FIELD

[0001] This disclosure relates to fusion proteins comprising a CD83 binding domain and methods of use.

CROSS REFERENCE TO PRIORITY APPLICATION AND INCORPORATION BY REFERENCE

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63/399,432, filed August 19, 2022, which is hereby incorporated by reference.

[0003] Incorporated by reference in its entirety is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: XML file named "58055_Seqlisting.xml", 40,926 bytes, created August 14, 2023.

BACKGROUND

[0004] CD83 is a 45 kDa, type-l membrane glycoprotein which is a member of the immunoglobulin superfamily. CD83 is expressed on the surface of activated human dendritic cells (DCs) as well as allo-activated T cells and some cancer cells. As such, CD83 is an attractive target for delivery of payload to target cells. Therapeutics which target CD83 show great promise in the treatment and prevention of cancer and immunological disorders, including Graft versus Host Disease (GvHD) and other autoimmune conditions.

SUMMARY

[0005] The disclosure provides a fusion protein comprising (a) a CD83 binding domain comprising the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 23, and SEQ ID NO: 24 and (b) an Fc domain. In various aspects, the CD83 binding domain comprises the amino acid sequences of SEQ ID NOs: 1-3 or the amino acid sequences of SEQ ID NOs: 11-13. Optionally, the CD83 binding domain comprises the amino acid sequence of SEQ ID NO: 25, such as the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 14; and/or the CD83 binding domain comprises the amino acid sequence of SEQ ID NO: 5; and/or the CD83 binding domain comprises the amino acid sequence of SEQ ID NO: 26, such as the amino acid sequence of SEQ ID NO: 6 or the amino acid sequence of SEQ ID NO: 16; and/or the CD83 binding domain comprises the amino acid sequence of SEQ ID NO: 7. For example, the disclosure provides a CD83 binding protein comprising the amino acid sequence of SEQ ID NO: 8. The disclosure further provides a fusion protein comprising a CD83 binding domain comprising the amino acid sequence of SEQ ID NO: 18. Optionally, the Fc domain comprises the amino acid sequence set forth in SEQ ID NO: 32. In various aspects, the fusion protein is conjugated to an effector moiety, such as a therapeutic moiety or a detectable moiety.

[0006] A nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein, a host cell comprising the nucleic acid, a method of making the fusion protein, and a composition comprising the fusion protein also are provided. [0007] It should be understood that, while various embodiments in the specification are presented using "comprising" language, under various circumstances, a related embodiment may also be described using "consisting of" or "consisting essentially of" language. The disclosure contemplates embodiments described as "comprising" a feature to include embodiments which "consist of" or "consist essentially of" the feature. The term "a" or "an" refers to one or more. For example, "a multispecific construct" is understood to represent one or more multispecific constructs. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein. The term "or" should be understood to encompass items in the alternative or together, unless context unambiguously requires otherwise.

[0008] It should also be understood that when describing a range of values, the disclosure contemplates individual values found within the range. In any of the ranges described herein, the endpoints of the range are included in the range. However, the description also contemplates the same ranges in which the lower and/or the higher endpoint is excluded. When the term "about" is used, it means the recited number plus or minus 5%, 10%, or more of that recited number. The actual variation intended is determinable from the context.

[0009] Additional features and variations of the invention will be apparent to those skilled in the art from the entirety of this application, including the figures and detailed description, and all such features are intended as aspects of the invention. Likewise, features of the invention described herein can be re-combined into additional embodiments that also are intended as aspects of the invention, irrespective of whether the combination of features is specified as an aspect or embodiment of the invention. All method steps described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0010] The entire document is intended to be related as a unified disclosure, and it should be understood that all combinations of features described herein (even if described in separate sections) are contemplated, even if the combination of features is not found together in the same sentence, or paragraph, or section of this document. Section headings are provided only for readability of the disclosure, and are not meant to be limiting. Also, only such limitations which are described herein as critical to the invention should be viewed as such; variations of the invention lacking limitations which have not been described herein as critical are intended as aspects of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 is listing of amino acid sequences and nucleic acid sequences referenced in the application. Sequences are provided for two representative CD83 binding domains (referenced as C10 and H1 in the figure). In SEQ ID NOs: 8, 9, 18, and 19, bolded regions correspond to CDR sequences. The CDR sequences were determined using the Kabat classification (numbering) scheme. In SEQ ID NOs: 9 and 19, the italicized region corresponds to a 6X His tag, and the underlined and italicized region corresponds to an HA tag. The disclosure contemplates sequences similar to SEQ ID NOs: 9 and 19 lacking the tags. In SEQ ID NOs: 34, 35, 37, and 38, the italicized region corresponds to the CD83 binding portion and the bold region corresponds to the linker. In SEQ ID NOs: 35 and 38, the underlined sequence corresponds to a signal sequence. The disclosure contemplates peptides comprising the sequence of SEQ ID NOs: 35 and 38 without the signal sequence.

[0012] Figures 2A-2D are schematics of different potential configurations for a complex comprising fusion proteins comprising a binding domain and an Fc domain. Fc domains of two fusion proteins may dimerize to form a complex comprising two binding domains (referenced as "nb #1” and "nb #2” in the figure). In various aspects of the disclosure, each "arm” of the complex may comprise a different binding domain (e.g., a different CD83 binding domain) (Figure 2C), although this is not required. Also, optionally, a binding domain may be fused to the N- terminus and the C-terminus of the Fc domain. These binding domains may be the same or may be different (see Figure 2D). Fc domains of multiple fusion proteins may associate, creating a complex with increased valency, such as three or four binding domains for a target (e.g., three or four CD83 binding domains).

[0013] Figure 3 illustrates bio-layer interferometry results demonstrating the affinity of a representative fusion protein of the disclosure, C10-Fc, for human CD83.

[0014] Figures 4A and 4B illustrate flow cytometry data confirming that a representative fusion protein of the disclosure, C10-Fc, binds CD83. Figure 4A illustrates flow cytometry data confirming binding of membrane bound CD83 on the U937 cell line. Figure 4B illustrates flow cytometry data collected during titration of C10-Fc binding of U937 cells.

[0015] Figures 5A and 5B illustrate flow cytometry data confirming that a representative fusion protein of the disclosure, C10-Fc, binds CD83. Figure 4A illustrates flow cytometry data confirming binding of membrane bound CD83 on the THP-1 cell line. Figure 4B illustrates flow cytometry data collected during titration of C10-Fc binding of THP-1 cells.

[0016] Figures 6A and 6B illustrate flow cytometry data confirming that a representative fusion protein of the disclosure, C10-Fc, binds CD83. Figure 4A illustrates flow cytometry data confirming binding of membrane bound CD83 on the OCI-AML3 cell line. Figure 4B illustrates flow cytometry data collected during titration of C10-Fc binding of OCI-AML3cells.

[0017] Figure 7 illustrates flow cytometry data confirming that a representative tetravalent fusion protein complex of the disclosure comprising four C10 binding domains binds CD83.

[0018] Figure 8 illustrates anti-CD83 C10-Fc uptake in non-specific organs and tumor over 48 h following administration of the fusion protein to mice bearing U937 subcutaneous xenografts. The %ID/g is provided on the y-axis, and the tissues tested are provided on the x-axis. Each tissue was tested for the presence of C10-Fc at four hours (right bar), 24 hours (middle bar), and 48 hours (left bar) following administration. C10-Fc displayed rapid and prolonged tumor localization DETAILED DESCRIPTION

[0019] The disclosure provides a fusion protein comprising a CD83 binding domain and an Fc domain. CD83 is expressed on myeloid and lymphoid malignancies. CD83 is also expressed on alloreactive T cells, as well as mature or activated myeloid cells. The fusion protein provided herein is useful in a variety of contexts where binding to CD83 is desired. For example, the fusion protein may be conjugated to a detectable label and, as such, is useful in diagnostic or laboratory research contexts. The fusion protein also may be conjugated to a therapeutic agent, thereby providing a means of effectively delivering the therapeutic agent to a cell displaying CD83, such as CD83+ hematologic malignancies (including, but not limited to, acute myeloid leukemia, T or B cell acute lymphoblastic leukemia/lymphoma, diffuse large B cell lymphoma, mantle cell lymphoma, chronic lymphocytic leukemia, myeloma, and Hodgkin lymphoma) and CD83+ solid malignancies (including, but not limited to, breast cancer, melanoma, lung cancer, ovarian cancer, cervical cancer, and colorectal cancer).

[0020] The fusion protein comprises a CD83 binding domain comprising the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 23, and SEQ ID NO: 24. In various aspects, the CD83 binding domain is a nanobody, also known as a single domain antibody or VHH, comprising a heavy chain complementarity determining region (HCDR)-1 comprising SEQ ID NO: 1, an HCDR2 comprising SEQ ID NO: 23, and an HCDR3 comprising SEQ ID NO: 24. Nanobodies have the benefit of being small (15 kDa), exhibiting greater tissue infiltration and target binding compared to larger constructs, and surviving acidic environments (such as, for instance, the leukemia niche) better than full length human antibodies. SEQ ID NO: 23 comprises the amino acid sequence INX1X2GTTN, wherein Xi is H or Y and X2 is E or D. Thus, in various aspects, the CD83 binding domain comprises an HCDR2 comprising SEQ ID NO: 2 or SEQ ID NO: 12. SEQ ID NO: 24 comprises the amino acid sequence X3ARSIFGNS, wherein X3 is I or N. In various aspects, the CD83 binding domain comprises an HCDR3 comprising SEQ ID NO: 3 or SEQ ID NO: 13. The disclosure provides a fusion protein comprising a CD83 binding domain comprising the amino acid sequences of SEQ ID NO: 1 for HCDR1, SEQ ID NO: 2 for HCDR2, and SEQ ID NO: 3 for HCDR3. Alternatively, the disclosure provides a fusion protein comprising a CD83 binding domain comprising the amino acid sequences of SEQ ID NO: 11 for HCDR1, SEQ ID NO: 12 for HCDR2, and SEQ ID NO: 13 for HCDR3.

[0021] Generally, nanobody structure entails CDR sequences interspersed with framework region (FR) sequences. A general nanobody structure comprises FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR1 to FR4 refer to framework regions 1 to 4, respectively. Optionally, the binding protein comprises an FR1 amino acid sequence of SEQ ID NO: 25 (QVQLVESGGGLVQAGGSLTLSCAAX4, wherein X4 is S or F), such as the amino acid sequence of SEQ ID NO: 4 or the amino acid sequence of SEQ ID NO: 14. In various aspects, the CD83 binding domain comprises an FR2 amino acid sequence of SEQ ID NO: 5. In various aspects, the CD83 binding domain comprises an FR3 amino acid sequence of SEQ ID NO: 26 (YKDX ₅ VKGRFAISRDNAKNTVSLQMNSLX ₆ PEDTAVYFC, wherein X ₅ and X ₆ is S or T), such as the amino acid sequence of SEQ ID NO: 6 or the amino acid sequence of SEQ ID NO: 16. Optionally, the CD83 binding domain comprises an FR4 amino acid sequence of SEQ ID NO: 7. [0022] In exemplary aspects, the disclosure provides a fusion protein comprising a CD83 binding domain comprising the amino acid sequence of SEQ ID NO: 8. In alternative aspects, the disclosure provides a fusion protein comprising a CD83 binding domain comprising the amino acid sequence of SEQ ID NO: 18. The fusion protein may comprise all or part of the amino acid sequences of SEQ ID NOs: 9 or 18.

[0023] The CD83 binding domain of the fusion protein binds CD83, preferably binding more frequently, more rapidly, with greater duration and/or with greater affinity to CD83 than it does with alternative targets (e.g., other cell surface proteins). For example, the CD83 binding domain preferably binds CD83 with greater affinity, avidity, more readily, and/or with greater duration than it binds to other non-CD83 proteins. It is also understood that, for example, a CD83 binding domain which specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. In general, under designated assay conditions, the CD83 binding domain binds preferentially to a CD83 and does not bind in a significant amount to other components present in a test sample.

[0024] A variety of assay formats may be used to select or characterize a CD83 binding domain. For example, solid-phase ELISA immunoassay, immunoprecipitation, BIAcore™ (GE Healthcare, Piscataway, NJ), fluorescence-activated cell sorting (FACS), Octet™ (ForteBio, Inc., Menlo Park, CA) and Western blot analysis are among many assays that may be used to characterize binding to CD83. Typically, a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 times background. Optionally, the CD83 binding domain binds CD83 with a KD (Equilibrium Dissociation Constant = Antibody Off Rate I Antibody On Rate) of less than about 500 nM, such as less than about 200 nM, less than about 150 nM, less than about 100 nM, less than about 50 nM, less than about 10 nM (such as between about 10 nM and about 5 nM, or less than about 5 nM (e.g., between about 5 nM and 1 about nM). For example, the CD83 binding domain may bind CD83 with a KD of about 1 .5 nM to about 3.5 nM.

[0025] The CD83 binding domain is fused to an Fc domain. An "Fc region" or "Fc domain" is a polypeptide comprising the constant region of an antibody, e.g., at least the second and third constant region domains of an antibody (CH2 and CH3 regions), and optionally comprising at least a portion of the first constant region domain (CH1), and/or in some cases, all or part of the hinge. Thus, an Fc domain can refer to the last two antibody constant region domains (e.g., CH2 and CH3) of IgA, IgD, and IgG, the last three antibody constant region domains of IgE and IgM, and the hinge N-terminal to these domains, in various aspects of the disclosure. For IgA and IgM, the Fc domain may include the J chain. For IgG, the Fc domain may comprise immunoglobulin domains Cy2 and Cy3 and the lower hinge region between Cyl and Cy2. In some aspects of the disclosure, the Fc domain comprises the CH2 and CH3 domains with a truncated CH1 domain. Merely to illustrate, the human IgG Fc region is usually defined to include residues E216 or C226 or P230 and extend to the C-terminus (numbering is according to the EU index as in Kabat); CH1 generally corresponds to positions 118-220 according to the EU index as in Kabat, CH2 generally corresponds to positions 237-340 according to the EU index as in Kabat, and CH3 generally corresponds to positions 341-447 according to the EU index as in Kabat, all in the context of IgG. The Fc domain is preferably derived from an IgG Fc domain, e.g., lgG1, lgG2, lgG3 or lgG4 Fc domain. In an exemplary aspect of the disclosure, the Fc domain is an lgG1 Fc domain. A representative Fc domain of the disclosure is provided as SEQ ID NO: 32. The Fc domain optionally comprises an amino acid sequence at least about 90% identical (e.g., at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100%) identical to SEQ ID NO: 32. The disclosure contemplates a fusion protein which is associated with another fusion protein of the disclosure to form an antibody-like structure. In this aspect, two CD83 binding domains are each fused to an Fc domain, and the Fc domains associate to form an antibody-like structure (or "complex”) wherein the CD83 binding domains are fused to, e.g., each CH2-CH3 arm and are available for bivalent binding. It will be appreciated that disclosure herein relating to, e.g., nucleic acids, host cells, methods of manufacture, kits, and methods of use of the fusion protein also are applicable to complexes comprising the fusion protein.

[0026] The CD83 binding domain and the Fc domain are optionally fused via a domain linker. A "domain linker” is an amino acid sequence which links the CD83 binding domain to the Fc domain. Any suitable linker can be used so long as the CD83 binding domain retains the ability to bind CD83. The linker may be any length, e.g., 1-60 amino acids, such as 1-5, 1-10, 2-18, 4-30, 5-15, or 10-25 amino acids, in length. A suitable linker comprises the sequence GPGGP (SEQ ID NO: 31). Suitable linkers also include, but are not limited to, glycineserine polymers, including for example (GS)n, (GGGGS)n (also referenced as "G4S,” SEQ ID NO: 27), (SGGGG)n (SEQ ID NO: 28), (GSSSS)n (SEQ ID NO: 29), and (GGGS)n (also referenced as "G3S,” SEQ ID NO: 30), where n is an integer of at least one (e.g., one, two, three, four, five, six, seven, eight, nine, or ten). Suitable linkers also include glycine-proline polymers, such as SEQ ID NO: 31, as well as glycine-proline polymers that also comprise glutamine, such as GPGGQ (SEQ ID NO: 39).

[0027] The disclosure provides a fusion protein comprising the amino acid sequence set forth in SEQ ID NO: 34. The disclosure also provides a fusion protein comprising the amino acid sequence set forth in SEQ ID NO: 37. Also provided is a fusion protein comprising the amino acid sequence of SEQ ID NO: 35 or 38.

[0028] A binding domain (e.g., the CD83 binding domain) can be attached in multiple orientations at either the N terminus or C terminus of an Fc domain. Attaching at one terminus results in a bivalent construct (two binding domains) when Fc domains associate in an antibody-type structure. Binding domains (e.g., CD83 binding domains) can be attached simultaneously to both termini. When Fc domains associate to create an antibody-like complex, the result is a tetravalent construct with four total binding domains. A fusion protein comprising a binding domain fused to an Fc domain may alternatively form a complex with a fusion protein comprising an Fc domain with binding domains fused at both termini, thereby forming a trivalent complex.

[0029] In various aspects of the disclosure, the fusion protein is conjugated to an effector moiety. By "effector moiety” is meant a moiety which conveys some attribute to the fusion protein, such as a biological activity or the ability to detect the fusion protein. Effector moieties include, but are not limited to, detectable moieties, therapeutic moieties (e.g., cytotoxic moieties), moieties that impact pharmacokinetic properties, and moieties that facilitate purification.

[0030] Therapeutic moieties include, but are not limited to, chemotherapeutics, radionuclides, antibiotics, immunosuppressants, antimicrobials, enzyme inhibitors, opioids, antihistamines, cell growth inhibitors, cytotoxic agents, analgesics, anti-inflammatory agents, anti-angiogenic factors, anti-secretory factors, small molecule inhibitors, toxins, and the like. Small molecule drugs (e.g., chemical compounds with biological activity having a molecular weight (MW) less than or equal to about 5 kDa, such as less than or equal to about 1 .5 kDa) are contemplated, as well as biologies and metals and other particles.

[0031] Examples of small molecule inhibitors include, but are not limited to, immunosuppressive agents such as tacrolimus and sirolimus; Janus kinase (JAK) inhibitors such as ruxolitinib, baricitinib, fedratinib, and tofacitinib; Aurora kinase A inhibitors such as alisertib, Bruton's tyrosine kinase inhibitors such as Ibrutinib, Rho- associated coiled-coil kinase 2 inhibitors such as belumosudil (KD025), JAK2/Aurora kinase A dual inhibitors such as AJI-100 and AJI-214, and STAT3 inhibitors such as S3I-201. The structures and activity of AJI-100, AJI- 214, and S3I-201 are further described in Yang et al., Oncotarget. 2014 May; 5(10): 2947-2961; and Siddiquee et al., PNAS 2007; 104(18): 7391-7396, and the structures are provided below:

S3I-201

[0032] In various aspects, the effector moiety is a chemotherapeutic such as, but not limited to, cyclophosphamide, doxorubicin, paclitaxel, arabinosylcytosine, venetoclax, decitabine, azacitabine, methotrexate, Vinca alkaloids, idarubicin, and melphalan. In various aspects, the effector moiety is a topoisomerase inhibitor, such as a topoisomerase I inhibitor (e.g., exatecan, indotecan, indimitecan, or topotecan) or a topoisomerase II inhibitor (e.g., PNU-159682, etoposide, doxorubicin, amonafide, or mitoxantrone). In various aspects, the effector moiety may be a DNA alkylator (e.g., pyrrolobenzodiazepine). [0033] In various aspects, the effector moiety is a toxin (such as a mitotic inhibitor). Exemplary toxins include, but are not limited to, Diphtheria toxin, Monomethyl auristatin E, Monomethyl auristatin F, Pseudomonas exotoxin (e.g., PE38), Mertansine, Calicheamicins, Deruxtecan, Govitecan, and Tesirine.

[0034] The effector moiety is, in various aspects of the disclosure, a detectable moiety. A detectable moiety is an agent detectable, for example, by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Examples of detection methods include, but are not limited to, magnetic resonance imaging (MRI) scans, x-radiographic imaging, computed tomographic (CT) scans, and positron emission tomography (PET) scans. Examples of detectable moieties (also referenced as imaging agents) include, but are not limited to, fluorescent dyes, chemiluminescent compounds, radioisotopes, electron-dense reagents, paramagnetic metal ions, positron emitting metals, enzymes, colored particles, biotin, and dioxigenin. Examples of enzymes suitable for use as a detectable moiety include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and acetylcholinesterase. Examples of fluorescent materials suitable for use as detectable moieties include, but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin. Examples of bioluminescent materials include, but are not limited to, luciferin and aequorin. Examples of radioactive material suitable for use as detectable moieties include, but are not limited to, radioactive iodine ( ¹¹¹ l, ¹²¹ l, ¹²³ l, ¹²⁴ l, ¹²⁵ l, and ¹³¹ l), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H), indium ( ¹¹¹ ln, ¹¹² ln, ^113m ln, and ^115m ln), technetium ("Tc and ^99m Tc), thallium ( ²⁰¹ Ti), gallium ( ⁶⁸ Ga and ⁶⁷ Ga), palladium ( ¹⁰³ Pd), molybdenum ( ⁹⁹ Mo), xenon ( ¹³³ Xe), fluorine ( ¹⁸ F), ²²⁵ Ac, ¹⁹⁸ Au, ²¹¹ At, ²¹² Bi, 2i3 _Bij i53 _{S rrij} 177 _LU , i59 _{Gd j} wpm, i4o _a , i75 _Y b, ¹⁸⁸ Ho, ⁸⁸ Y, ⁹⁰ Y, ⁴⁷ Sc, ²²³ Ra, ¹⁸⁸ Re, ¹⁸⁸ Re, ¹⁴² Pr, ¹⁰⁵ Rh, ⁸⁴ Cu, ⁸⁷ Cu, ⁸⁹ Zr, and ⁹⁷ Ru. In various aspects, the effector moiety is a |3-ray emitting metal (e.g., ⁹⁰ Y, ¹⁷⁷ Lu, ¹⁸⁸ Re, ¹⁹⁸ Au, ⁶⁴ Cu, or ¹⁶⁶ Ho). In various aspects, the effector moiety is an a-ray emitting metal (e.g., ²¹¹ At, ²¹² Bi, ²¹³ Bi, ²²³ Ra, or ²²⁵ Ac). In various aspects, the effector moiety is a PET radioisotope (e.g., ¹⁸ F, ⁶⁴ Cu, ⁶⁸ Ga, ⁸⁶ Y, ⁸⁹ Zr, or ¹²⁴ l) or a SPECT radioisotope (e.g., ⁶⁷ Cu, ^99m Tc, or ¹¹¹ 1 n). Optionally, the fusion protein comprises a chelate group selected from Desferoxamine (DFO); 2,2',2”-(1 ,4,7-triazacyclononane-1 ,4,7-triyl)triacetic acid (NOTA); and 2,2',2",2"'-(1 ,4,7,10-Tetraazacyclododecane-1,4,7, 10-tetrayl)tetraacetic acid (DOTA).

[0035] Exemplary therapeutic effector moieties include photosensitizing agents. Photosensitizing agents are agents (e.g., chemical compounds) that require a secondary, physical intervention in order to activate its cytotoxic properties. Merely to illustrate, a photosensitizing compound in its singlet state absorbs a photon of light at a specific wavelength, resulting in a short-lived excited singlet state, which can be converted by intersystem crossing to a longer-lived triplet state. This photosensitizing agent in a triplet state can exhibit cytotoxic properties due to photooxidation by radicals, singlet oxygen, and photoreaction not involving oxygen.

[0036] Techniques for conjugating moieties, such as those described above, to Fc domains are well-known. Chemical conjugation of a moiety to an Fc domain may include use of, e.g., an optionally substituted alkylene (e.g., Ci-Ce alkylene), optionally substituted heteroalkylene (Ci-Ce heteroalkylene), optionally substituted alkenylene (e.g., C2-C6 alkenylene), optionally substituted heteroalkenylene (e.g., C2-C6 heteroalkenylene), optionally substituted alkynylene (e.g., C2-C6 alkynylene), optionally substituted heteroalkynylene (e.g., C2-C6 heteroalkynylene), optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, optionally substituted heteroarylene, a peptide (e.g., a dipeptide), -(C=O)-, a disulfide, a hydrazone, a -(CF^CFWJp- group (wherein p is an integer from 1-6), a ((CH2) _m O) _n (CH2)m-- group (where n and each m are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10), or a combination thereof. An exemplary conjugation method involves attaching a moiety via a lysine residue in an antibody using any of a number of coupling chemistries (active ester, isothiocyanate, isocyanates, acyl azides, sulphonyl chloride, carbonyl, epoxide, carbonates, fluorobenzene deriv., imidoesters, carbodiimides, or anhydrides). While lysine is a common amino acid on the antibody surface used for conjugation, attachment to other amino acids also is appropriate (e.g., arginine, cysteine, glutamine, asparagine, glutamate, aspartate, serine, and/or tyrosine). Conjugation of metal ions to antibodies is further described in, e.g., U.S. Patent No. 4,741 ,900. Methods of conjugating drugs to antibody fragments also are described in, e.g., International Patent Publication Nos. WO 2014/068443, WO 2014/134457, WO 2013/082254, WO 2012/104344, WO 2016/045674, as well as U.S. Patent Publication No. 2010/0136033. Additional conjugation methods are further described in, e.g., Ducry, L, (Ed) (2013), Antibody-Drug Conjugates book, Methods in Molecular Biology volume 1045, Chapters 9-12, Humana Press; and Hermanson, G (2013) Bioconjugate Techniques book, Chapters 2-6, Academic Press; Hackenberger & Schwarzer, Angew Chem Int Ed Engl. 2008; 47: 10030-74 (native chemical ligation); Behrens & Liu, MAbs. 2014, 6: 46-53 (enzyme mediated conjugation); Castaneda et al., Chem Comm, 2013; 49: 8187-8189 (disulphide bridging technologies); Badescu et al., Bioconjug Chem. 2014; 25: 1124-36 (disulphide bridging technologies); Barbas et al., Bioconjugate Chemistry 2014; 25: 1402-1407 (use of methylsulphonylphenyloxadiazole reactive linkers to form thioethers), and Barbas et al., Bioconjugate Chemistry 2013; 24: 520-532 (tyrosine-click reaction).

[0037] Nucleic acids, host cells

[0038] The disclosure further provides nucleic acid molecules comprising nucleic acid sequences encoding all or part of the fusion protein described herein. The nucleic acid molecule may be provided in an expression construct or vector, e.g., a plasmid, cosmid, YAC, or a viral vector. Expression vectors provide for expression in vitro and/or in vivo (e.g., in a suitable host cell or organism). Many expression vectors are commercially available. Suitable viral vectors include, for example, retrovirus, adenovirus, parvovirus (for example, adeno- associated viruses), coronavirus, negative strand RNA viruses such as ortho-myxovirus (for example, influenza virus), rhabdovirus (for example, rabies and vesicular stomatitis virus), paramyxovirus (for example, measles and Sendai), picornavirus, alphavirus, herpesvirus (for example, Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), lentivirus, and poxvirus (for example, vaccinia, fowlpox, and canarypox). Vector components may include an origin of replication, one or more marker genes, a multiple cloning site containing recognition sequences for restriction endonucleases, enhancer elements, promoters, transcription termination sequences, and the like. Indeed, the nucleic acid of the disclosure may be operably linked to one or more regulatory elements, such as a promoter, enhancer, and/or terminator.

[0039] The disclosure further provides a host cell comprising the nucleic acid or the expression vector described herein. A "host cell" refers to a cell (e.g., prokaryotic or eukaryotic) into which exogenous nucleic acid has been introduced, including the progeny of such cells. A host cell may be a bacterial cell, a yeast cell, an insect cell, or a mammalian cell. In various aspects, the cell is a eukaryotic cell, such as a mammalian cell (e.g., a human cell; or cell from a non-human primate such as ape, chimpanzee, monkey, or orangutan; or cell from a domesticated animal, such as a dog or cat; or cell from livestock, such as a horse, cow, pig, sheep, or goat; or cell from another mammalian species including, without limitation, mice, rats, guinea pigs, rabbits, hamsters, birds (e.g., chicken, duck, goose, quail or pheasant) and the like). Examples of human cells include, but are not limited to, PER.C6 cells (described in, e.g., International Patent Publication No. WO 01/38362), MRC-5 (ATCC CCL-171), WI-38 (ATCC CCL-75), HEK-293 cells (ATCC CRL-1573), HeLa cells (ATCC CCL2), and fetal rhesus lung cells (ATCC CL-160). Examples of non-human primate cells are Vero cells (ATCC CCL81), COS-1 cells (ATCC CRL-1650), and COS-7 cells (ATCC CRL-1651). Examples of dog cells are MDCK cells (ATCC CCL-34). Examples of rodent cells are hamster cells, such as BHK21-F, HKCC cells, or Chinese hamster ovary (CHO) cells. Examples of insect cells include, but are not limited to, SF9 cells (ATCC CRL-1711), Sf21 cells (IPLB- Sf21), MG1 cells (BTI-TN-MG1), and High Five™ cells (BTI-TN-5B1-4).

[0040] A method of making the fusion protein described herein is provided. The method comprises culturing a host cell comprising a nucleic acid encoding the fusion protein under conditions which allow expression, and recovering the product. Culture conditions and methods for generating recombinant proteins are known in the art. Similarly, protein purification methods are known in the art and utilized herein for recovery of recombinant proteins from cell culture media. In some aspects, methods for protein purification include filtration, affinity column chromatography, cation exchange chromatography, anion exchange chromatography, and concentration. Optionally, the method comprises formulating the recovered product.

[0041] Compositions, Methods of use

[0042] The disclosure provides a composition comprising the fusion protein described herein, including compositions comprising a physiologically acceptable carrier. Physiologically acceptable carriers and excipients are preferably nontoxic to recipients under the administration conditions selected for a particular subject. Acceptable carriers and excipients include, for example, buffers (such as phosphate, citrate, HEPES, and TAE), antioxidants (such as ascorbic acid and methionine), preservatives (such as hexamethonium chloride, octadecyldimethylbenzyl ammonium chloride, resorcinol, and benzalkonium chloride), proteins (such as human serum albumin), hydrophilic polymers (such as polyvinylpyrrolidone), amino acids (such as glycine, glutamine, histidine, and lysine), and carbohydrates (such as glucose, mannose, sucrose, and sorbitol). Physiologically acceptable carriers also include, e.g., sterile water and physiological saline. Physiological/pharmaceutical compositions are preferably sterile and stable under conditions of manufacture and storage. Sterile solutions may be prepared, for example, by filtration through sterile filtration membranes.

[0043] The fusion protein (optionally conjugated to an effector moiety and/or optionally in the form of a complex of two fusion proteins) may be administered by any acceptable route, including parenteral and subcutaneous routes of administration. Suitable routes of administration include intravenous, intradermal, intramuscular, intraperitoneal, intranodal and intrasplenic, for example. In exemplary aspects, the fusion protein is provided via systemic (e.g., intravenous) administration.

[0044] The disclosure provides a method comprising administering to a subject in need thereof the fusion protein of the disclosure, optionally conjugated to any one or more of the effector moieties described herein and/or optionally complexed with another fusion protein of the disclosure. The amount of fusion protein (optionally conjugated to any one or more of the effector moieties described herein and/or optionally complexed with another fusion protein of the disclosure) provided should be sufficient to achieve a desired goal (e.g., detection of CD83-expressing cells or a biological effect, such as cell death) in a clinically relevant period of time. An amount of fusion protein of the disclosure may comprise, e.g., from about 100 ng/kg to about 50 mg/kg administered to the subject, including all integer values within those ranges, although in some embodiments the methods may be performed using a dose outside this range.

[0045] A method of treating cancer in a subject also is provided. The method comprises administering to the subject the fusion protein described herein (optionally in the form of a complex) conjugated to an effector moiety that directly or indirectly induces tumor cell death (e.g., a chemotherapeutic agent, a toxin, and the like). In various aspects, the cancer is a cancer which expresses CD83, such as CD83+ hematologic malignancies and CD83+ solid malignancies (i.e., solid tumors). CD83 may be detected in a variety of ways. In various aspects, any method of the disclosure may comprise detecting CD83 expression in a target cell (e.g., cancer cell or immune cell). Methods of determining protein levels or identifying expression of proteins in a target cell type are well known in the art. Optionally, CD83 protein levels are determined via western blot or dot blot analysis, immunohistochemistry (IHC, e.g., quantitative immunohistochemistry), flow cytometry, immunocytochemistry, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunosorbent spot (ELISPOT; Coligan, J. E., et al., eds. (1995) Current Protocols in Immunology. Wiley, New York), radioimmunoassay, or chemiluminescent immunoassay. CD83 protein levels also may be measured via geometric mean fluorescence intensity (gMFI). CD83 may be quantified and monitored in real-time on cells of interest (e.g., cancer cells, CD4+ T cells, Tfh, or B cells) by, for example, peripheral blood draws and measured by flow cytometry. CD83 RNA is optionally measured via real time polymerase chain reaction (qRT-PCR) or RNA sequencing. The sequence of CD83 is known in the art. See, e.g., Genbank Accession Nos. NM_001040280 and NM_004233. It will be appreciated that the methods described above which utilize an antigen-binding protein to detect CD83 may be employed using the fusion protein described herein.

[0046] The cancer is optionally prostate cancer, lung cancer, colon cancer, rectum cancer, urinary bladder cancer, melanoma, kidney cancer, renal cancer, oral cavity cancer, pharynx cancer, pancreas cancer, uterine cancer, thyroid cancer, skin cancer, head and neck cancer, cervical cancer, ovarian cancer, or hematopoietic cancer. In various aspects, the cancer is a CD83+ hematologic malignancy, such as but not limited to, acute myeloid leukemia, T or B cell acute lymphoblastic leukemia/lymphoma, diffuse large B cell lymphoma, mantle cell lymphoma, chronic lymphocytic leukemia, myeloma, and Hodgkin lymphoma. In various aspects, the cancer is a CD83+ solid malignancy, such as but not limited to, breast cancer, melanoma, lung cancer, ovarian cancer, cervical cancer, and colorectal cancer. When appropriate, the method may comprise administering the fusion protein conjugated to a therapeutic agent prior to, simultaneously with, or following, a different therapy, such as chemotherapy, surgical resection of a tumor, or radiation therapy.

[0047] In a representative aspect, the cancer is acute myeloid leukemia (AML). The fusion protein described herein has significant advantages compared to currently available therapeutics for AML. For example, existing anti-AML CAR (e.g., which target CD33 or CD123) involve a risk of myeloid aplasia and often require follow-up allogeneic hematopoietic cell transplantation a few weeks after cell infusion. In contrast, CD83 is significantly reduced or absent from healthy hematopoietic stem cells, myeloid precursors in the marrow, and steady state circulating neutrophils, and therefore is not associated with the risks of existing treatment options.

[0048] The disclosure also provides a method of treating or reducing the risk of developing an alloimmune or autoimmune condition in a subject in need thereof. The method comprises administering to the subject the fusion protein described herein (optionally in the form of a complex) conjugated to a therapeutic agent. Autoimmunity is an immune response to self-antigens, which results in the body attacking normal cells and tissues. There are more than 80 types of autoimmune diseases that present unique clinical symptoms and affect different parts of the body. In contrast, alloimmunity is an immune response to antigens from different members of the same species. Alloimmunity is triggered primarily by differences in the major histocompatibility complex between individuals within a species. The body's response to alloimmune conditions and autoimmune conditions mimics the response against infection, involving activation of immune cells, inflammation, and tissue damage. In various aspects, the alloimmune or autoimmune condition is Graft versus Host Disease (GvHD), systemic lupus erythematosus (SLE), multiple sclerosis, Sjogren's syndrome, systemic sclerosis/scleroderma, cutaneous sclerosis, ulcerative colitis, inflammatory bowel disease, or rheumatoid arthritis.

[0049] Graft versus Host Disease (GvHD) occurs when donor immune cells transferred to an allogeneic recipient attack tissues in the recipient. The skin, liver, and intestinal tract are often affected, resulting in significant damage and morbidity. GvHD is a common complication of allogeneic hematopoietic cell transplantation (HOT), but also may develop after solid organ transplantation. In various aspects, the alloimmune or autoimmune condition is GvHD. The GvHD may be acute GvHD or may be chronic GvHD. While the symptoms of acute and chronic GvHD overlap, acute and chronic GvHD are immunologically distinct complications (e.g., complications of alloHCT). Acute GvHD is primarily mediated by alloreactive T cells. Acute GvHD is potentiated by early tissue damage during the conditioning regimens (e.g., radiation and/or chemotherapy) used to prepare patients to receive allogeneic hematopoietic cells. Acute GvHD typically impacts the skin, gut, and liver and often occurs before day 100 post-transplant. Chronic GvHD stems from a unique triad of thymic impairment, auto- and alloanti body production by reactive B cells, and systemic tissue fibrosis. Chronic GvHD is a systemic disease affecting host mucocutaneous tissues, musculoskeletal system, gastrointestinal tract, liver, lungs, immune system, and cardiovascular system. Chronic GvHD typically occurs later after transplantation (e.g., after day 100 post alloHCT), and can essentially develop at any time, even decades after the procedure. [0050] The disclosure provides a method for treating or preventing rejection after solid organ or vascularized composite allotransplantation (e.g., heart, lung, liver, kidney, face, upper extremity, or abdominal wall tissue or organ transplantation) or rejection associated with a cell therapy (including, e.g., banked, off-the-shelf, allogeneic cell therapy (such as chimeric antigen receptor T cell or regulatory T cell therapy)), where CD83+ al loreactive T cells could be tolerized or eliminated with a CD83 nanobody fusion Fc, optionally conjugated to an immunosuppressant, toxin, or radio-isotope. In yet another aspect of the disclosure, the disclosure provides a method for preventing or treating acute or chronic GvHD after allogeneic hematopoietic cell transplantation. The method comprises administering to the subject in need thereof the fusion protein described herein conjugated to a therapeutic moiety, such as a moiety described herein. In various aspects of the disclosure, the anti-CD83 binding domain-Fc domain fusion protein is optionally conjugated to an imaging or PET label, which allows in vivo monitoring of GVHD or organ rejection.

[0051] The term "treat," as well as words related thereto, does not necessarily imply 100% or complete treatment. Rather, there are varying degrees of treatment of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the methods of treating a condition or disease of the present disclosure can provide any amount or any level of treatment. Furthermore, the treatment provided by the method may include treatment of one or more conditions or symptoms or signs of the disease being treated. For instance, the treatment method of the present disclosure may inhibit one or more symptoms of the disease. Also, the treatment provided by the methods of the present disclosure may encompass slowing the progression of the disease. The treatment provided by the presently disclosed method may delay the onset or reoccurrence/relapse of the disease being treated. For example, a therapeutic response would refer to one or more of the following improvements in a disease or condition: (1) a reduction in the number of neoplastic or unwanted immune cells; (2) an increase in target (e.g., neoplastic) cell death; (3) inhibition of target (e.g., neoplastic) cell survival; (4) inhibition (i.e., slowing to some extent, preferably halting) of tumor growth or appearance of new lesions in the context of cancer; (5) slowing of disease progression; (6) an increased patient survival rate; (7) downgrade of stage of a disease (e.g., Stage 2 to Stage 1) and/or (8) some relief from one or more symptoms associated with the disease or condition. Disease states may be monitored by, e.g., clinical examination, X-ray, computerized tomography (CT, such as spiral CT), magnetic resonance imaging (MRI), positron emission tomography (PET), ultrasound, endoscopy and laparoscopy, tumor marker levels in the context of cancer (e.g., carcinoembryonic antigen (CEA)), cytology, histology, biopsy sampling, and/or counting of target cells in circulation. These methods also are typically used to diagnose and stage, e.g., cancer. The term "effective," in various aspects of the disclosure, refers to a sufficient quantity of a composition which ameliorates one or more causes or symptoms of a condition or disease.

[0052] In certain aspects, the method of treating a disease or condition may be regarded as a method of inhibiting the disease or condition or a symptom thereof. As used herein, the term "inhibit" and words stemming therefrom may not be a 100% or complete inhibition or abrogation. Rather, there are varying degrees of inhibition of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. The presently disclosed methods may inhibit the onset or re-occurrence of the condition or a symptom thereof to any amount or level.

[0053] When appropriate, the method may comprise administering the fusion protein prior to, simultaneously with, or following, a secondary treatment, such as immunotherapy, chemotherapy, surgical resection of a tumor, radiation therapy, and the like. For example, the fusion protein when conjugated to a detectable moiety may be useful in characterizing the effectiveness of a therapy seeking to kill CD83-expressing cells. In this regard, the fusion protein may be administered to a subject after one or more courses of the therapy, to determine the existence or amount of CD83-bearing cells following the treatment. For example, the disclosure contemplates a method wherein the fusion protein conjugated to a detectable moiety is administered following surgical tumor resection and detecting the detectable moiety, which allows determination of whether tumor cells remain after resection. Alternatively, a fusion protein conjugated to a detectable moiety may be administered to a subject prior to a therapy in order to determine if a subject has CD83-bearing cells and should undergo a course of treatment.

[0054] In some embodiments, the method(s) described herein further comprises administration of one or more other therapeutic agents. In this regard, the method optionally comprises administering to the subject a second therapeutic agent selected from the group consisting of a corticosteroid, methotrexate, cyclosporine, mycophenolate mofetil, tacrolimus, sirolimus, everolimus, antithymocyte globulin, alemtuzumab, dexamethasone, cyclophosphamide, ibrutinib, imatinib, infliximab, etanercept, tocilizumab, alemtuzumab, basiliximab, daclizumab, rituximab, denileukin diftitox, pentostatin, ruxolitinib, belumosudil, abatacept, cyclosporine, thalidomide, bortezomib, lenalidomide, halofuginone, hydroxychloroquine, mesenchymal stem cells, type 2 innate lymphoid cells, and regulatory T cells (or any combination thereof, potentially in further combination with other therapeutics). In various aspects of the disclosure, the methods described herein further comprise administering chemotherapy to the subject; optionally the chemotherapy is altretamine, amsacrine, L-asparaginase, colaspase, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytophosphane, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fluorouracil, fludarabine, fotemustine, ganciclovir, gemcitabine, hydroxyurea, idarubicin, ifosfamaide, irinotecan, lomustine, melphalan, mercaptopurine, methotrexate, mitoxantrone, mitomycin C, nimustine, oxaliplatin, paclitaxel, pemetrexed, procarbazine, raltitrexed, temozolomide, teniposide, tioguanine, thiotepa, topotecan, vinblastine, vincristine, vindesine, or vinorelbine. The fusion protein (optionally conjugated to an effector moiety) may be used in combination with radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAM PATH, anti-CD3 antibodies or other antibody therapies, cytotoxin, fludarabine, rapamycin, mycophenolic acid, steroids, FR901228, and/or cytokines.

[0055] While the methods described herein are described as methods of treatment, it will be appreciated that the disclosure further contemplates use of the fusion protein (optionally in the form of a complex) in the treatment of any of the diseases, disorders, or conditions described herein. Use of the fusion protein described herein (optionally in the form of a complex) in the manufacture of a medicament for the treatment of any of the diseases, disorders, or conditions described herein also is provided, as is the fusion protein (optionally in the form of a complex) for use in the treatment of any of the diseases, disorders, or conditions described herein.

[0056] The terms "subject in need” or those "in need of treatment" include subjects already afflicted with the disorder, as well as those in which the disorder is to be prevented. The "subject in need” or "patient" includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment. The subject is a mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits, mammals from the order Carnivora, including Felines (cats) and Canines (dogs), mammals from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). In some aspects, the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). Preferably, the mammal is a human.

[0057] In another aspect, the disclosure provides a method of detecting a CD83-expressing cell. The method comprises contacting a cell with the fusion protein of the disclosure conjugated to a detectable moiety, and detecting the detectable moiety. The method may be performed in vivo or ex vivo {in vitro). Detectable moieties are described above, as well as methods of detecting various detectable moieties.

[0058] As an additional aspect, provided herein are kits which comprise the fusion protein described herein packaged in a manner which facilitates its use. In one embodiment, such a kit includes a composition described herein, packaged in a container such as a sealed bottle, vessel, single-use or multi-use vial, prefilled syringe, or prefilled injection device, optionally with a label affixed to the container or included in the package that describes use of the formulation in practicing the method. In one aspect, the formulation is packaged in a unit dosage form. The kit may further include a device suitable for administering the formulation according to a specific route of administration. In another aspect, the kit comprises the fusion protein conjugated to a detectable moiety (or provided separately from a detectable moiety in different containers intended to mix prior to use) and one or more reagents to detect the detectable moiety. Preferably, the kit contains a label that describes use of the composition described herein.

EXAMPLE

[0059] The affinity of a representative fusion protein described herein was examined. A nanobody comprising the sequence set forth in SEQ ID NO: 33 (comprising the CDR sequences of SEQ ID NOs: 1-3) was fused to an Fc domain comprising SEQ ID NO: 32, resulting in a CD83 binding domain-Fc fusion protein comprising SEQ ID NO: 34 (C10-Fc). Bio-layer interferometry (BLI) measurements were obtained using a Sartorius Octet R8. BLI experiments followed established protocols. Gang et al., eLife 10:e64815 (2001); Hintz et al., Bioconjug Chem. 15;30(5): 1466-1476 (2019). Biotinylated human CD83 protein was captured on hydrated SAX (high precision streptavidin) biosensors. An assay buffer of phosphate buffered saline (PBS) with 1% bovine serum albumin (BSA) was used for all steps including dilutions. Hydrated SAX biosensors were equilibrated for 30 seconds in the assay buffer before the biotinylated CD83 was captured for 45s. A second equilibration step was carried out for 30s. Serial dilutions of C10-Fc were then exposed to the CD83 loaded SAX biosensor for 180 - 240s, followed by a dissociation step in assay buffer for an equivalent amount of time. Two separate controls were run to evaluate non-specific binding. One control included a CD83 loaded SAX biosensor exposed to assay buffer in both association and dissociation steps. A second control included a SAX biosensor with no CD83 loaded exposed to the highest concentration of C10-Fc. The "no antibody” control was subtracted from the data before modeling and the dissociation constant KD was calculated.

[0060] The results of the BLI assay are illustrated in Figure 3. C10-Fc demonstrated a KD of 2.6 nM, a 15 fold improvement over the C10 monomer construct (the CD83 binding domain without the Fc domain).

[0061] The ability of C10-Fc to bind its cognate antigen in cell-based system was assessed by flow cytometry against the acute myeloid leukemia cell lines U937, THP-1 , and OCI-AML3. Cells were harvested and washed with DPBS. One million cells were stained with C10-Fc per concentration and incubated in staining buffer (eBioscience) for 1 hour on ice. Cells were washed and stained with 5 pig/mL Goat anti-human IgG Fc, PE for 1 h on ice. Cells were washed three times and resuspended in staining buffer. Data was collected with the Attune Flow Cytometer (ThermoFischer), collecting a minimum of 100,000 events. Data was analyzed using the software, FlowJo.

[0062] The results of the cell-based flow cytometry study are illustrated in Figures 4A-4B (U937), 5A-5B (THP- 1), and 6A-6B (OCI-AML3). C10-Fc was found to potently bind to all cell lines with picomolar affinities.

[0063] A tetravalent construct comprising a CD83 binding protein of the disclosure also was constructed and examined. A tetravalent structure is illustrated in Figure 2D. In this study, four anti-CD83 nanobodies comprising the CDR sequences of SEQ ID NOs: 1-3 were incorporated into a tetrameric construct (C10 tetravalent construct). U937 cells were harvested and washed with DPBS. One million cells were stained with the C10 tetravalent construct per concentration and incubated in staining buffer (eBioscience) for 1 hour on ice. Cells were washed and stained with 5 pig/mL Goat anti-human IgG Fc, PE for 1 h on ice. Cells were washed three times and resuspended in staining buffer. Data was collected with the Attune Flow Cytometer (ThermoFischer), collecting a minimum of 100,000 events. Data was analyzed using the software, FlowJo.

[0064] The results of the study are illustrated in Figure 7. The anti-CD83 C10 tetravalent construct potently binds the acute myeloid leukemia cell line, U937, as demonstrated by flow cytometry.

[0065] The ability of a representative fusion protein of the disclosure to home to target cells in vivo also was assessed. In this respect, PET/CT was performed using ⁸⁹ Zr labeled C10-Fc on mice bearing subcutaneous U937 xenografts. This study evaluated the in vivo kinetics, tissue specificity, and tumor uptake and retention of CW-Fc.

[0066] All animal studies were performed in 3- to 4-week-old athymic nude-Foxn1nu mice (Envigo). For subcutaneous tumor implantation, cells suspended in a 1 :1 mixture of PBS and Matrigel (Corning) were injected into the shoulders of the mice using a 22-gauge needle. For the U937 cell line model, animals received subcutaneous injections of U937 cells (1 x 10 ⁶ cells in 150 pL). Tumor volumes were measured twice weekly with calipers and tumors were allowed to grow to a size of 100 to 300 mm ³ before nuclear imaging and biodistribution experiments.

[0067] For nuclear imaging studies, the C10-Fc was site specifically conjugated to DI BO modified Deferoxamine (DFO, Macrocyclics). Conjugation was performed by enzymatic reactions using GlyCLICK® DFO obtained by Genovis. Zirconium-89 [ ⁸⁹ Zr] was purchased from the University of Wisconsin Medical Physics Department (Madison, Wl). [ ⁸⁹ Zr]Zr-oxalate (3 mCi) in 1 .0 mol/L oxalic acid (500 pL) was adjusted to pH 7.5 with 1.0 mol/L Na2CO3. To radiolabel C10-Fc, the DFO-C10-Fc conjugate in 0.5 mol/L HEPES (pH 7.5) was added to the neutralized [ ⁸⁹ Zr]Zr-oxalate solution and incubated at room temperature with rotation for 1 hour. The labeled product was purified using a size-exclusion PD-10 column preequilibrated with PBS buffer and evaluated via radioactive TLC for purity. Size-exclusion HPLC analysis with a radioactive detector was not performed on any of the radiolabeled products.

[0068] All microPET/CT studies were performed on an Inveon PET/CT Scanner (Siemens Medical Solutions). Mice were injected with ⁸⁹ Zr C10-Fc via tail vein injection. Four hours post injection mice were anesthetized via inhalation of 2.5% isoflurane and PET/CT images were recorded at 24-hour time points out to 96 hours post injection. PET list mode data were acquired for 80 million counts using a gamma ray energy window of 350-650 KeV and a coincidence timing window of 3.438 ns. A CT-based attenuation correction was performed for approximately 10 minutes with 80 kVp, 1mA, 220 rotation degrees in 120 rotation steps, 250ms exposure time, and subsequently reconstructed using a Shepp-Logan filter with 210 micron isotropic voxels. Scans were reconstructed using 3-dimensional ordered-subset expectation maximization (2 iterations, 16 subsets) with a maximum a posteriori probability algorithm (OSEM3DMAP). Two-dimension (2D) images and maximum intensity projections (MIPs) were prepared in Inveon Research Workplace and ROIs were manually drawn and quantified in Inveon Research Workspace.

[0069] The results of the study are illustrated in Figure 8. C10-Fc displayed rapid and prolonged tumor localization, as compared to non-specific organs. Tissues were harvested and C10-Fc was found to home to tumor tissue. PET/CT imaging of mice for C10-Fc uptake in U937 subcutaneous xenografts confirmed that the fusion protein of the disclosure targeted tumor tissue in vivo. The study confirms the ability of the fusion protein of the disclosure to serve as an imaging agent, which can quickly detect CD83-expressing tumor cells to, e.g., assess disease burden and/or response to therapeutics (e.g., CD83 targeted therapeutics).

[0070] In addition, the ability of a representative fusion protein of the disclosure to deliver a cytotoxic agent to target cells and mediate cell death was evaluated. The C10-Fc fusion protein described herein was conjugated to PNU-159682, a metabolite of the anthracycline nemorubicin, a DNA topoisomerase II inhibitor, or monomethyl auristatin E (MMAE), a mitotic inhibitor. C10-Fc PNU and C10-Fc MMAE were tested at concentrations starting at 1 uM to 6 pM in a 1 :2 dilution series to obtain an IC50. Cytotoxicity of unconjugated C10-Fc and free payload drug were tested in parallel using equivalent molar concentrations. U937 cells were plated at a density of 5x10 ³ cells per well on a 96 well tissue culture plate and cultured for 24 hours prior to drug conjugate treatment for 72 hours. Quantification of cell viability was determined by using Cell Titer Blue reagent (Promega).

[0071] Representative results of the study are illustrated in Figure 9. The cell viability assays demonstrated a cytotoxic effect for both anti-CD83 fusion protein conjugates in the CD83 positive cell line. IC50 values (half- maximal inhibitory concentrations; results of three replicate measures) were determined for the U937 cell line treated with the conjugates. The IC50 for the C10-Fc conjugated to PNU was 308 pM (R ² 0.9814). The IC50 for the C10-Fc conjugated to MMAE was 73.8 nM (R ² 0.9787). The results demonstrate that conjugates comprising an anti-CD83 binding domain of the disclosure fused to an Fc domain and carrying a cytotoxic agent effective kills target cells.

[0072] Additional studies to examine in vivo activity of the anti-CD83 fusion protein include administration of the fusion conjugated to a cytotoxic agent in clinically relevant models of graft versus host disease (GvHD) and human myeloid or lymphoid leukemia. With respect to GvHD, a xenogeneic graft versus host disease model is employed. Target organs of this model include the lungs, skin, Gl tract, and liver, replicating human pathology. NSG mice will receive 10-30x10e6 human peripheral blood mononuclear cells (PBMC) on day 0 by i.p. injection to induce xenogeneic GVHD. Groups of mice will also receive up to 5 mg/kg anti-CD83 binding domain-Fc with PNU conjugated to the Fc, anti-CD83 binding domain-Fc (no drug), or saline once within days 0-14 by i.p. injection. Mice will be followed twice a week for clinical GVHD (skin, fur, posture, mobility, and weight) up to day +80. Animals will be monitored and euthanized in cases of distress or if pre-moribund. The clinical endpoint is premoribund state. In cases of distress (including but not limited to hypoactivity, failure to respond to stimuli, vocalizations, shivering, ataxia, pale mucous membranes, labored breathing, and/or paresis, paralysis), loss of >20% body weight, or moribund state, animals will be immediately euthanized. Kaplan Meier curves will be constructed to determine survival. With respect to human myeloid or lymphoid leukemia models, NSG mice will receive 0.1-5x10e6 luciferase-transduced Raji (lymphoid leukemia) or MV411 (myeloid leukemia) or U937 (myeloid leukemia) cells by i.p. injection or tail vein injection. Groups of mice will also receive up to 5 mg/kg anti- CD83 binding domain-Fc with PNU conjugated to the Fc, anti-CD83 binding domain-Fc (no drug), or saline once within days 0-14 by i.p. injection. Controls groups will receive leukemia alone. BLI assessments will be performed twice a week (luciferin 150 mg/kg) up to day +60. During these experiments using live tumor cells, animals will be euthanized in cases of distress (including but not limited to hypoactivity, failure to respond to stimuli, vocalizations, shivering, ataxia, pale mucous membranes, labored breathing, and/or paresis, paralysis); if tumor grows >15 mm, ulcerates, restricts locomotion, or causes >10% body weight gain; or if premoribund. Any visually observed tumors will be measured twice a week with calipers. The endpoints for these experiments will be tumor growth by BLI. Mice will be euthanized if a tumor endpoint is met (including strong BLI). Non lethal, saphenous vein blood draws may be performed every 2 weeks (volume up to 1% mouse body weight) to enumerate any circulating tumor cells. [0073] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.