence.

Further, the present invention also envisages expression vectors comprising nucleic acid sequences encoding any of the immunoglobulin single variable domains of the invention as well as host cells expressing such expression vectors. Suitable expression systems include constitutive and inducible expression systems in bacteria or yeasts, virus expression systems, such as baculovirus, semliki forest virus and lentiviruses, or transient transfection in insect or mammalian cells. The cloning, expression and/or purification of the immunoglobulin single variable domains of the invention can be done according to techniques known by the skilled person in the art. Thus, the present invention encompasses a cell or a culture of cells expressing an immunoglobulin single variable domain of the invention that is directed against and/or capable of specifically binding to CD74. The cells according to the present invention can be of any prokaryotic or eukaryotic organism. Preferably, cells are eukaryotic cells, for example yeast cells, or insect cells, or cultured cell lines, for example mammalian cell lines, preferably human cell lines, that endogenously or recombinantly express CD74. The nature of the cells used will typically depend on the ease and cost of producing the native protein(s), the desired glycosylation properties, the origin of the target protein, the intended application, or any combination thereof. Eukaryotic cell or cell lines for protein production are well known in the art, including cell lines with modified glycosylation pathways, and non-limiting examples will be provided hereafter. Animal or mammalian host cells suitable for harboring, expressing, and producing proteins for subsequent isolation and/or purification include Chinese hamster ovary cells (CHO), such as CHO-K1 (ATCC CCL-61), DG44 (Chasin et al., 1986, Som. Cell Molec. Genet, 12:555-556; and Kolkekar et al., 1997, Biochemistry, 36:10901-10909), CHO-K1 Tet-On cell line (Clontech), CHO designated ECACC 85050302 (CAM , Salisbury, Wiltshire, UK), CHO clone 13 (GEIMG, Genova, IT), CHO clone B (GEIMG, Genova, IT), CHO-K1/SF designated ECACC 93061607 (CAMR, Salisbury, Wiltshire, UK), RR-CHOK1 designated ECACC 92052129 (CAMR, Salisbury, Wiltshire, UK), dihydrofolate reductase negative CHO cells (CHO/-DHFR, Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA, 77:4216), and dpl2.CHO cells (U.S. Pat. No. 5,721,121); monkey kidney CV1 cells transformed by SV40 (COS cells, COS-7, ATCC CRL- 1651); human embryonic kidney cells (e.g., 293 cells, or 293T cells, or 293 cells subcloned for growth in suspension culture, Graham et al., 1977, J. Gen. Virol., 36:59, or GnTI KO HEK293S cells, Reeves et al. 2002, PNAS, 99: 13419); baby hamster kidney cells (BHK, ATCC CCL-10); monkey kidney cells (CV1, ATCC CCL-70); African green monkey kidney cells (VERO-76, ATCC CRL-1587; VERO, ATCC CCL-81); mouse Sertoli cells (TM4, Mather, 1980, Biol. Reprod., 23:243-251); human cervical carcinoma cells (HELA, ATCC CCL-2); canine kidney cells (MDCK, ATCC CCL-34); human lung cells (W138, ATCC CCL-75); human hepatoma cells (HEP-G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL-51); buffalo rat liver cells (BRL 3A, ATCC CRL-1442); TRI cells (Mather, 1982, Annals NYAcad. Sci., 383:44-68); MCR 5 cells; FS4 cells. According to a particular embodiment, the cells are mammalian cells selected from Hek293 cells or COS cells.

Exemplary non-mammalian cell lines include, but are not limited to, Sf9 cells, baculovirus-insect cell systems (e.g. review Jarvis, Virology Volume 310, Issue 1, 25 May 2003, Pages 1-7), plant cells such as tobacco cells, tomato cells, maize cells, algae cells, or yeasts such as Saccharomyces species, Schizosaccharomyces species, Hansenula species, Yarrowia species or Pichia species. According to particular embodiments, the eukaryotic cells are yeast cells from a Saccharomyces species (e.g. Saccharomyces cerevisiae), Schizosaccharomyces sp. (for example Schizosaccharomyces pombe), a Hansenula species (e.g. Hansenula polymorpha), a Yarrowia species (e.g. Yarrowia lipolytica), a Kluyveromyces species (e.g. Kluyveromyces lactis), a Pichia species (e.g. Pichia pastoris), or a Komagataella species (e.g. Komagataella pastoris). According to a specific embodiment, the eukaryotic cells are Pichia cells, and in a most particular embodiment Pichia pastoris cells.

Transfection of target cells (e.g. mammalian cells) can be carried out following principles outlined by Sambrook and Russel (Molecular Cloning, A Laboratory Manual, 3 ^rd Edition, Volume 3, Chapter 16, Section 16.1-16.54). In addition, viral transduction can also be performed using reagents such as adenoviral vectors. Selection of the appropriate viral vector system, regulatory regions and host cell is common knowledge within the level of ordinary skill in the art. The resulting transfected cells are maintained in culture or frozen for later use according to standard practices.

Further, a pharmaceutical composition comprising any of the immunoglobulin single variable domains as described hereinbefore and optionally, at least one of a pharmaceutically acceptable carrier, adjuvant or diluent are also envisaged here. A 'carrier', or 'adjuvant', in particular a 'pharmaceutically acceptable carrier' or 'pharmaceutically acceptable adjuvant' is any suitable excipient, diluent, carrier and/or adjuvant which, by themselves, do not induce the production of antibodies harmful to the individual receiving the composition nor do they elicit protection. So, pharmaceutically acceptable carriers are inherently non-toxic and nontherapeutic, and they are known to the person skilled in the art. Suitable carriers or adjuvantia typically comprise one or more of the compounds included in the following non- exhaustive list: large slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles. Carriers or adjuvants may be, as a non-limiting example, Ringer's solution, dextrose solution or Hank's solution. Non aqueous solutions such as fixed oils and ethyl oleate may also be used. A preferred excipient is 5% dextrose in saline. The excipient may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, including buffers and preservatives.

The administration of an immunoglobulin single variable domain as described herein or a pharmaceutically acceptable salt thereof may be by way of oral, inhaled or parenteral administration. In particular embodiments the immunoglobulin single variable domain is delivered through intrathecal or intracerebroventricular administration. The active compound may be administered alone or preferably formulated as a pharmaceutical composition. An amount effective to treat a certain disease or disorder that express the antigen recognized by the immunoglobulin single variable domain depends on the usual factors such as the nature and severity of the disorder being treated and the weight of the mammal. However, a unit dose will normally be in the range of 0.01 to 50 mg, for example 0.01 to 10 mg, or 0.05 to 2 mg of immunoglobulin single variable domain or a pharmaceutically acceptable salt thereof. Unit doses will normally be administered once or more than once a day, for example 2, 3, or 4 times a day, more usually 1 to 3 times a day, such that the total daily dose is normally in the range of 0.0001 to 1 mg/kg; thus a suitable total daily dose for a 70 kg adult is 0.01 to 50 mg, for example 0.01 to 10 mg or more usually 0.05 to 10 mg. It is greatly preferred that the compound or a pharmaceutically acceptable salt thereof is administered in the form of a unit-dose composition, such as a unit dose oral, parenteral, or inhaled composition. Such compositions are prepared by admixture and are suitably adapted for oral, inhaled or parenteral administration, and as such may be in the form of tablets, capsules, oral liquid preparations, powders, granules, lozenges, reconstitutable powders, injectable and infusable solutions or suspensions or suppositories or aerosols. Tablets and capsules for oral administration are usually presented in a unit dose, and contain conventional excipients such as binding agents, fillers, diluents, tabletting agents, lubricants, disintegrants, colourants, flavourings, and wetting agents. The tablets may be coated according to well-known methods in the art. Suitable fillers for use include cellulose, mannitol, lactose and other similar agents. Suitable disintegrants include starch, polyvinylpyrrolidone and starch derivatives such as sodium starch glycolate. Suitable lubricants include, for example, magnesium stearate. Suitable pharmaceutically acceptable wetting agents include sodium lauryl sulphate. These solid oral compositions may be prepared by conventional methods of blending, filling, tabletting or the like. Repeated blending operations may be used to distribute the active agent throughout those compositions employing large quantities of fillers. Such operations are, of course, conventional in the art. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups, or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example, almond oil, fractionated coconut oil, oily esters such as esters of glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and if desired conventional flavouring or colouring agents. Oral formulations also include conventional sustained release formulations, such as tablets or granules having an enteric coating. Preferably, compositions for inhalation are presented for administration to the respiratory tract as a snuff or an aerosol or solution for a nebulizer, or as a microfine powder for insufflation, alone or in combination with an inert carrier such as lactose. In such a case the particles of active compound suitably have diameters of less than 50 microns, preferably less than 10 microns, for example between 1 and 5 microns, such as between 2 and 5 microns. A favored inhaled dose will be in the range of 0.05 to 2 mg, for example 0.05 to 0.5 mg, 0.1 to 1 mg or 0.5 to 2 mg. For parenteral administration, fluid unit dose forms are prepared containing a compound of the present invention and a sterile vehicle. The active compound, depending on the vehicle and the concentration, can be either suspended or dissolved. Parenteral solutions are normally prepared by dissolving the compound in a vehicle and filter sterilising before filling into a suitable vial or ampoule and sealing. Advantageously, adjuvants such as a local anaesthetic, preservatives and buffering agents are also dissolved in the vehicle. To enhance the stability, the composition can be frozen after filling into the vial and the water removed under vacuum. Parenteral suspensions are prepared in substantially the same manner except that the compound is suspended in the vehicle instead of being dissolved and sterilised by exposure to ethylene oxide before suspending in the sterile vehicle. Advantageously, a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the active compound. Where appropriate, small amounts of bronchodilators for example sympathomimetic amines such as isoprenaline, isoetharine, salbutamol, phenylephrine and ephedrine; xanthine derivatives such as theophylline and aminophylline and corticosteroids such as prednisolone and adrenal stimulants such as ACTH may be included. As is common practice, the compositions will usually be accompanied by written or printed directions for use in the medical treatment concerned.

The efficacy of the immunoglobulin single variable domains of the invention, and of compositions comprising the same, can be tested using any suitable in vitro assay, cell-based assay, in vivo assay and/or animal model known per se, or any combination thereof, depending on the specific disease or disorder involved.

Certain of the above-described immunoglobulin single variable domains may have therapeutic utility and may be administered to a subject having a condition in order to treat the subject for the condition. Thus, according to yet another aspect, the immunoglobulin single variable domains as described hereinbefore can be used as a medicine. More specifically, the immunoglobulin single variable domains may be very useful for the prevention and/or treatment of an inflammatory disease.

In particular, the invention relates to a method of preventing and/or treating an inflammatory disease, comprising administering a therapeutically effective amount of an immunoglobulin single variable domain of the invention or a pharmaceutical composition derived thereof to a subject in need thereof.

As used herein, the term "preventing an inflammatory disease" means inhibiting or reversing the onset of the disease, inhibiting or reversing the initial signs of the disease, inhibiting the appearance of clinical symptoms of the disease. As used herein, "treating an inflammatory disease" or "treating a subject or individual having an inflammatory disease" includes substantially inhibiting the disease, substantially slowing or reversing the progression of the disease, substantially ameliorating clinical symptoms of the disease or substantially preventing the appearance of clinical symptoms of the disease. A treatment is considered therapeutic if there is a decrease in mortality and/or morbidity, and may be performed prophylactically, or therapeutically. A variety of subjects or individuals are treatable. Generally the "subjects" are mammals or mammalian, where these terms are used broadly to describe organisms which are within the class mammalia, including the orders carnivore (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In many embodiments, the subjects will be humans.

In its broadest sense, the term "an inflammatory disease" refers to all inflammation-associated disease processes including autoimmune diseases (rheumatoid arthritis, atherosclerosis, asthma, inflammatory bowel and Crohn's disease and Alzheimer's disease), metabolic disorders (diabetes and obesity), systemic infections (inflammation-associated anemia (i.e. ACD)), renal diseases (e.g. diabetic kidney disease), as well as sepsis and cancer. According to a preferred embodiment, the immunoglobulin single variable domains are used for the prevention and/or treatment of cancer, e.g. breast cancer, ovarian cancer, cervical cancer, glioblastoma, leukemia, lymphoma, prostate cancer, Burkitt's lymphoma, head and neck cancer, colon cancer, colorectal cancer, non-small cell lung cancer, small cell lung cancer, gastric cancer, as well as other types of cancer.

In a specific embodiment, it should be clear that the therapeutic method of the present invention against an inflammatory disease can also be used in combination with any other therapy known in the art.

Another application that is envisaged here is the use of any of the immunoglobulin single variable domain as described hereinbefore for stabilizing and subsequently crystallizing a target CD74 protein in complex with an immunoglobulin single variable domain of the invention. Determining the crystal structure may be done by a biophysical method such as X-ray crystallography, which is a known technique by the person skilled in the art.

Further, the immunoglobulin single variable domains according to the invention can also be used for detecting a protein, in particular a CD74 protein, optionally with an interacting partner, which might find applications in the diagnosis, prognosis, or monitoring the status of an inflammatory disease (as described above), or for use in assessing the impact of a therapy on an inflammatory disease (as described above). In particular, the protein detection may occur in vitro or in vivo according to techniques known in the art. According to a specific embodiment, the detection may occur via in vivo imaging.

The immunoglobulin single variable domains of the present invention can also be used to modulate CD74 receptor signaling, including abolishing CD74 receptor signaling. The terms "modulating", "modulation", "modulated" means an increase or decrease in activity of a protein, in particular of a CD74 protein. In particular, the immunoglobulin single variable domains of the present invention can be orthosteric modulators or orthosteric inhibitor or alternatively, allosteric modulators or allosteric inhibitors. The terms "orthosteric modulator" or orthosteric inhibitor" in the context of the present invention refer to competitive modulators or inhibitors, which exert their effect by binding at the active site of the receptor. The terms "allosteric modulator" or "allosteric inhibitor" in the context of the present invention refer to noncompetitive modulators or inhibitors, which exert their effect by binding to a site other than the active site of the receptor, and modulate the activity of the receptor or render the receptor ineffective in terms of signal transduction. A "positive allosteric modulator (PAM)" increases signal transduction, whereas a "negative allosteric modulator (NAM)" reduces signal transduction. In particular, an allosteric inhibitor may also abolish signal transduction. Assays to evaluate the modulation in CD74 signaling by the immunoglobulin single variable domains of the invention are as described hereinbefore.

In that regard, according to a specific embodiment, the immunoglobulin single variable domains of the present invention can also be useful for lead identification and the design of peptidomimetics. Using a biologically relevant peptide or protein structure as a starting point for lead identification represents one of the most powerful approaches in modern drug discovery. Peptidomimetics are compounds whose essential elements (pharmacophore) mimic a natural peptide or protein in 3D space and which retain the ability to interact with the biological target and produce the same biological effect. Peptidomimetics are designed to circumvent some of the problems associated with a natural peptide: for example stability against proteolysis (duration of activity) and poor bioavailability. Certain other properties, such as receptor selectivity or potency, often can be substantially improved.

In still another aspect, the invention also encompasses a method of screening for immunoglobulin single variable domains directed against and/or specifically binding to CD74 comprising the steps of: a) Providing a plurality of immunoglobulin single variable domains, and

b) Screening said plurality of immunoglobulin single variable domains for an immunoglobulin single variable domain that binds to CD74, and

c) Isolating the immunoglobulin single variable domain that binds to CD74.

As described herein, immunoglobulin single variable domains can be generated in many ways. Typically, immunization of an animal will be done with a target CD74 as described hereinbefore (e.g. for V _H H sequences, as a non-limiting example) and also exemplified further herein.

For the immunization of an animal with a target CD74, the target protein may be produced and purified using conventional methods that may employ expressing a recombinant form of said protein in a host cell, and purifying the proteins using affinity chromatography and/or antibody-based methods. In particular embodiments, the baculovirus/Sf-9 system may be employed for expression, although other expression systems (e.g., bacterial, yeast or mammalian cell systems) may also be used. Other immunization methods include, without limitation, the use of complete cells expressing a target CD74 or membrane preparations derived thereof.

Any suitable animal, e.g., a warm-blooded animal, in particular a mammal such as a rabbit, mouse, rat, camel, sheep, cow or pig or a bird such as a chicken or turkey, may be immunized using any of the techniques well known in the art suitable for generating an immune response. The screening for immunoglobulin single variable domains specifically binding to a target CD74 may for example be performed by screening a set, collection or library of cells that express the immunoglobulin single variable domains on their surface (e.g. B-cells obtained from a suitably immunized Camelid), by screening of a (naive or immune) library of immunoglobulin single variable domains, or by screening of a (naive or immune) library of nucleic acid sequences that encode amino acid sequences of the immunoglobulin single variable domains, which may all be performed in a manner known per se, and which method may optionally further comprise one or more other suitable steps, such as, for example and without limitation, a step of affinity maturation, a step of expressing the desired amino acid sequence, a step of screening for binding and/or for activity against the desired antigen, a step of determining the desired amino acid sequence or nucleotide sequence, a step of introducing one or more humanizing substitutions, a step of formatting in a suitable multivalent and/or multispecific format, a step of screening for the desired biological and/or physiological properties (i.e. using a suitable assay known in the art), and/or any combination of one or more of such steps, in any suitable order.

In another aspect, the present invention also relates to a method for producing an immunoglobulin single variable domain according to the invention, said method comprising the steps of:

- expressing, in a suitable expression system, a nucleic acid sequence encoding an immunoglobulin single variable domain according to the invention; and optionally

- isolating and/or purifying said immunoglobulin single variable domain.

The above methods for isolating and/or purifying immunoglobulin single variable domains include, without limitation, affinity-based methods such as affinity chromatography, affinity purification, immunoprecipitation, protein detection, immunochemistry, surface-display, amongst others, and are all well-known in the art.

Yet another aspect of the invention relates to a kit comprising an immunoglobulin single variable domain according to the invention. The kit may further comprise a combination of reagents such as buffers, molecular tags, vector constructs, reference sample material, as well as a suitable solid supports, cells, nucleic acids, and the like. Such a kit may be useful for any of the applications of the present invention as described herein.

Also encompassed within the scope of the present invention is a solid support comprising an immunoglobulin single variable domain according to the invention. Non-limiting examples of suitable solid supports include beads, columns, slides, chips or plates. More specifically, the solid supports may be particulate (e. g. beads or granules, generally used in extraction columns) or in sheet form (e. g. membranes or filters, glass or plastic slides, microtiter assay plates, dipstick, capillary fill devices or such like) which can be flat, pleated, or hollow fibers or tubes. The following matrices are given as examples and are not exhaustive, such examples could include silica (porous amorphous silica), i. e. the FLASH series of cartridges containing 60A irregular silica (32-63 um or 35-70 um) supplied by Biotage (a division of Dyax Corp.), agarose or polyacrylamide supports, for example the Sepharose range of products supplied by Amersham Pharmacia Biotech, or the Affi-Gel supports supplied by Bio- ad. In addition there are macroporous polymers, such as the pressure-stable Affi-Prep supports as supplied by Bio-Rad. Other supports that could be utilised include; dextran, collagen, polystyrene, methacrylate, calcium alginate, controlled pore glass, aluminium, titanium and porous ceramics. Alternatively, the solid surface may comprise part of a mass dependent sensor, for example, a surface plasmon resonance detector. Further examples of commercially available supports are discussed in, for example, Protein Immobilisation, R.F. Taylor ed., Marcel Dekker, Inc., New York, (1991). Immobilization may be either non-covalent or covalent, using techniques known in the art.

Finally, a last aspect of the invention is the use of any immunoglobulin single variable domain according to the invention to isolate amino acid sequences that are responsible for specific binding to a target CD74 protein and to construct artificial immunoglobulin single variable domains based on said amino acid sequences. It will be appreciated that in the immunoglobulin single variable domains according to the invention, the framework regions and the complementarity determining regions are known, and the study of derivatives of the immunoglobulin single variable domains, binding to the same epitope of a target CD74 protein, will allow deducing the essential amino acids involved in binding said conformational epitope. This knowledge can be used to construct a minimal immunoglobulin single variable domain and to create derivatives thereof, which can routinely be done by techniques known by the skilled in the art.

The following examples more fully illustrate preferred features of the invention, but are not intended to limit the invention in any way. Those having ordinary skill in the art and access to the teachings herein will recognize additional modifications and embodiments within the scope thereof. Therefore, the present invention is limited only by the claims attached herein. All of the starting materials and reagents disclosed below are known to those skilled in the art, and are available commercially or can be prepared using well-known techniques. EXAMPLES

Example 1. Camelid immunization and Nanobody repertoire cloning

In order to generate human CD74 (SEQ I D NO: 1) antigen specific Nanobodies (VHHs), an alpaca was immunized with the human monocytic cell line TH P-1. This cell line was originally used by Leng et al., 2003 to identify the MI F receptor CD74 and was found to be positive for CD74 expression via flow- cytometry. An alpaca underwent six rounds of immunization with 10 ⁷ non-stimulated TH P-1 cells, as well as 10 ⁷ LPS (^g/ml) stimulated (48h) TH P-1 cells. Following immunization, peripheral blood was collected, the immune response of the alpaca was assessed (data not shown) and a Nanobody library was created. Therefore, lymphocytes of the immunized alpaca were isolated from peripheral blood using Lymphoprep™ (Axis-Shield) in accordance to the manufacturer's instructions. The TH P-1 VH H library was constructed by first extracting total NA from the peripheral blood lymphocytes using the TRIzol ^® RNA extraction protocol (Invitrogen), followed by first strand cDNA synthesis with oligo (dT) primer using Superscript I I Reverse Transcriptase (Invitrogen), all according to the manufacturer's instructions. Via nested PCR, the Nanobody repertoire was amplified and cloned as Pstl-BstEW fragments (Conrath et al. 2001) into phagemid vector pMES4 (GenBank GQ907248), resulting in a phage display library harboring the His ₆ -tagged Nanobody as a genel l l fusion. The size of the library was determined to be about 3.2 x 10 ^s individual colonies with around 62% of the colonies having the correct insert size.

Example 2. Selecting and screening for CD74 specific Nanobodies In order to enrich for CD74-specific Nanobodies expressed on the phage surface, human CD74 ^{73 232} (SEQ I D NO: 2) was coated on Maxisorp (Nunc) 96-well plates. Phage libraries were rescued according to standard protocols and phage particles were allowed to bind to the coated human CD74 ^{73 232} . After thorough washing to remove aspecific binding, phage was eluted via trypsin treatment and re-infected into E. coli TG I for a consecutive selection round. Five rounds of panning on recombinant human CD74 (CD74 ^{73 232} ; SEQ ID NO: 2) were performed. We screened 287 colonies from round 2, 96 colonies from round 3 and 72 colonies from round 4. It was determined that 0/287 colonies were positive from round 2, 61/96 colonies were positive from round 3 and 54/72 were positive from round 4. Based on these results, colony PCR was performed on 47 positive clones from round 3 and the PCR products were subsequently sequenced. From the sequencing results, one unique Nanobody against human CD74 was generated. The Nanobody, named "N bhCD74n49" and hereafter referred to as "Nb_49", has an amino acid sequence as defined in SEQ I D NO: 12, and a nucleotide sequence as defined in SEQ I D NO: 25. The Nanobody was purified via immobilized metal affinity chromatography and passed over a size exclusion chromatography (Superdex75(16/600) column, AktaXpress) after expression in E.coli WK6 cells (Conrath et al. 2001). Subsequent sequence analysis confirmed the correct amino acid sequence of N b_49 (SEQ I D NO: 12; Table 7). The VH H gene was recloned into the expression vectors pH EN6c and pMECS using the restriction enzymes PPUM I and Pstl.

Based on the amino acid sequence of N b_49, the protein parameters (Isoelectric point (pl)/extinction coefficient and MW) could be determined using ExPASY software (see Table 1). In addition, the average production yield as well as the potential LPS-contamination (LAL-assay, Cambrex) was determined (see Table 1).

In order to determine the affinity of the Nanobody for its antigen, the N b_49 binding kinetic parameters were determined using BIAcore. To this end, biotinylated N b_49 (Biotinylation EZ-link kit, Thermo Scientific) was coupled to a streptavidin sensor chip (GE Healthcare Sensor Chip SA certified, Series S) (146 RU). Su bsequently, varying concentrations ranging from 0.05 till 500 nM of recombinant human CD74 ^{73 232} was added (at a flow rate of 20μΙ/ιτιϊη) followed by dissociation of the complex by washing with H BS-EP buffer for 600 seconds. The results from the BIAcore are summarized in Table 1.

Next, the immune phage library was used again to pan against recombinant human CD74 ^{73 232} which allowed retrieving another anti-CD74 N b (i.e. N bhCD74n95, herein referred by as N b_95). The anti- CD74 N b_95 was isolated from the library in a similar way as described above and its amino acid sequence was determined. According to the plasmid sequencing results it appeared that there was a frame shift in framework 1. In order to correct this, a new primer was designed: 5'- CAGCTGCAGGAGTTGGGGGAGGCTCTGGTGCAGCCTGGGG-3' (forward primer; SEQ ID NO: 100). Following colony PGR with this primer and the Gi l l primer, the PGR fragment was digested with Pstl and Bstell and ligated in the pH EN6c vector. After purification of the plasmid and sequencing we could conclude that the frame shift has been corrected. The corrected N b_95 amino acid sequence was defined in SEQ I D NO: 14, and a nucleotide sequence was defined in SEQ I D NO: 27. The characteristics of N b_95 were identified in similar way as described above, and are summarized in Table 1. The VH H gene was also recloned into the pMECS expression vector using the restriction enzymes Pstl and Bstell.

Table 1: Characteristics of Nb 49 and Nb 95

Parameter Values Nb_49 Values Nb_95 Test

LPS content 8.11 pg^g 8 pg^g LAL test (Lonza)

Ka (1/Ms) 1.015E+5 / BIAcore

Kd (1/s) 5.814E-4 /

KD(M) 5.726E-9 /

Expression vector pMECS and pMECS and

pHEN6c pHEN6c

Example 3. Bivalent constructs

Due to the fact that Nb_49 contains BstEII and EcoRI restriction sites in its DNA sequence we preferred to remove them in order to facilitate the generation of derivative constructs of the Nanobodies. Therefore, two consecutive splice overlap extension (SOE) reactions were performed using pHEN6cNb49 as template. In the first SOE reaction M13F (5'-TTCCCAGTCACGAC-3'; SEQ ID NO: 101)/Nb49BstEII F (5'-CTGTGCGCGCGTAGGCGACCCCAGTGGTGGC-3'; SEQ ID NO: 102) and Nb49BstEII R (5'-GCCACCACTGGGGTCGCCTACGCGCGCACAG-3'; SEQ ID NO: 103)/M13R (5'- CACACAGGAAACAGCTATGAC-3'; SEQ ID NO: 104) primer pairs were used to remove a BstEII site (t303- >c303). The resulting fragment was used for removal of EcoRI (t225->c225) site with a second SOE reaction using primer pairs M 13 F/Nb49 EcoRI F (5'-TATCTCCCGGGAGAACTCCAACAACACGGTG-3'; SEQ ID NO: 105) and M13R/Nb49EcoRI R (5'-CACCGTGTTGTTGGAGTTCTCCCGGGAGATA-3'; SEQ ID NO: 106). The resulting Nb49b DNA was cut using Pstl and BstEII restriction enzymes. Empty pHEN6c and pMECS vectors were opened using the same enzymes. The Nb49b gene was ligated into both vectors, resulting in pHEN6cNb49b and pMECSNb49b.

Bivalent constructs (Nb_49b-Nb_49b; Nb_49b-anti-Albumin(SAl); Nb_49b-anti-Albumin(SA16) have been created in order to increase the avidity/half-life extension. The Nb_49b-Nb_49b construct was cloned in pHEN6c and pMECS, while the Nb_49b-anti-Albumin(SAl and SA16) were only cloned in pHEN6c. Bivalent Nanobodies were generated by recombinantly attaching a linker sequence 3' of the VHH sequence using PCR primer biNbFvl (5'-TACCATGGCCCAGGTGCAGCTTCAGGAGTCYGGRGGAGG-3'; SEQ ID NO: 107) and primer biNbGSR (5'-

ATTCCTGCAGCTGCACCTGACTACCGCCGCCTCCGGAACCTCCACCGCCGGATCCGC CTCCGCCTGAGGAGACCG TGACCTGGGTBCC-3'; SEQ ID NO: 108), which code for a (G4S)3 (GGGGSGGGGSGGGGS; SEQ ID NO: 109) linker. For the bivalent constructs this PCR fragment was inserted 5' of the Nb49b gene in the pHEN6cNb49b and pMECSNb49b vectors with a Ncol/PstI restriction digest. The serum albumin binding constructs were made by inserting the Nb49b PCR fragment 5' of the NbSAl or NbSA16 gene in the pHEN6cNbSAl and pHEN6cNbSA16 vectors with a Ncol/PstI restriction digest. After ligation, the resulting bivalent Nanobody vector was expressed as described above. A summary of the bivalent Nbs generated and their respective amino acid sequences is provided in Table 7.

Example 4. Evaluation of cross-reactivity of anti-CD74 na nobodies

Both Nbs (Nb_49 and Nb_95) were also evaluated if they cross-react with human and mouse CD74. This was performed in first instance via ELISA, whereby a fixed amount (l^g/ml) of recombinant human CD74 ^{73 232} or mouse CD74 ^56"215 was coated. A titration of Nb_49 or Nb_95 starting at ^g/well followed by 1/3 serial dilution was added to the plate. Rabbit anti-VHH was used as the primary antibody, anti-rabbit HRP was used as the secondary antibody and the plate was developed using TMB substrate. Of note, the plate was washed between each step with 0.1%Tween20/PBS. The results for recombinant human CD74 ^{73 232} and mouse CD74 ^56"215 are shown in Figure 8. The results show that both Nb_49 and Nb_95 bind to human as well as mouse CD74 and thus can be considered cross-reactive. Yet, the binding signal of Nb_95 for both human and mouse CD74 is less strong in comparison to the original Nb_49.

Next, the BLItZ Biacore system (http://www.blitzmenow.com/ PALL Life Sciences) was used to confirm the ELISA results to determine the binding potential of the different anti-CD74 Nbs for both human and mouse CD74. To this end, biotinylated Nb_49 or Nb_95 were coated to a streptavidin sensor chip at a concentration of 25μg/ml. Both recombinant human CD74 ^73"232 and mouse CD74 ^56"215 were used at a concentration of 25 μg/ml, whereby the dilutions were made in PBS. Both Nb_49 and Nb_95 are able to bind recombinant human CD74 ^73"232 , whereby the binding signal is much lower for Nb_95 compared to Nb_49 (i.e. similar as ELISA results, see Figure 8). Yet, only Nb_49 is able to bind recombinant mouse CD74 ^56"215 while Nb_95 does not give any signal. The discrepancy between ELISA and BIAcore might reside in the fact that these are two completely different approaches to evaluate antigen-antibody interactions, whereby in the first situation the CD74 is coated while in the second case the Nb is coated. In the latter situation the Nb has to be able to capture the antigen. Hence, these results suggest that Nb_95 is only able to detect although with very low affinity and not capture mouse CD74, while Nb_49 is able to detect as well as capture mouse CD74.

Example 5. Expanding the repertoire of anti-CD74 Nbs

In order to expand the repertoire of anti-CD74 Nbs, a new library was generated following immunization of an alpaca with recombinant human CD74 ^72"232 rather than intact CD74 expressing cells (i.e. THP-1). The same panning procedure against recombinant human CD74 ^73"232 as described above was used. The results of the panning (3 rounds) and screening are shown in Table 2. Herein, 9 positive clones have been identified. Following colony PCR and sequencing we could confirm that 2 out of the 9 clones did not give any reliable Nanobody sequence, while 7 new Nanobodies against CD74 could be confirmed. From these 7 new binders, clone 3 and 7 belong to the same family and are cross-reactive with mouse CD74 in ELISA. In addition, clone 6 and 9 also belong to the same family. The newly generated Nanobodies, named "NbhCD74nl, NbhCD74n3, NbhCD74n4, NbhCD74n5, NbhCD74n6, NbhCD74n7, NbhCD74n9 and hereafter referred to as "Nb_01", "Nb_03", "Nb_04", "Nb_05", "Nb_06", "Nb_07", "Nb_09", have an amino acid sequence as defined in SEQ ID NOs: 15 to 21 (Table 7).

Table 2: Results from panning and screening.

Example 6. Anti-CD74 Nanobody binding studies on intact cells The next stage was to determine whether the anti-CD74 Nanobodies can bind its intact antigen on the original cell line (THP-1) and in addition, to determine if it cross-reacts with CD74 expressing murine cells. This was tested via flow-cytometry (FACS) using Alexa488 labeled Nanobody (Alexa Fluor ^® 488 Protein Labeling kit, Invitrogen). In particular, Nb_49 was tested here. 6.1 Binding Studies on THP1 Cells

The first FACS analysis was aimed to determine if Nb_49 can bind its intact antigen (CD74) on the surface of the original cell line from which it was created (THP-1). As shown in Figure 1, we first set a live gate (Figure 1A) and subsequently confirmed that CD74 is present on the THP-1 cells as evidenced by the right shift in the peak using a commercial monoclonal antibody against CD74 (green) in comparison to the isotype control antibody (red) (Figure IB). As shown in Figure 1C, Nb_49 could also bind to CD74 on the cells (blue), whereby the green peak which is indicative of the negative control (i.e. an irrelevant Nanobody recognizing an antigen that is not present on the cells) shows negligible movement from the red peak representing the cells without any antibody. Considering the signal of the Alexa488 labeled Nb_49 (blue), there is a positive right shift observed in comparison to the controls (i.e. no staining or the irrelevant Nanobody). From these results we can conclude that Nb_49 can indeed bind to CD74 on the surface of THP-1 cells. These results can also be expressed as mean fluorescence intensity, thereby giving numerical value to the results (Table 3).

Table 3: FACS results indicating Nb_49 binding as well as monoclonal antibody binding to CD74 on the surface of the THP-1 cell line. Results are shown as mean fluoresence intensities (arbitrary units).

6.2 Binding Studies on PECs

The same experiment as above was performed using mouse peritoneal exudate cells (PECs) to determine whether the Nanobody is cross-reactive with mouse cells. Flow-cytometry was performed on PECs derived from both naive as well as T.brucei brucei (ITG, Antwerp) infected (day 18 p.i.) WT C57Black/6 mice (Figure 2 and 3, respectively). In first instance a live gate was used to select for myeloid (CDllb ⁺ ) cells (panel A in Figure 2 and 3). Next, the expression of CD74 was determined on this selected population. Considering the naive mouse cells, as shown in Figure 4B, we confirmed that CD74 is present on the cells in the population. This is evidenced by the right shift in the observed peak when looking at a commercial monoclonal antibody against CD74 (blue) in comparison to the isotype control IgG (red). With this positive control, the next step was to determine if the Nb_49 could also bind to CD74 on the cells. As shown in Figure 3C, the signal indicative of the Nanobody (blue) indicates that there is a positive weak right shift observed in comparison to the irrelevant Nanobody (red). From these results we can conclude that Nb_49 can indeed bind to CD74 on the surface of naive mouse PECs. The same conclusion can be drawn from the infected PECs (Figure 3). To gain a clearer indication of the overall results, the binding signals are also shown as mean fluorescence intensity (Table 4).

Table 4: FACS results indicating Nb_49 binding as well as monoclonal antibody binding to CD74 on the surface of mouse PECs. Results are shown as mean fluoresence intensities (arbitrary units) for gated CDllb-positive cells (to select the myeloid cells among the total PECs). These results are representative for 1 out of 3 independent experiments.

Overall, from these results we can conclude that Nb_49 can indeed bind to CD74 on the surface of mouse PECs and is therefore cross-reactive. In addition, it seems that the CD74 expression on PECs decreases upon infection. 6.3 Material and Methods for binding studies

PECs were obtained from 3 naive (wild type (WT)) and T. brucei brucei (AnTatl.lE) infected mice that were euthanized via C0 ₂ in accordance to ethical commission standards (Ethical Commission Number 08-220-8). The peritoneal cavity was flushed with 10ml of ice cold PBS whereby PBS was injected and subsequently withdrawn from the cavity. The sample was then centrifuged (Eppendorf Centrifuge 5810 ) at 1400rpm for 7 minutes at 4°C. The supernatant was discarded and the cells were re- suspended in 2ml of RPMI 1640 (Invitrogen). The cell concentration was determined by adding 10μΙ of the cell suspension to 90μΙ of Turk's solution (Merck) and subsequently adding 10μΙ to a counting chamber (Assistant, Germany) and observed using light microscopy (Olympus CK2). The cells were adjusted with RPMI 1640 media to have a final concentration of 2*10 ⁶ cells/ml (stock solution). The Nb binding study experimental setup consisted of using five different incubation conditions, whereby in each condition 2*10 ⁵ cells (ΙΟΟμΙ of the stock 2*10 ⁶ cells/ml) were used. In the first condition the cells were incubated alone on ice for 30 minutes. In the second condition the cells were first treated with ^g of FC blocking IgG (2.4G2, VUB) for 30 minutes while on ice. Following treatment 0.2 μg of monoclonal anti-CD74 Ab (BD Pharmingen) was added and incubated on ice for 30 minutes. The third condition was also treated with ^g FC blocking IgG for 30 minutes on ice followed by the addition of 0.2 μg isotype control IgG. The fourth condition consisted of incubating cells along with 10μg (0.6 nM) of our Alexa488 labeled anti-CD74 Nb on ice for 30 minutes. The final condition consisted of incubating cells along with ^g (0.6 nM) of an irrelevant Alexa488 labeled nanobody (Nb_BCII10, VUB). Of note, each condition also had anti-CDllb-PE Cy7 (BD Pharmingen) and anti-B220-PerCP-Cy5.5 (BD Pharmingen) (0.2μΙ/10 ⁶ cells) added thereby allowing us to gate on Myeloid cells for binding analysis. In order to wash out unbound antibodies, all conditions were washed with 2ml of ice cold PBS and centrifuged (Eppendorf Centrifuge 5810R) at 1400rpm for 7 minutes at 4°C. The samples were then measured via FACS (BD FACS Canto™ II). Data was analyzed using FLOWJO 7.5 software.

THP-1 cell line culture was centrifuged (Eppendorf Centrifuge 5810R) at 1400rpm for 7 minutes at 4°C. The supernatant was discarded and the cells were re-suspended in 2ml of RPMI 1640. The cell concentration was determined by adding 10μΙ of the cell suspension to 90μΙ of Turk's solution and subsequently adding 10μΙ to a counting chamber (Assistant, Germany) and observed using light microscopy (Olympus CK2). The cells were adjusted with RPMI 1640 media to have a final concentration of 2*10 ⁶ cells/ml. In order to determine if our anti CD74 Nb is able to bind to its receptor on TH P-1 cells (the original cell line from which it was created), FACS analysis was completed. The experimental setup consisted of using five different incu bation conditions similar as described for PECs, with the exception of using anti-CDllb-PE Cy7 or anti-B220-PerCP-Cy5.5 since this is a human cell line and the antibodies are directed against murine cells. Example 7. Competition binding studies between anti-CD74 Nanobody and MIF for CD74

Next, we wanted to determine if the anti-CD74 Nanobodies are able to compete with MIF binding to its receptor. We focussed on using N b_49 and PECs (isolated as previously described) to address this question. In addition to WT mice, we also used M I F ^7" mice (Fingerle- owson et al. 2003) (all in C57Black/6 background) to avoid the presence of endogenous MI F produced by the PECs. Furthermore, ISO-1 (synthetic MI F inhibitor; Santa Cruz Biotechnology) was used as a positive control as it is documented to prevent the MI F/CD74 interaction (Al-Abed et al., 2005). The experimental setup consisted of using four different incubation conditions. In the first condition recombinant human MIF (SEQ I D NO: 9) which was labeled with APC (Lightning-link, Innova Biosciences) and added to PECs (2* 10 ⁵ cells) derived from WT or M I F ^7" C57Black/6 mice at a concentration of 200ng and incubated on ice for 30 minutes. In the second condition both cell types were incubated alone on ice for 30 minutes. The third condition consisted of first pre-incubating labeled MI F (200ng) with ISO-1 (5 g or 20nM) at 37°C for 30 minutes after which the mixture was added to both cell types and likewise incubated on ice for 30 minutes. The final condition consisted of adding both labeled MI F (200ng) as well as N b_49 (5 g or 0.3nM) to both cell types followed with incubation on ice for 30 minutes. In order to wash out unbound antibodies all conditions were washed with 2ml of ice cold PBS and centrifuged (Eppendorf Centrifuge 5810R) at 1400rpm for 7 minutes at 4°C. The samples were then measured via FACS (BD FACS Canto™ I I) and the data was analyzed using FLOWJO 7.5 software.

A representative FACS profile showing the difference in signal (indicated by the scatter plot) when comparing cells alone to cells where APC-labeled MI F has been added to PECs derived from naive WT mice is shown in Figure 4. Hereby, we can observe a significant shift/binding of APC-labeled MI F to PECs. Hence, a reduction in the shift in the APC signal is indicative for competition with MI F binding to the cells. The experiment as a whole is represented by mean fluorescence intensities in the APC channel, reflecting binding of MI F to the cells, are shown in Table 5.

Table 5: Summary of the FACS results indicating MIF binding/inhibition upon the addition of ISO-1 or Nb_49. Results are shown as mean fluorscence intensity of APC-labeled MIF bound to the cells.

Source of PECs No MI F added + MI F (200ng) + MI F (200ng) + MI F (200ng)

+ISO-1 ^g or 20 + N B_49 (5 g or nM) 0.3 nM)

WT mice 22.4 64.84 49.93 46.23 Source of PECs No MIF added + MIF (200ng) + MIF (200ng) + MIF (200ng)

+ISO-1 ^g or 20 + NB_49 (5μg or nM) 0.3 nM)

MIF-/- mice 25.6 119.5 60.97 69.37

Considering the results shown in Table 5, upon the addition of ISO-1 to WT PECs, we observed as expected that the MIF binding signal is reduced. This suggests that MIF binding does indeed occur via CD74. Upon the addition of Nb_49 we can also observe a reduced MIF signal, comparable to the reduction due to the addition of ISO-1. This suggests that the Nb_49 is able to bind the CD74 receptor and prevent directly or indirectly (via sterical hindrance) binding of MIF to its receptor CD74. Of note, we have to take into consideration that some of the loss of the MIF signal could be as a result of competition from endogenous MIF. This is illustrated by the fact that there is an increase in the detection of APC-labeled MIF binding to the surface of the cells in the MIF ^7" mice in comparison to the WT. Considering the results using PECs derived from MIF ^7" mice and addition of both ISO-1 and Nb_49 to, we also see a comparable reduction in the percentage of bound MIF. Overall it can be concluded from these results that Nb_49 appears to be a successful inhibitor of MIF binding to CD74 on mouse PECs, which is particularly obvious in MIF ^7" PECs where the results are not confounded by the presence of endogenous MIF. These observations were complemented by evaluating via ELISA if binding of Nb_49 to CD74 could be inhibited using recombinant human MIF (R&D systems). Herein a fixed amount of recombinant human CD74 (^g/ml) was coated on an ELISA plate. A titration of Nb_49 starting at 10μg/ml, followed by a 1/3 serial dilution was added to the plate in presence or absence of MIF (200ng/well). Mouse anti-HA (Covance, Brussels) was used as the primary antibody, goat anti-mouse-HRP (Sigma-Aldrich) was used as the secondary antibody and the plate was developed using TMB substrate. Of note, the plate was washed between each step with 0.1% Tween20/PBS. As shown in Figure 9, we were able to outcompete Nb_49 binding efficiently (50% reduction in signal) starting from a concentration of O.l^g/ml of Nb_49. From these results as well as FACS data (see above) we can indeed conclude that Nb_49 can interfere with MIF binding to CD74. Example 8. Competition binding studies between anti-CD74 Nanobody and D-DT (MIF2) for CD74

Given that D-dopachrome tautomerase (D-DT) is a mammalian structural MIF homologue able to bind, although with lower affinity, to CD74 and trigger similar transduction pathways, we evaluated whether the anti-CD74 Nb_49 was able to compete with D-DT binding on intact cells using flow-cytometry. The same experimental setup as in Example 6 was used, whereby PECs from naive MIF ^7" mice were used and either incubated with nothing, 200ng APC-labeled D-DT (ProSpec (www.prospecbio.com)) alone or in presence of 200ng APC-labeled D-DT + 5μg Nb-49. To asses binding we gated on CDllb+F4/80+ cells as being myeloid cells. The resulting AMFI (median fluorescence intensity) was calculated by subtracting the MFI of cells incubated with D-DT or D-DT+Nb_49 by the MFI of PECs alone (Figure 10). Thus, it can be concluded that Nb_49 can also interfere with D-DT binding to CD74. Example 9. Effect ofanti-CD74 Nanobody on LPS-induced TNF induction

9.1 Material and Methods for inhibition studies

To determine if the anti-CD74 Nanobodies could inhibit MIF-induced TNF-a production by myeloid cells following LPS stimulation, inhibition studies were performed on both mouse and human cells. Next, the culture supernatants were evaluated in a TNF-a ELISA (R&D systems) in accordance to the manufactures instructions. The samples were prepared for ELISA in accordance to the following procedure:

2*10 ⁵ THP-1 cells (ΙΟΟμΙ of a stock of 2*10 ⁶ cells/ml of ME medium prepared as described above) were incubated in a Nunc Maxisorp 96 well flat bottom tissue culture plate either alone, in combination with lOng of LPS, in combination with lOng of LPS and lC^g (0.6 nM) of our anti-CD74 Nb_49, or in combination with lOng of LPS and 10μg (0.6 nM) of an irrelevant Nanobody. The incubation time for each of the conditions was 3 hours at 37°C. After incubation the supernatant was collected for use in the human TNF-a ELISA (R&D systems). ELISA plates were read using an EL _X 808 Ultra micro plate reader spectrophotometer using Gen5 1.08 software.

- 2*10 ⁵ PECs cells (ΙΟΟμΙ of a stock of 2*10 ⁶ cells/ml of ME medium) previously isolated (as described above) from WT, MIF ^7" , and CD74 ^7" C57Black/6 mice were incubated in a Nunc Maxisorp 96 well flat bottom tissue culture plate either alone, in combination with lOOng of LPS, in combination with lOOng of LPS and 10μg (0.6 nM) of our anti-CD74 Nb_49, or in combination with 5μg (20nM) of ISO-1. The incubation time for each of the conditions was 3 hours at 37°C. After incubation the supernatant was collected for use in a mouse TNF-a ELISA (R&D systems). ELISA plates were read using EL _X 808 Ultra micro plate reader spectrophotometer using Gen5 1.08 software.

9.2 Results

The first set of experiments was conducted to determine whether the Nb_49 on itself has an effect on TNF-a induction by the cells, which would suggest that the Nb_49 would function as a MIF agonist. To this end, cells were incubated for 3 hours with Nb_49 or irrelevant Nb. Next, a TNF-a ELISA (R&D Systems) was conducted on the supernatant of both THP-1 cells and mouse PEC cultures, in accordance to the manufactures instructions. There was no significant induction of TNF-a produced when Nb_49 was added to the cell cultures in comparison to the non-stimulated cells (p value=0.6476 and 0.1946 respectively) (data not shown). Collectively, these results show that Nb_49 is not inducing any cell activation with respect to TNF-a induction.

The next set of results aims to determine whether the Nb_49 could reduce the MIF-induced TNF-a production in both THP-1 cells and mouse PECs that have been stimulated with LPS. In these assays, stimulation of the macrophages with LPS results in MIF secretion, which in turn acts on MIF receptors on the cells to induce secretion of TNF-a (Calandra and Roger, 2003). Successful inhibition of MIF binding to the receptor is in that case expected to reduce the resulting TNF-a secretion, which can be detected using ELISA. As shown in Figure 5, Nb_49 was also able to reduce the TNF-a production by 51% when 10μg was used. There was no effect observed when ^g of Nb_49 was used (data not shown). As expected the irrelevant Nanobody showed no inhibitory effect on the TNF-a production following LPS-stimulation. From these results we can conclude that the Nb_49 can significantly (p-value= 0.0157) inhibit MIF- mediated TNF-a induction following LPS-stimulation. The same experimental setup as described for the THP-1 cells was used for PECs from naive wild type (WT) mice, CD74 ^7" mice (gift from Richard Bucala, Yale University, New Haven, CT), and MIF ^7" mice (see Figure 6). The positive control, i.e. ISO-1 shows a 52% reduction in the amount of TNF-a produced following LPS-stimulation. Interestingly, Nb_49 was also able to significantly reduce, albeit borderline, the amount of TNF-a produced following LPS-stimulation (p-value=0.0498). These results suggest that Nb_49 is at least as potent as ISO-1 to block MIF-induced TNF-a induction following LPS-stimulation (and this using 0.6 nM Nb_49 versus 20 nM ISO-1). The level of TNF-a produced by PECs from MIF ^7" mice as well as CD74 ^7" mice stimulated with LPS in the presence of Nb_49 was comparable to that of PECs stimulated with LPS alone (p-value=0.1797 and 0.1946, respectively) (Figure 6B and C). This latter result confirms that the Nb_49 is only involved in inhibiting the binding of MIF to CD74. Example 10. African Trypanosomiasis as a model to study role of MIF/CD74 in the development of anemia of chronic disease

Previous research from our laboratory has shown that one of our in house models, African

Trypanosomiasis (T. brucei brucei, AnTatl.lE strain), exhibits characteristics of ACD and therefore can be used as a model to study the underlying mechanisms of ACD (Stijlemans et al., 2008). As demonstrated by this study, the infection is characterized by a high (most prominent) parasitemia peak which is controlled by a strong type-l immune response during the early stages of infection. This results in a rapid decrease in the amount of red blood cells (RBC) giving rise to the acute phase of anemia, which is followed by a partial recovery phase. The persistence of the pro-inflammatory immune response during the later stages (chronic phase) of infection results in a progressive and increased severity of anemia (data not shown). Macrophage hyper activation (as a result of the persistently high pro-inflammatory environment) has been proposed to be the main mechanism involved in destruction of red blood cells (via massive erythrophagocytosis) in the spleen and the liver of trypanosome infected hosts leading to anemia (one of the most important infection-associated immunopathological features (Stijlemans et al., 2008)) during the chronic phase of infection. Subsequent studies by Stijlemans et al., have unraveled that the anemia (due to the imbalance between erythrophagocytosis and erythropoiesis) is due to iron sequestration by the hyper activated macrophages thereby limiting iron availability to the host (Stijlemans et al., 2008). Interestingly, past laboratory analysis in the form of a comparative gene analysis between trypano-susceptible animals (exhibiting severe ACD) and trypano-tolerant animals (exhibiting reduced ACD) and focusing on molecules playing a key role in regulation of the inflammatory immune response allowed the identification of MIF as a potential candidate. It is yet unknown if CD74 and subsequently the MIF/CD74 interaction influences the development of ACD. Therefore, and first of all, it was determined whether MIF/CD74 interaction plays a key role in the anemia (ACD) development using the experimental African Trypanosomiasis model.

Initial steps were undertaken to establish whether the MIF/CD74 axis plays a role in experimental African Trypanosomiasis, which is used as a model to study anemia of inflammatory response (Al). This study is of importance for future studies whereby we aim at using the anti-CD74 Nb in vivo in a model of inflammation-associated pathology. As such, the infection parameters (parasitemia, anemia and survival) were determined for WT C57Black/6 mice as well as MIF and CD74 transgenic mice infected with the pleomorphic strain of T.brucei brucei (AnTatl.lE) (Figure 7).

7-8 weeks old WT, CD74 ^7" and MIF ^7" C57Black/6 mice (6 animals/group) were injected intra peritonealy (i.p) with 5000 T. brucei brucei parasites in a volume of 200μΙ. On days 0, 4, 5, 6, 7, 10, 14, 21, 28, 35 and 42 blood was collected (2.5μΙ) from the tip of the tail and diluted 1/200 using PMI + 5% FCS. 10μΙ was placed directly on a counting chamber and subsequently the number of parasites as well as the number of red blood cells present in the sample was counted using light microscopy (Olympus CK2). The data was analyzed using GraphPad Prism software. Following day 42 post infection the mice were left untouched and monitored for survival. As far as anemia is concerned, we observed a "comparable" drop in RBC percentages in all mouse strains during the early/acute phase of infection, followed by an initial recovery. This recovery is more pronounced in MIF ^7" and CD74 ^7" mice as compared to WT mice. Once entering into the chronic phase of infection, the WT mice become more anemic, while both the CD74 and MIF transgenic mice progressively recover although not reaching completely the level of naive mice (Figure 7B). Of note, statistically there is a significant difference in the red blood cell count (p-values < 0.01) between the WT mice in comparison to the MIF ^7" and CD74 ^7" mice each day during the chronic phase with the exception of day 28. Considering parasitemia (Figure 7A) there appears to be no significant difference in first peak parasitemia between WT and MIF ^7" mice. However, there seems to be a significantly higher first peak parasitemia in CD74 ^7" mice as compared to the WT and MIF ^7" mice (p-value <0.05). With respect to survival (Figure 7C), there does not seem to be any significant difference between the three types of mice. Collectively, these data suggest that blocking of/interfering with the MIF/CD74 interaction might be beneficial in preventing/alleviating Al development in this experimental model. Interestingly, we also observed that MIF ^7" mice exhibited greatly reduced liver pathogenicity (as evidenced morphologically and by reduced serum AST/ALT levels as read-out parameters for liver pathology) during the later stages of infection as compared to WT mice, suggesting that the MIF/CD74 axis might also play a role in the development of inflammation-associated liver pathology. Thereto, the anti-CD74 Nbs are evaluated in a Concanavalin A (Con-A) induced liver model. Concanavalin A (Con-A) injection into mice provides a model for the investigation of T-cell activation-associated diseases of the liver, such as autoimmune hepatitis. Investigation into the role of MIF in Con-A induced liver pathology has led to the conclusion that lack of MIF protects mice against Con-A induced liver injury (Nakajima et al., 2006). This suggests that the MIF/CD74 axis plays an important role in the development of the pathology. Hence, the Con-A induced liver model is scrutinized to validate the in vivo applicability of the anti-CD74 Nbs.

Example 11. Evaluation of anti-CD74 Nbs in tumor models.

The involvement of the MIF/CD74 in cancer, i.e. tumor progression and metastasis, has been widely accepted (Borghese & Clanchy 2011). Moreover, given that CD74 is preferentially expressed in hematopoietic cancers and some solid tumors and has a fast internalization, makes CD74 an ideal target for cancer therapy (Govindan et al. 2013). Using the MC38 colon carcinoma model we could establish that it indeed expressed CD74 (Figure 11). To this end, 2*10E5 cells were incubated for 30 minutes with 5μg of either Alexa-488 labeled Nb_49 (orange line) or Nb-BCIIlO (blue line), while one ice. As negative control we also used cells without adding anything (red line). We could conclude that Nb_49 can recognize tumor cells, as evidenced by the right shift in signal (left panel) or expressed in median fluorescence intensity (right panel). Similar results were obtained on a Lewis Lung carcinoma model (3LL ). Overall, these results reveal that Nb_49 allows to detect its antigen (i.e. CD74 receptor) on the surface of intact tumor cells. Example 12: Evaluation of the internalization potential of the anti-CD74 Nbs.

To determine if Nb_49 and Nb_95 are internalized following binding to CD74 and therefore could be used as targeting entities to deliver toxin, an internalization assay was performed. For this experiment, a Raji cell line (i.e. Burkitt's lymphoma) was used. First, 20C^g of both Nanobodies were labeled using the Ph Rhodo labeling kit (Molecular Probes). Subsequently, 2*10 ⁵ Raji cells were incubated with 2^g of either labeled Nb_49, Nb_95 or irrelevant Nb (BC-II10) at 37°C overnight and 5% C0 ₂ . Following incubation the cells were washed with PBS and measured via FACS. If the dye is internalized (assuming the dye is exposed to a pH drop) then fluorescence would be observed. As shown in Figure 12 (left panel) we can observe a slight shift in the peaks corresponding to either Nb_95 or Nb_49 (blue line) compared to untreated cells ((orange line) and cells incubated with Nb-BCIIlO (red line). This indicates that indeed the Nb_49 can be internalized. In Figure 12 (right panel), the results are also presented in median fluorescence intensity (MFI), whereby an increase in intensity could be observed when cells are incubated with Nb_49. Collectively, these results indicate that the Nb_49 can be internalized following binding to CD74 on the surface of tumor cells (i.e. Raji cell line) and hence might be a valuable tool to be used as drug-delivery entity to target cancer cells, which intrinsically have a higher CD74 turnover.

Table 6. Amino acid sequences of human and mouse CD74, human MIF and human MIF2 (D-DT) (uniprot identifier)

Name SEQ ID NO Amino acid sequence

Human CD74 1

(P04233: HG2A_HUMAN) 296 AA MHRRRSRSCREDQKPVMDDQRDLISNNEQLPMLGRRPGAPES

KCSRGALYTGFSILVTLLLAGQATTAYFLYQQQGRLDKLTVTSQNL

QLENLRMKLPKPPKPVSKMRMATPLLMQALPMGALPQGPMQ

NATKYGNMTEDHVMHLLQNADPLKVYPPLKGSFPENLRHLKNT

METIDWKVFESWMHHWLLFEMSRHSLEQKPTDAPPKVLTKCQ

EEVSHIPAVHPGSFRPKCDENGNYLPLQCYGSIGYCWCVFPNGT

EVPNTRSRGHHNCSESLELEDPSSGLGVTKQDLGPVPM

Recombinant human CD74 ^73"232 2 YPYDVPDYAQQQGRLDKLTVTSQNLQLENLRMKLPKPPKPVSK (R&D systems Catalog Number: MRMATPLLMQALPMGALPQGPMQNATKYGNMTEDHVMHLL 3590-CD; NP_004346.1; QNADPLKVYPPLKGSFPENLRHLKNTMETIDWKVFESWMHHW Includes N-terminal LLFEMSRHSLEQKPTDAPPKESLELEDPSSGLGVTKQDLGPVPM hemagglutinin tag (bold

underlined))

Recombinant human CD74 3 HHHHHHQQQGRLDKLTVTSQNLQLENLRMKLPKPPKPVSKMR (Sino Biologicals, Catalog MATPLLMQALPMGALPQGPMQNATKYGNMTEDHVMHLLQN Number: 11091-H07H derived ADPLKVYPPLKGSFPENLRHLKNTMETIDWKVFESWMHHWLLF from the human CD74 isoform b EMSRHSLEQKPTDAPPKESLELEDPSSGLGVTKQDLGPVPM (NP_004346.1) and fused with a

polyhistidine tag at the N- terminus (bold underlined) Name SEQ ID NO Amino acid sequence

Mouse CD74 4

(P04441: HG2A_MOUSE) 279 AA MDDQRDLISNHEQLPILGNRPREPERCSRGALYTGVSVLVALLLA

GQATTAYFLYQQQGRLDKLTITSQNLQLESLRMKLPKSAKPVSQ

MRMATPLLMRPMSMDNMLLGPVKNVTKYGNMTQDHVMHL

LTRSGPLEYPQLKGTFPENLKHLKNSMDGVNWKIFESWMKQW

LLFEMSKNSLEEKKPTEAPPKVLTKCQEEVSHIPAVYPGAFRPKCD

ENGNYLPLQCHGSTGYCWCVFPNGTEVPHTKSRGRHNCSEPLD

MEDLSSGLGVTRQELGQVTL

Recombinant mouse CD74 (R&D 5 QQQGRLDKLTITSQNLQLESLRMKLPKSAKPVSQMRMATPLLM Systems, Catalog Number: RPMSMDNMLLGPVKNVTKYGNMTQDHVMHLLTRSGPLEYPQ NP_034675.1, Gln56-Leu215, LKGTFPENLKHLKNSMDGVNWKIFESWMKQWLLFEMSKNSLE with an N-terminal HA-tag (bold EKKPTEAPPKEPLDMEDLSSGLGVTRQELGQVTLYPYDVPDYA underlined)

Extracellular domain of human 6

CD74: AA 73-296 QQQGRLDKLTVTSQNLQLENLRMKLPKPPKPVSKMRMATPLL

MQALPMGALPQGPMQNATKYGNMTEDHVMHLLQNADPLKV

YPPLKGSFPENLRHLKNTMETIDWKVFESWMHHWLLFEMSRHS

LEQKPTDAPPKVLTKCQEEVSHIPAVHPGSFRPKCDENGNYLPLQ

CYGSIGYCWCVFPNGTEVPNTRSRGHHNCSESLELEDPSSGLGV

TKQDLGPVPM

Extracellular domain of mouse 7

CD74: AA 56-279 QQQGRLDKLTITSQNLQLESLRMKLPKSAKPVSQMRMATPLLM

RPMSMDNMLLGPVKNVTKYGNMTQDHVMHLLTRSGPLEYPQ LKGTFPENLKHLKNSMDGVNWKIFESWMKQWLLFEMSKNSLE EKKPTEAPPKVLTKCQEEVSHIPAVYPGAFRPKCDENGNYLPLQC HGSTGYCWCVFPNGTEVPHTKSRGRHNCSEPLDMEDLSSGLGV TRQELGQVTL

Human MIF (P14174; 8 MPMFIVNTNVPRASVPDGFLSELTQQLAQATGKPPQYIAVHVV MIF_HUMAN) PDQLMAFGGSSEPCALCSLHSIGKIGGAQNRSYSKLLCGLLAERLR

ISPDRVYINYYDMNAANVGWNNSTFA

Recombinant human MIF ^{2 115} 9 PMFIVNTNVPRASVPDGFLSELTQQLAQATGKPPQYIAVHVVPD (R&D systems Catalog Number: QLMAFGGSSEPCALCSLHSIGKIGGAQNRSYSKLLCGLLAERLRIS 289-MF) PDRVYINYYDMNAASVGWNNSTFA

Human MIF2 D-Dopachrome 10

Tautomerase (D-DT) (Q53Y51; MPFLELDTNLPANRVPAGLEKRLCAAAASILGKPADRVNVTVRP Q53Y51_HUMAN) GLAMALSGSTEPCAQLSISSIGVVGTAEDNRSHSAHFFEFLTKELA

LGQDRILIRFFPLESWQIGKIGTVMTFL

Human MIF2 D-Dopachrome 11

Tautomerase (D-DT) ordered MGSSHHHHHHSSGLVPRGSHMPFLELDTNLPANRVPAGLEKRL from ProSpec CAAAASILGKPADRVNVTVRPGLAMALSGSTEPCAQLSISSIGVV

(www.prospecbio.com) including GTAEDNRSHSAHFFEFLTKELALGQDRILIRFFPLESWQIGKIGTV a 20 aa N-terminal Histidine Tag MTFL

(bold underlined) Table 7. List of nanobodies (monovalent and bivalent format)

Nanobody Nanobody SEQ ID NO

reference short

number notation

N b_07 20 QVQLQESGGGLVQPGGSLRLSCAASGFTFSSYVMSWVR

QAPGKGPEWVSAVNGGGSATYADSVKGRFTISRDNVK

NTLYLQMNSLKPEDTAVYYCATVAWNWGQGTQVTVSS

N b_09 21 QVQLVESGGGSVQPGGSLRLSCAASGFTFSSSVMSWVR

QAPGKGLEWVSSIYSYSYNTYYADSVKGRFTISTDNAKNT LYLQMNSLKS E DTAVYYC AYG E D FSS WGQGTQVTVSS

Bivalent

(linker is

underlined)

N b49b- 22 QVQLQESGGGSVQAGGSLRLSCVASGLVYSLKAYDWG N b49b WARQAPGKECELVGRI PNGATVYI DSVKGRFIISRENS

N NTVYLQM NSLKFE DTAVYYCARVG DPSGGWCKGQGT

QVTVSSGGGGSGGGGSGGGGSQVQLQESGGGSVQAG

GSLRLSCVASGLVYSLKAYDWGWARQAPGKECE LVGRI

MPNGATVYI DSVKGRFIISRENSNNTVYLQMNSLKFEDT

AVYYCARVGDPSGGWCKGQGTQVTVSS

N b49b- 23 QVQLQESGGGSVQAGGSLRLSCVASGLVYSLKAYDWG anti- WARQAPGKECELVGRIM PNGATVYI DSVKGRFIISRENS al bumin N NTVYLQM NSLKFE DTAVYYCARVG DPSGGWCKGQGT (SA1) QVTVSSGGGGSGGGGSGGGGSQVQLQESGGGLVQAG

GSLRLSCAASGRN ISEYVMGWFRQAPGKEREFVAAISW

SAGNIYYADSVKGRFTISRDNAKNTVHLQMNTLRPEDTA

VYYCAAGRYSAWYVAAYEYDYWGQGTQVTVSS

N b49b- 24 QVQLQESGGGSVQAGGSLRLSCVASGLVYSLKAYDWG anti- WARQAPGKECE LVGRIM PNGATVYI DSVKGRFIISRENS al bumin N NTVYLQM NSLKFE DTAVYYCARVG DPSGGWCKGQGT (SA16) QVTVSSGGGGSGGGGSGGGGSQVQLQESGGGLVQAG

GSLRLSCAASGRNISEYVMGWFRQAPGKEREFVAAISW

SSH NTYYADSVKGRFTISRDNAKNTVH LQMNTLRPEDTA

VYYCAAGRYSAWYVAAYEYDYWGQGTQVTVSS Nanobody Nanobody SEQ ID NO

reference short

number notation

Nucleotide sequence

CA4435 N b_49 25 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTA

CAGGCTGGAGGGTCTCTGAGACTCTCCTGTGTCGCCTC

TG G ATTAGTCTAC AG CCTC AA AG CCTATG ACTG G G G CT

GGGCCCGTCAGGCTCCGGGGAAGGAGTGCGAATTGG

TCG G C AG A ATTATG CCTA ATG GTG CG AC AGTCTATATC

GACTCCGTAAAGGGCCGATTCATTATCTCCCGGGAGA

ATTCCAACAACACGGTGTATCTACAAATGAATAGCCTG

AAATTTGAGGACACGGCCGTGTATTACTGTGCGCGCG

TAGGTGACCCCAGTGGTGGCTGGTGCAAGGGCCAGG

GGACCCAGGTCACCGTCTCCTCA

N b_49b 26 C AG GTG C AG CTG C AG G AGTCTG G G G G AG G CTCG GTA

CAGGCTGGAGGGTCTCTGAGACTCTCCTGTGTCGCCTC

TG G ATT AGTCTAC AG CCTC AA AG CCTATG ACTG G G G CT

GGGCCCGTCAGGCTCCGGGGAAGGAGTGCGAATTGG

TCG G C AG A ATTATG CCTA ATG GTG CG AC AGTCTATATC

GACTCCGTAAAGGGCCGATTCATTATCTCCCGGGAGA

ACTCCAACAACACGGTGTATCTACAAATGAATAGCCTG

AAATTTGAGGACACGGCCGTGTATTACTGTGCGCGCG

TAGGCGACCCCAGTGGTGGCTGGTGCAAGGGCCAGG

GGACCCAGGTCACCGTCTCCTCA

N b_95 27 C AG GTG C AG CTG C AGG AGTTG G G G G AG GCTCTG GTG

C AG CCTG G G G G GTCC ATG AG ACTCTCCTGTG C AG CCT CTG G AA AT ATCTTC AGT ATC AATTCC ATG G CCTG GT AC CGCCAGCCTTCAGGGAAGCGGCGCGAGTTGGTCGCA G C ATTTACTCG CG GTG GTA AT AT AA ACT ATG C AG ACTC CGTGAAGGAGCGATTCACCATCTCCAGAGACAACGCC AAGAACACGATGTATCTGCAAATGAACAACCTGAAAC CCGAGGATACGGCCGTCTATTATTG 1 1 1 1 GTAGATTGG AGTGACTCCGACGATTCTGAGTATGAGGACTGGGGCC AGGGGACCCAGGTCACCGTCTCCTCA Table 8. Combinations of FRs and CDRs of nanobodies

Nanobody Short FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 reference notation

number

NbhCD74n4 Nb_04 QVQLVESG GFSFS MSWV INSGGG YYATSV TSQDWH WGKGTQ

GALVQPGG SYF RQAP ST KGRFTIS Y VTVSS

SL LSCAAS GKGLE RDNAK

(SEQID (SEQID (SEQID (SEQID WVSS NTLYLQ

(SEQID NO: NO: 41) NO: 59) NO: 77) NO: 86)

MNSLKP

32) (SEQID

EDTAVY NO: 50)

YC

(SEQID

NO: 68)

NbhCD74n5 Nb_05 QVQLQESG GFTFD IGWFR ISSSDGS YYADSV AALELPGI WGQGT

GGLVQAGG DYA QAPG T KGRFTIS KWGAETR QVTVSS

SLRLSCADS KEREG SDNAKN WDRWAE

(SEQID (SEQID (SEQID VSC TVYLQM YDY

(SEQID NO: NO: 42) NO: 60) NO: 87)

NSLKPE

33) (SEQID (SEQID

DTAVYY NO: 51) NO: 78)

C

(SEQID

NO: 69)

NbhCD74n6 Nb_06 QVQLQESG GFTFS MSWV IYSYTSN YYADSV AYGEDFS WGQGT

GGLVQPGG SSA RQAP T KGRFTIS S QVTVSS

SLRLSCAAS GKGLE TDNAKN

(SEQID (SEQID (SEQID (SEQID WVSS TLYLQM

(SEQID NO: NO: 43) NO: 61) NO: 79) NO: 88)

NSLKSE

34) (SEQID

DTAVYY NO: 52)

C

(SEQID

NO: 70)

NbhCD74n7 Nb_07 QVQLQESG GFTFS MSWV VNGGG TYADSV ATVAWN WGQGT

GGLVQPGG SYV RQAP SA KGRFTIS QVTVSS

(SEQID

SLRLSCAAS GKGPE RDNVK

(SEQID (SEQID NO: 80) (SEQID WVSA NTLYLQ

(SEQID NO: NO: 44) NO: 62) NO: 89)

MNSLKP

35) (SEQID

EDTAVY NO: 53)

YC

(SEQID

NO: 71) Nanobody Short FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 reference notation

number

NbhCD74n9 Nb_09 QVQLVESG GFTFS MSWV YSYSYNT YYADSV AYGEDFS WGQGT

GGSVQPGG SSV RQAP KGRFTIS S QVTVSS

(SEQID

SLRLSCAAS GKGLE TDNAKN

(SEQID NO: 63) (SEQID (SEQID WVSSI TLYLQM

(SEQID NO: NO: 45) NO: 81) NO: 90)

NSLKSE

36) (SEQID

DTAVYY NO: 54)

C

(SEQID

NO: 72)

REFERENCES

Abbas A., and Lichtman A. (2003). Cellular and Molecular Immunology. Philadelphia: Saunders, Elsevier Inc.

Al-Abed Y., Dabideen D., Aljabari B., Valster A., Messmer D., Ochani M.,....,Tracey K.(2005). ISOl Binding to the Tautomerase Active Site of MIF Inhibits Its Pro-inflammatory Activity and

Increases Survival in Severe Sepsis. The Journal of Biological Chemistry, 280(44), 36541-36544.

Berkova Z, Tao RH, Samaniego F. (2010). Milatuzumab - a promising new immunotherapeutic

agent. Expert Opin Investig Drugs, 19(1), 141-9

Borghese F and Clanchy F. (2011). CD74: an emerging opportunity as a therapeutic target in cancer and autoimmune disease. Expert Opin. Ther. Targets, 15(3), 237-251. Bozza M., Martins Y., Carneiro

L, Paiva C. (2011). Macrophage Migration Inhibitory Factor in Protozoan Infections. Journal of Parasitology Research, 2012, 1-12.

Calandra T., Roger T. (2003). Macrophage Migration Inhibitory Factor: A Regulator of Innate Immunity.

Nature Reviews, 3, 791-800. Cao and Suresh (2000), Journal of Drug Targetting, 8, 4, 257 .

Chen P., Luo Y., Wang W., Wang J., Lai W., Hu S., Cheng K., Al-Abed Y.(2010). ISO-1, a macrophage migration inhibitory factor antagonist, inhibits airway remodeling in a murine model of chronic asthma. Mol. Med., 16(9-10), 400-408.

Conrath K, Pereira AS, Martins CE, Timoteo CG, Tavares P, Spinelli S, Kinne J, Flaudrops C, Cambillau C, Muyldermans S, Moura I, Moura JJ, Tegoni M, Desmyter A. Camelid nanobodies raised against an integral membrane enzyme, nitric oxide reductase. Protein Sci. 2009 Mar;18(3):619-28.

Conrath K. E., M. Lauwereys, M. Galleni et al., Antimicrob Agents Chemother 45 (10), 2807 (2001).

Conroy H., Mawhinney L., Donnelly S.(2010). Inflammation and cancer: macrophage migration inhibitory factor (MIF)-the potential missing link. Q J Med, 103, 831-836. Daryadel A., Grifone r., Simon H., Yousefi S. (2006). Apoptotic neutrophils release macrophage migration inhibitory factor upon stimulation with tumor necrosis factor-alpha. J Biol Chem, 281(37), 27635-27661.

Desmyter A, Spinelli S, Payan F, Lauwereys M, Wyns L, Muyldermans S, Cambillau C (2002). Three camelid VHH domains in complex with porcine pancreatic alpha-amylase. Inhibition and versatility of binding topology. J Biol Chem. 277: 23645-50.

Desmyter A., Transue T., Ghahroudi M., Dao-Thi M., Poortmans F., Hamers R., Muyldermans S. & Wyns L, 1996, Nat. Struct. Biol., p. 803-811.

Fingerle-Rowson G, Petrenko O, Metz CN, Forsthuber TG, Mitchell R, Huss R, Moll U, Muller W, Bucala R. (2003). The p53-dependent effects of macrophage migration inhibitory factor revealed by gene targeting.Proc Natl Acad Sci U S A. 100: 9354-9. Flaster H., Bernhagen J., Calandra T., Bucala R. (2007). The Macrophage Migration Inhibitory Factor- Glucocorticoid Dyad: Regulation of Inflammation and Immunity. Molecular Endocrinology, 21(6), 1267-1280.

Govindan SV, Cardillo TM, Sharkey RM, Tat F, Gold DV, Goldenberg DM (2013). Milatuzumab-SN-38 conjugates for the treatment of CD74+ cancers. Mol Cancer Ther. 12:968-78.

Hamers-Casterman, C, T. Atarhouch, S. Muyldermans et al. Naturally occurring antibodies devoid of light chains. Nature 363, 446-448, doi:10.1038/363446a0 (1993).

Korotkov KV, Pardon E, Steyaert J, Hoi WG. Crystal structure of the N-terminal domain of the secretin GspD from ETEC determined with the assistance of a nanobody. Structure. 2009 Feb 13;17(2):255-65.

Lefranc, M. P., C. Pommie, et al. (2003). "IMGT unique numbering for immunoglobulin and T cell

receptor variable domains and Ig superfamily V-like domains." Developmental and

Comparative Immunology 27(1): 55-77.

Leng L, Metz CN, Fang Y, Xu J, Donnelly S, Baugh J, Delohery T, Chen Y, Mitchell RA, Bucala R.(2003).

MIF signal transduction initiated by binding to CD74. J Exp Med., 197(ll),1467-76

Leng L. and Bucala R. (2006). Insight into the biology of macrophage migration inhibitory factor (MIF) revealed by the cloning of its cell surface receptor. Cell Res., 16(2), 162-168

Leng L, Chen L., Fan J., Greven D., Arjona A., Du X., , Bucala R. (2010). A Small-Molecule Macrophage

Migration Inhibitory Factor Antagonist Protects against Glomerulonephritis in Lupus-Prone NZB/NZW Fl and MRL//pr Mice. The Journal of Immunology, 186(1), 527-538.

Morand E., Leech M., Bernhagen J. (2006). MIF: a New Cytokine Link Between Rheumatoid Arthritis and Atherosclerosis . Nature Reviews Drug Discovery, doi:10.1038/nrd2029.

Riechmann and Muyldermans J. Immunol. Methods 2000; 240: 185-195.

Saerens D., Ghassabeh G., Muyldermans S., 2008, Current Opinion in Pharmacology 8, p. 600-608. Stijlemans B., Vankrunkelsven A., Brys L., Magez S., De Baetselier P. (2008). Role of iron homeostasis in trypanosomiasis-associated anemia. Immunobiology, 213(2008), 823-83

Wesolowski et al. (2009) , Med. Microbiol. Immunol. 198:157.

Zhang Z, Chen X., Lin P., Jiang H., Xiong X., Zhu Y.(2009). Role of macrophage migration inhibitory factor in mice with sepsis and after effect of its intervention. Zhongguo Wei Zhong Bing Ji Jiu Yi Xue, 21(8), 481-484.