Anti-CD90 antibodies, binding fragments, and uses thereof

Abstract

Anti-CD90 antibodies, binding fragments, and uses thereof are described. The provided antibodies and binding fragments can be used to isolate CD90 cells or to target such cells ex vivo or in vivo for a research, a diagnostic, or a therapeutic purpose. Anti-CD90 antibodies and binding fragments thereof can also be used in engineered formats to create multi-domain binding molecules, antibody conjugates, recombinant receptors, or within a recombinant protein to pseudotype viral vectors.

Description

ANTI-CD90 ANTIBODIES, BINDING FRAGMENTS, AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 587,384 filed October 2, 2023, which is incorporated herein by reference in its entirety as if fully set forth herein.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under AH 35953 awarded by the National Institutes of Health. The government has certain rights in the invention.REFERENCE TO SEQUENCE LISTING

[0003] The Sequence Listing associated with this application is provided in XML format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the file containing the Sequence Listing is 3B49297.XML. The file is 231 ,218 bytes, was created on September 30, 2024, and is being submitted electronically via Patent Center.FIELD OF THE DISCLOSURE

[0004] The present disclosure provides anti-CD90 antibodies, binding fragments, and uses thereof. The provided antibodies and binding fragments can be used to isolate CD90 cells or to target such cells ex vivo or in vivo for research, diagnostic, or therapeutic purposes.BACKGROUND OF THE DISCLOSURE

[0005] CD90, also referred to as Thy-1 , is a 25-37 kDa glycosylphosphatidylinositol (GPI)- anchored glycoprotein. CD90 is expressed on hematopoietic stem cells (HSC) and mesenchymal stem / stromal cells (MSC), two therapeutically important cell types. For example, HSC are the basis for therapeutic cord blood, bone marrow and mobilized peripheral blood stem cell transplantation approaches, and blood stem cell gene therapy approaches. HSC transplantation can be curative in many non-malignant and malignant disorders including severe combined immune deficiency (SCID), Fanconi anemia (FA), and several blood cancers. MSC are adult, multipotent non-hematopoietic stem cells. MSC have demonstrated therapeutic effects in several diseases such as in liver diseases, kidney diseases, cardiovascular diseases, neurological diseases, and immune diseases.SUMMARY OF THE DISCLOSURE

[0006] The current disclosure provides antibodies and binding fragments thereof that bind CD90. Among other uses, the antibodies and binding fragments thereof can be used to target CD90- expressing cells, such as hematopoietic stem cells (HSC) and mesenchymal stem / stromal cells (MSC), in vivo or ex vivo for research, diagnostic, and therapeutic purposes.

[0007] In certain examples, the disclosed antibodies and binding fragments thereof are used to create single chain variable fragments (scFv). In other examples, the disclosed antibodies and binding fragments thereof can be used to enhance the cell specificity of viral vector-based nucleic acid delivery. For example, a binding fragment of the disclosed antibodies can be used to create a recombinant protein with a viral structural protein (e.g., the measles virus hemagglutinin protein or vesicular stomatitis virus G protein). This recombinant protein can be used to pseudotype other viral vectors, such as lentiviral vectors.

[0008] The antibodies and binding fragments thereof can also be engineered into numerous additional formats, such as multi-domain binding molecules, antibody-conjugates, and recombinant receptors.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0009] Some of the drawings submitted herein may be better understood in color. Applicant considers the color versions of the drawings as part of the original submission and reserves the right to present color images of the drawings in later proceedings.

[0010] FIG. 1. CD90 5E10 heavy and light chain sequences. Sequence based on Sanger Sequencing of PCR-amplified chain coding regions with complementarity determining region (CDR) encoding segments (according to IMGT CDR Definition) underlined. Sequences include naive CD905E10 heavy and light chain encoding sequences; codon-optimized CD90 5E10 heavy and light chain encoding sequences; codon-optimized CD90 5E10 heavy and light chain amino acid sequences; and codon-optimized CD90 5E10 with mutated unpaired cysteine residues for the light chain encoding sequence and associated protein sequence. Dunbar et al. Nucleic Acids Res. 2016, 44:w474-w478.

[0011] FIGs. 2A, 2B. Naive and codon optimized CD90 5E10 heavy chain (2A) and light chain (2B) encoding sequence alignments. Substituted base pairs are indicated, whereas homologous base pairs are indicated by a dot. Codon optimization performed with assistance from the Codon Usage Database and the GenScript® (GenScript Biotech Co., Piscataway, NJ) Codon Usage Frequency Table Tool.

[0012] FIG. 3. Identification of non-conserved cysteine residues in the light chain. Sequences were compared to the data repository to identify potential residues that cause problems during the folding and production of scFvs. Two cysteines at position 92 and 178 were identified that are predicted to not form di-sulfate bonds. Both cysteines were mutated into tyrosine and arginine as found in the other 5 reference sequences (see details in FIGs. 1 and 7).

[0013] FIGs. 4A-4D. (4A) CD90 scFv design KB1. Engineered codon optimized anti-CD90 scFv sequence (SEQ ID NO: 63) connecting the light chain to the heavy chain with theGGGGSGGGGSGGGGS (SEQ ID NO: 64) linker sequence. (4B) CD90 scFv design KB2. Engineered codon optimized with mutated cysteines anti-CD90 scFv sequence (SEQ ID NO: 156) connecting the light chain to the heavy chain with the GSDSNAGHASAGNTS (SEQ ID NO: 66) linker sequence. (4C) CD90 scFv design KB3. Engineered codon optimized with mutated cysteines anti-CD90 scFv sequence (SEQ ID NO: 157) connecting the heavy chain to the light chain with the GSDSNAGHASAGNTS (SEQ ID NO: 66) linker sequence. (4D) CD90 scFV design KB4 (SEQ ID NO: 197) connecting the light chain to the heavy chain with the GSDSNAGHASAGNTS (SEQ ID NO: 66) linker sequence. Light chain segments are italicized, CDRs are underlined, linker is in double underlined, and mutated unpaired cysteines are in bold.

[0014] FIGs. 5A-5C. Measles Hemagglutinin CD90 scFv Design. Engineered sequence of the CD90-targeted Measles-pseudotyped lentiviral envelope plasmid. Anti-CD90 scFvs shown in FIGs. 4A-4C are attached to the Hemagglutinin Endodomain connected through a linker sequence. Plasmids containing Measles Hemagglutinin provided by Els Verhoeyen at the Universite de Lyon (Frecha, et al., Blood 2008; 112 (13): 4843-4852). (5A) Measles Hemagglutinin KB1 scFv Design (SEQ ID NO: 158). (5B) Measles Hemagglutinin KB2 scFv Design (SEQ ID NO: 159). (5C) Measles Hemagglutinin KB3 scFv Design (SEQ ID NO: 160). Hemagglutinin ectodomain is in normal font (residues 1 -102), hemagglutinin endodomain is underlined (residues 103-1772), GGGGS linkers are in bold, CD90 scFv variable light chain is in italics, CD90 scFv variable heavy chain is double underlined, the tail is in bold italics (residues 2541 -2549), removed cysteine residues are in boxes, and knockout regions are in bold and underlined.

[0015] FIG. 6. Measles Production Schematic. For the production of Measles-pseudotyped lentiviral vectors (LV), HEK-293T cells are transfected with four plasmids, pRSCP newGFPwpre GFP plasmid(V), pCMVdelta8.74 Gag / POL(X), HA24(Y) and FA30(Z) in the ratio V(19.1 ):X(14.5):Y(7.5):Z(7.5). Transfected producer cells are cultured for 40 hours, supernatant harvested, and concentrated by centrifugation (6000G, 24Hrs). Viral production protocol is provided by Els Verhoeyen at the Universite de Lyon (Frecha, et al., Blood 2008; 112 (13): 4843- 4852). Vectors 115 pRSCP GFP and Plasmid #22 Gag / Pol are described in Trobridge, et al., Blood. 2008;111 (12):5537-43).

[0016] FIGs. 7A, 7B. CD90 scFv Thy-1 Docking Modeling. Computer generated structural prediction and interaction of the designed anti-CD90 scFv with CD90. Model generated in SABPred (Dunbar, et al., Nucleic Acids Res. 2016; 44. W474-W478), docking estimated with Zdock (Pierce, et al., Bioinformatics 2014; 30(12): 1771 -3), and visualized in Pymol (The PyMOL Molecular Graphics System, Version 2.0 Schrodinger, LLC.) (7A) Codon-optimized CD90 scFv.(7B) Codon-optimized CD90 with mutated cysteines. VL is in the left bottom, VH is in the right bottom, CDR regions are in the center between the VL and VH, Thy-1 / CD90 protein is at the top. Mutated cysteines are indicated.

[0017] FIGs. 8A, 8B. CD90ex-mCherry production for scFv testing. In order to test the functionality of CD90 scFvs and ability to bind CD90 protein, a custom fusion protein merging the extracellular domain of CD90 (CD90ex) with the fluorochrome mCherry (CD90ex-mCh) was created. Interaction of this fusion protein with anti-CD90 Fab2 fragments was confirmed by size exclusion chromatography (SEC, 8A) and SDS PAGE of the eluted peaks (8B). CD90ex-mCh successfully interacted with the anti-CD90 Fab2 fragments creating a complex (UV A200 cd90- mCherry-5E10-FAb) that showed increased size and was eluted in a fraction distinct from CD90ex-mCh (UV A200 cd90-mCherry) and CD90 Fab2s (UV A2005E10 Fab) alone. (7B) Lanes of SDS PAGE are: Lane 1 is Ez-prot-stan; Lane 2 is isolated 5E10 Fab (5E10 Fab treatment, 15.5ml peak); Lane 3 is isolated CD90-mcherry (CD90-mCherry treatment. 14.3 ml peak); Lane 4 is complex peak 1 (cd90-mCherry-5E10-Fab treatment, 12.7 ml peak); and Lane 5 is complex peak 2 (cd90-mCherry-5E10-Fab treatment, 14.3 ml peak).

[0018] FIGs. 9A, 9B. Binding of CD90ex-mCherry to CD90-targeted viruses. (9A) Schematic showing quality control of Measles virus. CD90-targeted viruses were incubated with CD90ex- mCh and the interaction measured with a Zetaviewer measuring the size of particles. (9B) Untargeted measles-pseudotyped lentiviral viruses were 100-140nm in size (panel 1 and panel 2; upper far left and lower far left graphs) and did not show a size increase when incubated with CD90ex-mCH (panel 2; lower far left graph). Virus design KB1 (without mutated cysteines) was similarly sized and did now show a size increase when incubated with CD90ex-mCh (panel 3 and panel 4; second from the left, upper and lower graphs). Virus design KB2 (panel 5 and panel 6; second from the right, upper and lower graphs) and KB3 (panel 7 and panel 8; upper far right and lower far right graphs) (mutated cysteines, VL-VH, VH-VL) both demonstrated interaction with CD90ex-mCh and the appearance of larger particles of 190-200nm in size (panel 6 and panel 8; lower second from right and lower far right graphs, arrows) indicating interaction of the CD90 scFvs with CD90 protein.

[0019] FIG. 10. Additional sequences supporting the disclosure. Primers used in sequencing CD90 Ab Variant Heavy and Variant Light Chains (von Boehmer et al., Nat Protoc. 2016 Oct;1 1 (10):1908-1923): forward primer for sequencing CD90 VL Ab Variant (SEQ ID NO: 70), reverse primer for sequencing CD90 VL Ab Variant (SEQ ID NO: 71), forward primer for sequencing CD90 VH Ab Variant (SEQ ID NO: 72), reverse primer for sequencing CD90 VH Ab Variant (SEQ ID NO: 73). Additional sequences include hemagglutinin (SEQ ID NO: 74),engineered CD90-targeted Measles-pseudotyped lentiviral envelope plasmid (SEQ ID NO: 161 ), engineered CD90-targeted Measles-pseudotyped Lentiviral Envelope with knockout regions (SEQ ID NO: 162), engineered CD90-targeted Measles-pseudotyped lentiviral envelope plasmid ectodomain (SEQ ID NO: 76), engineered CD90-targeted Measles-pseudotyped lentiviral envelope plasmid endodomain (SEQ ID NO: 77), engineered CD90-targeted Measles- pseudotyped lentiviral envelope plasmid endodomain with knockout regions (SEQ ID NO:163), linkers (SEQ ID NOs: 78, 164, and 165), and tail of engineered CD90-targeted Measles- pseudotyped lentiviral envelope plasmid.

[0020] Fl Gs. 11A-11 E. Design and computational validation of an anti-CD90 scFv. (1 1 A) Workflow to determine the variant heavy (VH) and variant light (VL) sequence from the hybridoma cell line producing the CD90 antibody clone 5E10. (11 B) Amino acid sequence of the initial scFv design connecting the VL and VH chain with a 3x(GGGGS) (SEQ ID NO: 64) linker (italicized and bolded) sequence. Complementarity-determining regions (CDRs, color coded) for the VH and VL sequence were determined using IMGT. (1 1 C) Predicted interaction of CD90 with the anti-CD90 scFv. CDRs of the scFv are shown. (11 D) Representative database crystal structure highlighting the regions of putative mismatched cysteines from our anti-CD90 scFv. (11 E) Sequence alignment of our anti-CD90 scFv with the reference database for highly homologous crystal structures. Mismatched cysteines in position 31 and 59 are shown enclosed in boxes.

[0021] FIGs. 12A-12J. Design and quality control of CD90-targeted measles-pseudotyped lentiviral vectors. (12A) Variant H1 WT measles. (12B) Summary of designed Measles envelopes and the expected antigen recognition. (12C) Variant H2 KO measles annotated sequence. (12D) Variant H3 WT measles and targeting domain annotated sequence. (12E) Variant H4 KO measles and targeting domain annotated sequence. (12F) Viral variants were tested on HT1080 cells demonstrating inability of the KO versions to fuse and transduce. (12G) Workflow for the hydrodynamic titration of engineered viral vectors using nanoparticle tracking analysis (NTA). (12H) Concentration of viral particles per ml culture medium harvested. (121) Concentration of RNA-loaded viral particles per ml culture medium measured with SYBR II. (12J) RNA loading efficiency calculated based on values from FIGs. 12H and 121.

[0022] FIGs. 13A-13C. Assessment of antigen recognition by CD90-targeted lentiviral vectors. (13A) Quality control of custom produced CD90-mCherry fusion protein. CD90 Fab (peak 2), CD90-mCherry fusionprotein (peak 3), and a mix of CD90 Fab with CD90-mCherry were analyzed by size exclusion chromatograph (line graph). Fractions / peaks indicated with numbers were collected and content validated by SDA PAGE (lower panel). (13B) Workflow for the assessment of CD90-mCherry interaction with CD90-targeted viral vectors using nanoparticle trackinganalysis (NTA). (13C) Size of viral particles measuring scattered light (scatter, top row). Size distribution of mRNA-loaded particles stained with SYBR II and measuring the fluorescence in the 500nm filter (+ SYBR II, middle row). SYBR II signal of mRNA-loaded viral particles coincubated with CD90-mCh measuring the fluorescence in the 500nm filter (+ SYBR II + CD90- mCh, bottom row). Numbers in each plot indicate the average size of numbered peaks.

[0023] FIGs. 14A-14C. Selective transduction of cell lines and HSCs with CD90-targeted viral vectors. (14A) Flow-cytometric assessment of the transduction efficiency of Jurkat cells using all 4 vector variants. Efficiencies are visualized relative to the efficiency of variant H1 (mean±SD). (14B) Correlation of the number of transduced cells with the number of mRNA-loaded viral particles using the WT variant H1. R2: Spearman’s rank correlation coefficient. (14C) Functional assessment of transduced human CD34+cells in colony-forming cell (CFG) assays. mScarlet expressing CD34+cells were FACS-purified and introduced into primary (1st) CFC assays (upper left graph). After 14 days, colonies were counted, all cells harvested, and 5% of all cells replated into secondary (2nd) CFC assays (upper right graph). Secondary CFCs were counted after 14 days, all cells harvested, the total number of WBCs counted (lower left graph), and the mScarlet expression measured by flow-cytometry (lower right graph).

[0024] FIGs. 15A-15E. Design and quality control of CD90-targeted vesicular stomatitis virus G (VSV-G)-pseudotyped lentiviral vectors. (15A) VSV-G variant V2 and V4 binder KO sequence. (15B) VSV-G variant V3 and V4 CD90 scFv annotated sequence. (15C) Summary of designed VSV-G envelopes and the expected antigen recognition. (15D) Concentration of total and RNA- loaded viral particles per ml culture medium measured with NTA (top two graphs). Calculated RNA loading efficiency (bottom graph). (15E) Transduction of 200.000 K562 cells with all four VSV-G variants at different doses as indicated below the graph. Transduction efficiency (mScarlet expression) measured by flow-cytometry.

[0025] FIGs. 16A-16C. On-target specificity of CD90-targeted vectors. (16A) Flow-cytometric assessment of CD90 expression on Raji and Jurkat cells. (16B) Transduction efficiency of mixed cultures containing GFP- Raji and GFP+Jurkat cells with either Variant V1 or V4. (16C) On target specificity of Measles- and VSV-G-based vector variants 1 and 4 determined by the ratio of mScarlet+GFP cells within total GFP Raji to mScarlet+GFP+cells within total GFP+Jurkat cells.

[0026] FIG. 17. Crystal structure of the isolated Fab from the murine 5E10 Anti-CD90 Antibody. The protein crystallized in the orthorhombic space group C222i with one copy in the asymmetric unit diffracting to 1 .85A resolution.

[0027] FIGs. 18A, 18B. Expression of recombinant 5E10 chimeric antibody in HEK cells. (18A) Cells were transfected with plasmids encoding 5E10 heavy chain (HC) and light chain (LC), wherethe HC variable region is identical, but 2 different LC variable sequences are used, the LC comprising the sequence as set forth in SEQ ID NO: 31 (left panel) or the LC comprising the sequence as set forth in SEQ ID NO: 190 (right panel). Transfectant supernatants were immunoprecipitated with protein A / G beads and separated on SDS-PAGE under reducing (R) conditions, stained with Coomassie brilliant blue. 2D5, a purified recombinant antibody is shown as control, HC and LC indicated. (18B) 10 pg of 5E10 with new LC was separated by SDS-PAGE under reducing or non-reducing conditions. 2D5 is shown as control.

[0028] FIG. 19. Testing 5E10 mAbs on CD90-positive Jurkat cells. Recombinant chimeric 5E10 v4 antibodies were generated using Fc regions from human lgG1 or lgG4 and IgK, and tested alongside the original mouse monoclonal 5E10 antibody. Non-binding control antibodies (13R4, G1 and G4 versions) or secondary alone are also shown.

[0029] FIGs. 20A,20B. Ex vivo transduction of human CD34+ cells (Granulocyte colonystimulating factor (G-CSF)-mobilized). No coating (e.g., Retronectine) or transduction enhancers were used for transduction. Comparison of VSV-G / Cocal pseudotyped lentiviral vectors with V2 binders encoding for mScarlet. mScarlet expression was measured by flow-cytometry 5 days post-tranduction. (20A) Similar to data shown in FIG. 16C, on-target specificity improves when CD90-binder is added on the Cocal envelope in comparison to random transduction with the wildtype virus. (20B) Cocal-pseudotyped, CD90-targeted viruses show greater overall transduction efficiency compared to VSV-G-pseudotyped, CD90-targeted viral particles.

[0030] FIGs. 21 A, 21 B. In vivo administration Cocal-pseudotyped viral vectors encoding mScarlet and decorated with either version (V)2 or V4 CD90 binders into humanized NBSGW mice (for details on the generation of humanized NBSGW mice see Choo et al, Blood Advances, 2023). Viral particles were either injected intraosseous (IO, into the bone marrow, max volume 15pl), or intravenous (IV, max volume 200pl). Transduction of human CD90+ HSCs located in the bone marrow of the mice was measured 2-4 weeks post-infusion of the virus by flow-cytometry. (21 A) Significantly greater in vivo transduction of human CD90+ cells was seen for viral vectors decorated with V4 CD90 binders in comparison to V2 CD90 binders, both, when virus was administered IO and IV. (21 B) Very high on-target specificity was seen for all tested conditions (IO / IV and V2 / V4) with on average 75% of mScarlet signal found on CD90+ human HSCs.DETAILED DESCRIPTION

[0031] CD90, also referred to as Thy-1 , is a 25-37 kDa glycosylphosphatidylinositol (GPI)- anchored glycoprotein. CD90 is expressed on hematopoietic stem cells (HSC) and mesenchymal stem / stromal cells (MSC), two therapeutically important cell types. For example, HSC are the basis for therapeutic cord blood, bone marrow and mobilized peripheral blood stem celltransplantation approaches, and blood stem cell gene therapy approaches. HSC transplantation or modification can be curative in immune deficiencies. More than 80 primary immune deficiency diseases are recognized by the World Health Organization. These diseases are characterized by an intrinsic defect in the immune system in which, in some cases, the body is unable to produce any or enough antibodies against infection. In other cases, cellular defenses to fight infection fail to work properly. Typically, primary immune deficiencies are inherited disorders.

[0032] Secondary, or acquired, immune deficiencies are not the result of inherited genetic abnormalities, but rather occur in individuals in which the immune system is compromised by factors outside the immune system. Examples include trauma, viruses, chemotherapy, toxins, and pollution. Acquired immunodeficiency syndrome (AIDS) is an example of a secondary immune deficiency disorder caused by a virus, the human immunodeficiency virus (HIV), in which a depletion of T lymphocytes renders the body unable to fight infection.

[0033] X-linked severe combined immunodeficiency (SCID-X1 ) is both a cellular and humoral immune depletion caused by mutations in the common gamma chain gene (yC), which result in the absence of T and natural killer (NK) lymphocytes and the presence of nonfunctional B lymphocytes. SCID-X1 is fatal in the first two years of life unless the immune system is reconstituted, for example, through bone marrow transplant (BMT) or gene therapy.

[0034] Fanconi anemia (FA) is an inherited blood disorder that leads to bone marrow failure. It is characterized, in part, by a deficient DNA-repair mechanism. At least 20% of patients with FA develop cancers such as acute myeloid leukemias, and cancers of the skin, liver, gastrointestinal tract, and gynecological systems. The skin and gastrointestinal tumors are usually squamous cell carcinomas. The average age of patients who develop cancer is 15 years for leukemia, 16 years for liver tumors, and 23 years for other tumors.

[0035] Cells from FA patients display a characteristic hypersensitivity to agents that produce interstrand DNA crosslinks such as mitomycin C or diepoxybutane. FA genes define a multicomponent pathway involved in cellular responses to DNA cross-links. Five of the FA genes (FANCA, FANCC, FANCE, FANCF and FANCG) have been cloned and the FANCA, FANCC, and FANCG proteins have been shown to form a molecular complex with primarily nuclear localization. A number of mutations in the FANCC gene have been identified which are correlated with FA of differing degrees of severity.

[0036] Mesenchymal stem / stromal cells (MSC) are adult, multipotent, non-hematopoietic stem cells. MSC have demonstrated therapeutic effects in several diseases and there are effects largely mediated by paracrine factors such as exosomes. Exosomes are 40-150 nm membranebound vesicles generated in multivesicular bodies and secreted when these compartments fusewith the plasma membrane. MSC-derived exosomes function in cell-cell communication as they can contain cytokines and growth factors, such as lipids, proteins, mRNAs, transfer RNA (tRNA), long noncoding RNAs (IncRNAs), microRNAs (miRNAs) and mitochondrial DNA (mtDNA) and once released into the extracellular space, they can be taken up by other cells or carried to distant sites via biological fluid.

[0037] Most exosomes include similar proteins such as tetraspanins (CD81 , CD63, and CD9), heat-shock proteins (HSP60, HSP70 and HSP90), ALIX and tumor susceptibility gene 101 (TSG101 ); and have tissue type-specific proteins according to their cell source. MSC-derived exosomes have been used to treat, for example, liver diseases (Li T, et al., Stem Cells Dev. 2013; 22(6):845-54; Tan, et al. Stem Cell Res Ther. 2014; 5(3):76; Fouraschen, et al., Stem Cells Dev. 2012; 21 (13):2410— 9; and Lou, et al., Exp Mol Med. 2017;49(6):e346), kidney diseases (Zou, etal., Am J Transl Res. 2016; 8(10):4289-99; Eirin A, etal., Kidney Int. 2017; 92(1 ):114-24; Bruno, et al., J Am Soc Nephrol. 2009; 20(5): 1053-67; Bruno, et al., PLoS One. 2012; 7(3):e33115; Zhang, et al., Kidney Blood Press Res. 2016; 41 (2):119-28; and Tomasoni, et al., Stem Cells Dev. 2013; 22(5):772-80), cardiovascular diseases (Suzuki, et al., Adv Exp Med Biol. 2017; 998:179-85; Cui, et al., J Cardiovasc Pharmacol. 2017; 70(4):225-31 ; Wang, et al., Stem Cells Transl Med. 2017; 6(1 ):209-22; Arslan, et al., Stem Cell Res. 2013;10(3):301— 12; Feng, et al., PLoS One. 2014; 9(2):e88685; Xiao, et al., Circ Res. 2018; 123(5):564-78; Mayourian, et al., Circ Res. 2018;122(7):933-44; and Liu, et al., Cell Physiol Biochem. 2017;43(1 ):52-68), neurological diseases (Luarte, et al., Stem Cells Int. 2016; 2016:5736059; Cooper, et al., Mov Disord. 2014; 29(12): 1476-85; Xin, et al., Stroke. 2017; 48(3):747-53; Katsuda, et al., Sci Rep. 2013; 3:1197; Yang, et al., J Mol Neurosci. 2018; 65(1 ):74-83; and Cui, et al., FASEB J. 2018; 32(2):654-68), and immune diseases (You, etal., J Hematol Oncol. 2017; 10(1 ):165; Lai, etal., J Hematol Oncol. 2018; 11 (1 ):135; Shigemoto-Kuroda, et al., Stem Cell Reports. 2017; 8(5):1214— 25; Du, et al., Exp Cell Res. 2018; 363(1 ):114-20; and Li, et al., EBioMedicine. 2016; 8:72-82).

[0038] The current disclosure provides antibodies and binding fragments thereof that bind CD90. Among other uses, the antibodies and binding fragments thereof can be used to target HSC and MSC in vivo or ex vivo for research, diagnostic, and / or therapeutic purposes.

[0039] Naive anti-CD90 antibody sequences were codon-optimized to provide the codon- optimized anti-CD90 antibody sequences and encoding sequences provided herein. This new, codon-optimized anti-CD90 antibody sequence is more efficient and more effective than prior anti-CD90 antibodies, and functions with virus types in addition to Measles virus.

[0040] In certain examples, the disclosed antibodies and binding fragments thereof are used to create single chain variable fragments (scFv). In other examples, the disclosed antibodies and binding fragments thereof can be used to enhance the cell specificity of viral vector-based nucleic acid delivery. For example, a binding fragment of the disclosed antibodies can be used to create a recombinant protein with a viral structural protein (e.g., the measles virus hemagglutinin protein or the vesicular stomatitis virus G (VSV-G) protein). This recombinant protein can be used to pseudotype other viral vectors, such as lentiviral vectors. In certain examples, the recombinant protein can be integrated into viral vectors to target cells ex vivo or in vivo for modification.

[0041] The antibodies and binding fragments thereof can also be engineered into numerous additional formats, such as multi-domain binding molecules, antibody conjugates, and recombinant receptors.

[0042] Having highlighted key aspects of the current disclosure, the following additional details and options to practice the disclosure are provided as follows: (I) CD90 Antibodies & Binding Fragments Thereof; (II) Antibody Variants; (III) Multi-Domain Binding Molecules; (IV) Expression of Recombinant Antibodies; (V) Anti-CD90 Antibody Conjugates; (VI) Antibody-Enhanced Viral Vector Delivery; (VII) Recombinant Receptors; (VIII) Ex Vivo Cell Manufacturing & Formulation; (IX) In Vivo or Ex Vivo Genetic Modification; (X) Compositions and Formulations for Administration; (XI) Kits; (XII) Methods of Use; (XIII) Exemplary Embodiments; (XIV) Experimental Example; and (XV) Closing Paragraphs. These headings are provided for organization purposes only and should not be construed to limit the teachings or interpretation of the current disclosure.

[0043] (I) CD90 Antibodies & Binding Fragments Thereof. The current disclosure provides anti- CD90 antibodies and binding fragments thereof. The anti-CD90 antibodies and fragments thereof bind CD90. In particular embodiments, CD90 [Homo sapiens] (GenBank: AAH65559.1 ) includes the sequence:MNLAISIALLLTVLQVSRGQKVTSLTACLVDQSLRLDCRHENTSSSPIQYEFSLTRETKKHVLFG TVGVPEHTYRSRTNFTSKYNMKVLYLSAFTSKDEGTYTCALHHSGHSPPISSQNVTVLRDKLVK CEGISLLAQNTSWLLLLLLSLSLLQATDFMSL (SEQ ID NO: 51 ).

[0044] Naturally occurring antibody structural units include a tetramer. Each tetramer includes two pairs of polypeptide chains, each pair having one light chain and one heavy chain. The aminoterminal portion of each chain includes a variable region that is responsible for antigen recognition and epitope binding. The variable regions exhibit the same general structure of relatively conserved framework regions (FR) joined by three hyper variable regions, also called complementarity determining regions (CDRs). The CDRs from the two chains of each pair arealigned by the framework regions, which enables binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chain variable regions include the domains FR1 , CDR1 , FR2, CDR2, FR3, CDR3 and FR4.

[0045] The assignment of amino acids to each domain is typically in accordance with the definitions of IMGT (Lefranc M P et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 January; 27(1 ):55-77 (“IMGT” numbering scheme)), Kabat numbering (Kabat et al. (1991 ), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (“Kabat” numbering scheme)); Chothia (Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme)), Martin (Abinandan etal., Mol Immunol. 45:3832-3839 (2008), “Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains”), Gelfand, Contact (MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745.” (Contact numbering scheme)), AHo (Honegger A and Pluckthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun. 8; 309(3) :657-70, (AHo numbering scheme)), North (North etal., J Mol Biol. 406(2) :228-256 (2011 ), “A new clustering of antibody CDR loop conformations”), or other numbering schemes.

[0046] Definitive delineation of a CDR and identification of residues including the binding site of an antibody can be accomplished by solving the structure of the antibody and / or solving the structure of the antibody-epitope complex. In particular embodiments, this can be accomplished by methods such as X-ray crystallography and cryoelectron microscopy. Alternatively, CDRs are determined by comparison to known antibodies (linear sequence) and without resorting to solving a crystal structure. To determine residues involved in binding, a co-crystal structure of the Fab (antibody fragment) bound to the target can optionally be determined. Software programs and bioinformatical tools, such as ABodyBuilder and Paratome can also be used to determine CDR sequences. Additionally, delineation of a CDR can be according to X-ray crystallography.

[0047] Referring to the antibodies provided herein, the CDR sequences are provided in Table 1 .Table 1.

[0048] CDR coding sequence can be determined by those skilled in the art by comparing the above amino acid CDRs with the corresponding coding sequence.

[0049] Alternatively, an anti-CD90 antibody (CD90 5E10 antibody) or fragment thereof includes a variable heavy chain including CDRs determined from the sequence as set forth in SEQ ID NO: 40 and a variable light chain including a CDRL1 sequence including RASQDISNYL (SEQ ID NO:194), a CDRL2 sequence including YTSRLHS (SEQ ID NO: 195), and a CDRL3 sequence including CQQGNTLPRT (SEQ ID NO: 196).

[0050] In particular embodiments, an anti-CD90 antibody (CD90 5E10 antibody) or fragment thereof includes a variable heavy chain having a sequence as set forth in SEQ ID NO: 40 and a variable light chain having a sequence as set forth in SEQ ID NO: 193.

[0051] In particular embodiments, an anti-CD90 antibody (CD90 5E10 antibody) or fragment thereof includes a variable heavy chain encoded by a sequence as set forth in SEQ ID NO: 22 and a variable light chain encoded by a sequence as set forth in SEQ ID NO: 190.

[0052] The carboxy-terminal portion of each chain defines a constant region, which can be responsible for effector function particularly in the heavy chain (the Fc). Examples of effector functions include: C1q binding and complement dependent cytotoxicity (CDC); antibodydependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B-cell receptors); and B-cell activation.

[0053] Human light chains are classified as kappa (IgK) and lambda (IgA) light chains.

[0054] In particular embodiments, a human IgK constant region includes the sequence: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 13).

[0055] In particular embodiments, a human IgA Fc region includes the sequence: GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSN NKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 14).

[0056] Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, lgG1 , lgG2, lgG3, and lgG4. IgM has subclasses including lgM1 and lgM2. IgA is similarly subdivided into subclasses including lgA1 and lgA2.

[0057] In particular embodiments, a human IgG 1 constant region includes the sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK (SEQ ID NO: 15).

[0058] In particular embodiments, a human lgG2 Fc region includes the amino acid sequence: PAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPRE EQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 16).

[0059] In particular embodiments, a human lgG3 Fc region includes the amino acid sequence: PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPR EEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQ QGNIFSCSVMHEALHNRFTQKSLSLSPGK (SEQ ID NO: 17).

[0060] In particular embodiments, a human lgG4 Fc region includes the amino acid sequence: PAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPR EEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQ EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 18).

[0061] In particular embodiments, a human lgG4_constant region with an S228P mutation includes the sequence:

[0062] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMH EALHNHYTQKSLSLSLGK (SEQ ID NO: 19).

[0063] The human IgD constant region typically includes the amino acid sequence: APTKAPDVFPIISGCRHPKDNSPVVLACLITGYHPTSVTVTWYMGTQSQPQRTFPEIQRRDSYY MTSSQLSTPLQQWRQGEYKCVVQHTASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKAT TAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFT CFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTL NHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQRE VNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSY VTDHGPMK (SEQ ID NO: 20).

[0064] The human IgE constant region typically includes the amino acid sequence: ASTQSPSVFPLTRCCKNIPSNATSVTLGCLATGYFPEPVMVTWDTGSLNGTTMTLPATTLTLSG HYATISLLTVSGAWAKQMFTCRVAHTPSSTDWVDNKTFSVCSRDFTPPTVKILQSSCDGGGHF PPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTY TCQVTYQGHTFEDSTKKCADSNPRGVSAYLSRPSPFDLFIRKSPTITCLVVDLAPSKGTVNLTW SRASGKPVNHSTRKEEKQRNGTLTVTSTLPVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTSGPRAAPEVYAFATPEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTK GSGFFVFSRLEVTRAEWEQKDEFICRAVHEAASPSQTVQRAVSVNPGK (SEQ ID NO: 21).

[0065] In particular embodiments, an anti-CD90 antibody (CD90 5E10 antibody) or fragment thereof includes an IgG 1 heavy chain and an IgK light chain. In particular embodiments, the anti- CD90 antibody or fragment thereof includes a heavy chain having a sequence as set forth in SEQ ID NO: 3 and a light chain having a sequence as set forth in SEQ ID NO: 7. In particular embodiments, the anti-CD90 antibody or fragment thereof includes a heavy chain encoded by a sequence as set forth in SEQ ID NO: 2 and a light chain encoded by a sequence as set forth in SEQ ID NO: 6.

[0066] In particular embodiments, an anti-CD90 antibody (CD90 5E10 antibody) or fragment thereof includes an lgG4 heavy chain with an S228P mutation and an IgK light chain. In particular embodiments, the anti-CD90 antibody or fragment thereof includes a heavy chain having a sequence as set forth in SEQ ID NO: 5 and a light chain having a sequence as set forth in SEQ ID NO: 7. In particular embodiments, the anti-CD90 antibody or fragment thereof includes a heavy chain encoded by a sequence as set forth in SEQ ID NO: 4 and a light chain encoded by a sequence as set forth in SEQ ID NO: 6.

[0067] In particular embodiments, the variable heavy chain, variable light chain, heavy chain, or light chain further includes a signal peptide. In particular embodiments, the signal peptide is selected from a sequence set forth in SEQ ID NOs: 191 , 192, 8, or 9.

[0068] Within full-length light and heavy chains, the variable and constant regions are joined by a “J” region of amino acids, with the heavy chain also including a “D” region of amino acids. See, e.g., Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)).

[0069] As indicated, antibodies bind epitopes on antigens. The term antigen refers to a molecule or a portion of a molecule capable of being bound by an antibody when in the non-blocked presence of the antibody. An epitope is a region of an antigen that is bound by the variable region of an antibody. Epitope determinants can include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three-dimensional structural characteristics, and / or specific charge characteristics. When the antigen is a protein or peptide, the epitope includes specific amino acids within that protein or peptide that contact the variable region of an antibody.

[0070] Unless otherwise indicated, the term “antibody” includes (in addition to naturally occurring antibodies having two full-length heavy chains and two full-length light chains as described above) variants, derivatives, and fragments thereof, examples of which are described below. Furthermore, unless explicitly excluded, antibodies can include monoclonal antibodies (mAbs),human or humanized antibodies, antibodies with multiple binding domains, bispecific antibodies, trispecific antibodies, tetraspecific antibodies, multi-specific antibodies, polyclonal antibodies, linear antibodies, minibodies, domain antibodies, synthetic antibodies, chimeric antibodies, antibody fusions, single chain variable fragments (scFvs), polyclonal antibodies, and fragments thereof, respectively. In particular embodiments, antibodies can include oligomers or multiplexed versions of antibodies.

[0071] A monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies including the population are identical and / or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which include different antibodies directed against different epitopes, each monoclonal antibody of a monoclonal antibody preparation is directed against a single epitope on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies can be made by a variety of techniques, including the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci.

[0072] A “human antibody” is one which includes an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-coding sequences.

[0073] A “human consensus framework” is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VLor VHsequences is from a subgroup of variable domain sequences. The subgroup of sequences can be a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91 - 3242, Bethesda Md. (1991 ), vols. 1 -3. In particular embodiments, for the VL, the subgroup can be subgroup kappa I as in Kabat et al. (supra). In particular embodiments, for the VH, the subgroup can be subgroup III as in Kabat et al. (supra).

[0074] A “humanized” antibody refers to a chimeric antibody including amino acid residues from non-human CDRs and amino acid residues from human FRs. In particular embodiments, a humanized antibody will include substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-humanantibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may include at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0075] Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633, 2008, and are further described, e.g., in Riechmann et al., Nature 332:323-329, 1988; Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033, 1989; U.S. Pat. Nos. 5,821 ,337, 7,527,791 , 6,982,321 , and 7,087,409; Kashmiri et al., Methods 36:25- 34, 2005 (describing SDR (a-CDR) grafting); Padlan, Mol. Immunol. 28:489-498, 1991 (describing “resurfacing”); Dall'Acqua etal., Methods 36:43-60,2005 (describing “FR shuffling”); and Osbourn etal., Methods 36:61 -68, 2005 and Klimka et al., Br. J. Cancer, 83:252-260, 2000 (describing the “guided selection” approach to FR shuffling). EP-B-0239400 provides additional description of “CDR-grafting”, in which one or more CDR sequences of a first antibody is / are placed within a framework of sequences not of that antibody, for instance of another antibody.

[0076] Human framework regions that may be used for humanization include: framework regions selected using the “best-fit” method (see, e.g., Sims et al. J. Immunol. 151 :2296, 1993); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al., Proc. Nati. Acad. Sci. USA, 89:4285, 1992; and Presta et al., J. Immunol., 151 :2623, 1993); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633, 2008); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684, 1997; and Rosok et al., J. Biol. Chem. 271 :22611 -22618, 1996).

[0077] (II) Antibody Variants. Antibodies disclosed herein can be utilized to prepare various forms of relevant binding domain molecules. For example, particular embodiments can include binding fragments of an antibody, e.g., Fv, Fab, Fab', F(ab')2, and single chain Fv fragments (scFvs) or any biologically effective fragments of an immunoglobulin that bind specifically to an epitope described herein.

[0078] In particular embodiments, an antibody fragment is used. An “antibody fragment” denotes a portion of a complete or full-length antibody that retains the ability to bind to an epitope. Antibody fragments can be made by various techniques, including proteolytic digestion of an intact antibody as well as production by recombinant host-cells (e.g., mammalian suspension cell lines, E. coli or phage), as described herein. Antibody fragments can be screened for their binding properties in the same manner as intact antibodies. Examples of antibody fragments include Fv, scFv, Fab,Fab', Fab'-SH, F(ab')2; diabodies; and linear antibodies.

[0079] A single chain variable fragment (scFv) is a recombinant protein of the variable regions of the heavy and light chains of immunoglobulins connected with a linker peptide. Fv fragments include the VLand VHdomains of a single arm of an antibody but lack the constant regions. Although the two domains of the Fv fragment, VL and VH, are coded by separate genes, they can be joined, using, for example, recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VLand VHregions pair to form monovalent molecules (single chain Fv (scFv)). For additional information regarding Fv and scFv, see e.g., Bird, et al., Science 242:423-426, 1988; Huston, et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988; Plueckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore (eds.), Springer- Verlag, New York), (1994) 269-315; WO 1993 / 16185; U.S. Pat. No. 5,571 ,894; and U.S. Pat. No. 5,587,458.

[0080] Linker sequences that are used to connect the VL and VH of an scFv or other forms of binding fragments are generally five to 35 amino acids in length. In particular embodiments, a VL- VH linker includes from five to 35, ten to 30 amino acids or from 15 to 25 amino acids. Variation in the linker length may retain or enhance activity, giving rise to superior efficacy in activity studies. Linker sequences of scFv are commonly Gly-Ser linkers. In particular embodiments, the linker sequence includes the sequence: GGGGSGGGGSGGGGS (SEQ ID NO: 64) or GSDSNAGHASAGNTS (SEQ ID NO: 66). Additional linker sequences that can be used include sets of glycine and serine repeats such as from one to ten repeats of (GlyxSery)n, wherein x and y are independently an integer from 0 to 10 provided that x and y are not both 0 and wherein n is an integer of 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10). Particular examples include (Gly4Ser)n(SEQ ID NO: 79), (Gly3Ser)n(Gly4Ser)n(SEQ ID NO: 80), (Gly3Ser)n(Gly2Ser)n(SEQ ID NO: 81 ), or (Gly3Ser)n(Gly4Ser)i (SEQ ID NO: 82). In particular embodiments, the linker is (Gly4Ser)4(SEQ ID NO: 83), (Gly4Ser)2(SEQ ID NO: 84), (Gly4Ser)i (SEQ ID NO: 85), (Gly3Ser)2(SEQ ID NO: 86), (Gly3Ser)i (SEQ ID NO: 87), (Gly2Ser)2(SEQ ID NO: 88) or (Gly2Ser)i, GGSGGGSGGSG (SEQ ID NO: 89), GGSGGGSGSG (SEQ ID NO: 90), or GGSGGGSG (SEQ ID NO: 91 ). In particular embodiments, the linker includes a Whitlow linker (GSTSGSGKPGSGEGSTKG, SEQ ID NO: 10).

[0081] In particular embodiments, an anti-CD90 scFv is shown in FIG. 4D (SEQ ID NO: 197). This scFv, referred to herein as KB4, includes (from N to C terminal) a CD90 5E10 heavy chain, GSDSNAGHASAGNTS (SEQ ID NO: 66) linker, and a CD90 5E10 codon-optimized light chain.

[0082] Another particular example of an anti-CD90 scFv is shown in FIG. 4A (SEQ ID NO: 63). This scFv, referred to herein as KB1 , includes (from N to C terminal) a CD90 5E10 light chain,GGGGSGGGGSGGGGS (SEQ ID NO: 64) linker, and a CD905E10 heavy chain.

[0083] Another particular example of an anti-CD90 scFv is shown in FIG. 4B (SEQ ID NO: 156). This scFv, referred to herein as KB2, includes (from N to C terminal) a CD90 5E10 light chain wherein the unpaired cysteines are mutated, GSDSNAGHASAGNTS (SEQ ID NO: 66) linker, and a CD905E10 heavy chain.

[0084] Another particular example of an anti-CD90 scFv is shown in FIG. 4C (SEQ ID NO: 157). This scFv, referred to herein as KB3, includes (from N to C terminal) a CD90 5E10 heavy chain, GSDSNAGHASAGNTS (SEQ ID NO: 66) linker, and a CD905E10 light chain wherein the unpaired cysteines are mutated.

[0085] Additional examples of antibody-based binding domain formats include scFv-based grababodies and soluble VH domain antibodies. These antibodies form binding regions using only heavy chain variable regions. See, for example, Jespers et al., Nat. BiotechnoL 22:1161 , 2004; Cortez-Retamozo etal., Cancer Res. 64:2853, 2004; Baral etal., Nature Med. 12:580, 2006; and Barthelemy et al., J. Biol. Chem. 283:3639, 2008.

[0086] A Fab fragment is a monovalent antibody fragment including VL, VH, CL and CH1 domains. A F(ab')2 fragment is a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region. For discussion of Fab and F(ab')2 fragments having increased in vivo half-life, see U.S. Patent 5,869,046. Diabodies include two epitope-binding sites that may be bivalent. See, for example, EP 0404097; WO1993 / 01161 ; and Holliger, et al., Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993. Dual affinity retargeting antibodies (DART™; based on the diabody format but featuring a C-terminal disulfide bridge for additional stabilization (Moore et al., Blood 117:4542-51 , 201 1 )) can also be used. Antibody fragments can also include isolated CDRs. For a review of antibody fragments, see Hudson, et al., Nat. Med. 9:129-134, 2003.

[0087] Variants of antibodies described herein are also included. Variants of antibodies can include those having one or more conservative amino acid substitutions or one or more nonconservative substitutions that do not adversely affect the binding of the protein. Options for conservative substitutions are described elsewhere herein.

[0088] In particular embodiments, a VL region can be derived from or based on a disclosed VL and can include one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions), or a combination of the above-noted changes, when compared with the disclosed VL. An insertion, deletion or substitution may be anywhere in the VLregion, including at the amino- or carboxy-terminus or both ends of this region, provided that each CDR includes zero changes or at most one, two, or three changes and provided an antibodyincluding the modified VLregion can still bind its target epitope with an affinity similar to the wild type binding domain.

[0089] In particular embodiments, a V region can be derived from or based on a disclosed VHand can include one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions or non-conservative amino acid substitutions), or a combination of the above-noted changes, when compared with the VH disclosed herein. An insertion, deletion or substitution may be anywhere in the VHregion, including at the amino- or carboxy-terminus or both ends of this region, provided that each CDR includes zero changes or at most one, two, or three changes and provided an antibody including the modified VHregion can still bind its target epitope with an affinity similar to the wild type binding domain.

[0090] In particular embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody, thereby generating an Fc region variant. The Fc region variant may include a human Fc region sequence (e.g., a human IgG 1 , lgG2, lgG3 or lgG4 Fc region) including an amino acid modification (e.g., a substitution) at one or more amino acid positions. Numerous Fc modifications are known in the art, and a representative sampling of such possible modifications are described herein.

[0091] In particular embodiments, variants (including Fc variants) have been modified from a reference sequence to produce an administration benefit. Exemplary administration benefits can include (1 ) reduced susceptibility to proteolysis, (2) reduced susceptibility to oxidation, (3) altered binding affinity for forming protein complexes, (4) altered binding affinities, (5) reduced immunogenicity; and / or (6) extended half-life. While the disclosure below describes these modifications in terms of their application to antibodies, when applicable to another particular anti- CD90 binding domain format (e.g., an scFv, bispecific antibody), the modifications can also be applied to these other formats.

[0092] In particular embodiments the antibodies can be mutated to increase their affinity for Fc receptors. Exemplary mutations that increase the affinity for Fc receptors include: G236A / S239D / A330L / I332E (GASDALIE). Smith et al., Proceedings of the National Academy of Sciences of the United States of America, 109(16), 6181-6186, 2012. In particular embodiments, an antibody variant includes an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 of the Fc region (EU numbering of residues). In particular embodiments, alterations are made in the Fc region that result in altered C1 q binding and / or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551 , WO 99 / 51642, and Idusogie et al., J. Immunol. 164: 4178-4184, 2000.

[0093] In particular embodiments, it may be desirable to create cysteine engineered antibodies, e.g., “thioMAbs,” in which one or more residues of an antibody are substituted with cysteine residues. In particular embodiments, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further below. In particular embodiments, residue 5400 (Ell numbering) of the heavy chain Fc region is selected. Cysteine engineered antibodies may be generated as described, e.g., in U.S. Pat. No. 7,521 ,541.

[0094] Antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from 1% to 80%, from 1 % to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008 / 077546, for example. Asn297 refers to the asparagine residue located at position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., W02000 / 61739; WO 2001 / 29246; W02002 / 031140; US2002 / 0164328; W02003 / 0851 19; W02003 / 084570; US2003 / 0115614; US2003 / 0157108; US2004 / 0093621 ; US2004 / 0110704; US2004 / 0132140; US2004 / 01 10282; US2004 / 0109865; W02005 / 035586; W02005 / 035778; W02005 / 053742; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); and Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Led 3 CHO cells deficient in protein fucosylation (Ripka etal. Arch. Biochem. Biophys. 249:533-545, 1986, and knockout cell lines, such as alpha- 1 ,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614, 2004; Kanda et al., Biotechnol. Bioeng., 94(4):680-688, 2006; and W02003 / 085107).

[0095] In particular embodiments, the Fc moiety of an antibody includes a substitution at positions CH2 4, CH2 5, or both. In general, the amino acid at positions 4 and 5 of CH2 of the wild-type IgG 1 and lgG3 is a leucine ("L"). In particular embodiments, the antibody includes an amino acid at position CH24, CH2 5, or both, that is not an L. In particular embodiments, an antibody includes an alanine ("A") at position CH2 4, or CH2 5, or both. In particular embodiments, the antibodyincludes both, a CH2 L4A and a CH2 L5A substitution. Such antibodies are referred to herein as a "LALA" variant. Interestingly, a "LALA" mutation in the Fc moiety does not only result in a lack of contribution of the respective antibody in antibody-dependent enhancement (ADE), but also blocks ADE.

[0096] In particular embodiments, an lgG4 Fc region is mutated to form the lgG4_S228P Fc region. lgG4 antibodies can undergo a process called Fab arm exchange which results in functionally monovalent, bispecific antibodies with unknown specificity and thus potentially reduced therapeutic efficacy. Mutating the wildtype lgG4 serine at position 228 within the corehinge region to a proline creates the lgG4_S228P mutant. In particular embodiments, the lgG4_S228P mutant prevents Fab arm exchange.

[0097] In particular embodiments, modified antibodies include those wherein one or more amino acids have been replaced with a non-amino acid component, or where the amino acid has been conjugated to a functional group or a functional group has been otherwise associated with an amino acid. The modified amino acid may be, e.g., a glycosylated amino acid, a PEGylated amino acid, a farnesylated amino acid, an acetylated amino acid, a biotinylated amino acid, an amino acid conjugated to a lipid moiety, an amino acid conjugated to a lipid nanoparticle, an amino acid conjugated to a polymer nanoparticle, or an amino acid conjugated to an organic derivatizing agent. Amino acid(s) can be modified, for example, co-translationally or post-translationally during recombinant production (e.g., N-linked glycosylation at N-X-S / T motifs during expression in mammalian cells) or modified by synthetic means. The modified amino acid can be within the sequence or at the terminal end of a sequence. Modifications also include nitrited constructs.

[0098] In particular embodiments, variants include glycosylation variants wherein the number and / or type of glycosylation site has been altered compared to the amino acid sequences of a reference sequence. In particular embodiments, glycosylation variants include a greater or a lesser number of N-linked glycosylation sites than the reference sequence. An N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X can be any amino acid residue except proline. The substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitutions which eliminate this sequence will remove an existing N-linked carbohydrate chain. Also provided is a rearrangement of N-linked carbohydrate chains wherein one or more N-linked glycosylation sites (e.g., those that are naturally occurring) are eliminated and one or more new N-linked sites are created. Additional antibody variants include cysteine variants wherein one or more cysteine residues are deleted from or substituted for another amino acid (e.g., serine) as compared to the reference sequence.These cysteine variants can be useful when antibodies must be refolded into a biologically active conformation such as after the isolation of insoluble inclusion bodies. These cysteine variants generally have fewer cysteine residues than the reference sequence, and typically have an even number to minimize interactions resulting from unpaired cysteines.

[0099] PEGylation particularly is a process by which polyethylene glycol (PEG) polymer chains are covalently conjugated to other molecules such as proteins. Several methods of PEGylating proteins have been reported in the literature. For example, N-hydroxy succinimide (NHS)-PEG was used to PEGylate the free amine groups of lysine residues and N-terminus of proteins; PEGs bearing aldehyde groups have been used to PEGylate the amino-termini of proteins in the presence of a reducing reagent; PEGs with maleimide functional groups have been used for selectively PEGylating the free thiol groups of cysteine residues in proteins; and sitespecific PEGylation of acetyl-phenylalanine residues can be performed.

[0100] Covalent attachment of proteins to PEG has proven to be a useful method to increase the half-lives of proteins in the body (Abuchowski, A. et al., Cancer Biochem. Biophys. ,1984, 7:175- 186; Hershfield, M. S. et al., N. Engl. J. Medicine, 1987, 316:589-596; and Meyers, F. J. et a / ., Clin. Pharmacol. Then, 49:307-313, 1991 ). The attachment of PEG to proteins not only protects the molecules against enzymatic degradation, but also reduces their clearance rate from the body. The size of PEG attached to a protein has significant impact on the half-life of the protein. The ability of PEGylation to decrease clearance is generally not a function of how many PEG groups are attached to the protein, but the overall molecular weight of the altered protein. Usually the larger the PEG is, the longer the in vivo half-life of the attached protein. In addition, PEGylation can also decrease protein aggregation (Suzuki et al., Biochem. Bioph. Acta 788:248, 1984), alter protein immunogenicity (Abuchowski etal., J. Biol. Chem. 252: 3582, 1977), and increase protein solubility as described, for example, in PCT Publication No. WO 92 / 16221).

[0101] Several sizes of PEGs are commercially available (Nektar Advanced PEGylation Catalog 2005-2006; and NOF DDS Catalogue Ver 7.1 ), which are suitable for producing proteins with targeted circulating half-lives. A variety of active PEGs have been used including mPEG succinimidyl succinate, mPEG succinimidyl carbonate, and PEG aldehydes, such as mPEG- propionaldehyde.

[0102] In particular embodiments, anti-CD90 antibodies or anti-CD90 binding fragments disclosed herein are conjugated to polyglutamic acid (PGA). Antibody-functionalized PGA can be useful because it shields the positive charge of polymeric particles (Moffett et al., Nature Communications 8 (389, 2017). PGA can be functionalized with antibodies using similar covalent attachment methods described for PEGylation. In particular embodiments,antibody-functionalized PGA can be encapsulated in polymer nanoparticles or lipid nanoparticles.

[0103] In particular embodiments, the antibody can be fused or coupled to an Fc polypeptide that includes amino acid alterations that extend the in vivo half-life of an antibody that contains the altered Fc polypeptide as compared to the half-life of a similar antibody containing the same Fc polypeptide without the amino acid alterations. In particular embodiments, Fc polypeptide amino acid alterations can include M252Y, S254T, T256E, M428L, and / or N434S and can be used together, separately or in any combination. For example, M428L / N434S is a pair of mutations that increase the half-life of antibodies in serum, as described in Zalevsky etal., Nature Biotechnology 28, 157-159, 2010. Other alterations that can be helpful are described in US Patent No. 7,083,784, US Patent No. 7,670,600, US Publication No. 2010 / 0234575, PCT / US2012 / 070146, and Zwolak, Scientific Reports 7: 15521 , 2017. In particular embodiments, any substitution at one of the following amino acid positions in an Fc polypeptide can be considered an Fc alteration that extends half-life: 250, 251 , 252, 259, 307, 308, 332, 378, 380, 428, 430, 434, 436. Each of these alterations or combinations of these alterations can be used to extend the half-life of an antibody as described herein.

[0104] (III) Multi-Domain Binding Molecules. Multi-domain binding molecules include at least two binding domains, wherein at least one binding domain includes an anti-CD90 antibody or fragment thereof disclosed herein. In particular embodiments, a multi-domain binding molecule includes at least one, at least two, at least, three, at least four binding domains that bind an epitope on CD90. In particular embodiments, all of the binding domains of a multi-domain binding molecule bind CD90.

[0105] In particular embodiments, a multi-domain binding molecule a multi-domain binding molecule can include binding domains that all bind the same epitope on the same antigen. In particular embodiments, a multi-domain binding molecule can include binding domains that all bind different epitopes on the same antigen. In particular embodiments, , a multi-domain binding molecule can include binding domains that bind different antigens. A multi-specific antibody has binding domains that bind different epitopes. In particular embodiments, a multi-specific antibody includes a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

[0106] Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (for example, F(ab')s bispecific antibodies). For example, WO 1996 / 016673 describes a bispecific anti-ErbB2 / anti-Fc gamma Rill antibody; US Pat. No. 5,837,234 describes a bispecific anti- ErbB2 / anti-Fc gamma Rl antibody; WO 1998 / 002463 describes a bispecific anti-ErbB2 / Fc alpha antibody; and US 5,821 ,337 describes a bispecific anti-ErbB2 / anti-CD3 antibody. In particular embodiments, a bispecific antibody can be in the form of a Bispecific T-cell Engaging (BiTE®)antibody.

[0107] Some additional exemplary bispecific antibodies have two heavy chains (each having three heavy chain CDRs, followed by (N-terminal to C-terminal) a CH1 domain, a hinge, a CH2 domain, and a CH3 domain), and two immunoglobulin light chains that confer antigen-binding specificity through association with each heavy chain. However, as indicated, additional architectures are envisioned, including bi-specific antibodies in which the light chain(s) associate with each heavy chain but do not (or minimally) contribute to antigen-binding specificity, or that can bind one or more of the epitopes bound by the heavy chain antigen-binding regions, or that can associate with each heavy chain and enable binding of one or both of the heavy chains to one or both epitopes.

[0108] scFv dimers or diabodies may be used, rather than whole antibodies. Diabodies and scFv can be constructed without an Fc region, using only variable domains (usually including the variable domain components from both light and heavy chains of the source antibody), potentially reducing the effects of anti-idiotypic reaction. Other forms of bispecific antibodies include the single chain “Janusins” described in Traunecker et al. (Embo Journal, 10, 3655-3659, 1991 ).

[0109] Exemplary bispecific antibodies with extended half-lives are described in, for example, US Patent No. 8,921 ,528 and US Patent Publication No. 2014 / 0308285.

[0110] Methods for making antibodies with two binding domains are known in the art. For example, traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (see, for example, Millstein et al. Nature 305:37-39, 1983). Similar procedures are disclosed in, for example, WO 1993 / 008829, Traunecker eta / ., EMBO J. 10:3655-3659, 1991 and Holliger & Winter, Current Opinion Biotechnol. 4, 446-449 (1993).

[0111] In particular embodiments, multi-domain binding molecules with two binding domains can be prepared using chemical linkage. For example, Brennan et al. (Science 229: 81 , 1985) describes a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent, sodium arsenite, to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated then are converted to thionitrobenzoate (TNB) derivatives. One of the Fab'- TNB derivatives then is reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the antibody having two binding domains.

[0112] Two or more binding domains can be linked through a linker to form a multi-domain binding molecule. Examples of linkers can be found in Chen et al., Adv Drug Deliv Rev. 2013 Oct 15;65(10): 1357-1369. Linkers can be flexible, rigid, or semi-rigid, depending on the desired functional domain presentation to a target.

[0113] Commonly used flexible linkers include linker sequences described elsewhere herein. Linkers that include one or more antibody hinge regions and / or immunoglobulin heavy chain constant regions, such as CH3 alone or a CH2CH3 sequence can also be used. Additional examples of linkers can be found in Chen et al., Adv Drug Deliv Rev. 2013 Oct 15; 65(10): 1357- 1369. Linkers can be flexible, rigid, or semi-rigid, depending on the desired functional domain presentation to a target.

[0114] In some situations, flexible linkers may be incapable of maintaining a distance or positioning of binding domains needed for a particular use. In these instances, rigid or semi-rigid linkers may be useful. Examples of rigid or semi-rigid linkers include proline-rich linkers. In particular embodiments, a proline-rich linker is a peptide sequence having more proline residues than would be expected based on chance alone. In particular embodiments, a proline-rich linker is one having at least 30%, at least 35%, at least 36%, at least 39%, at least 40%, at least 48%, at least 50%, or at least 51% proline residues. Particular examples of proline-rich linkers include fragments of proline-rich salivary proteins (PRPs).

[0115] Multi-domain binding molecules having three binding domains are artificial proteins that simultaneously bind to three antigens wherein at least one of the antigens is CD90. Anti-CD90 trispecific antibodies are artificial proteins that simultaneously bind to three different types of antigens, wherein at least one of the antigens is CD90. Tri-specific antibodies are described in, for example, WO2016 / 105450, WO 2010 / 028796; WO 2009 / 007124; WO 2002 / 083738; US 2002 / 0051780; and WO 2000 / 018806.

[0116] In particular embodiments, multi-domain binding molecules can include at least one binding domain disclosed herein and a second binding domain. In particular embodiments, the second binding domain can also be CD90. In particular embodiments, the second binding domain can include a binding domain that binds CD90. Other binding domains that bind CD90 are known in the art.

[0117] In particular embodiments, an anti-CD90 antibody (naive CD90 5E10 antibody) or fragment thereof includes a variable heavy chain including the sequence as set forth in SEQ ID NO: 40 and a variable light chain including the sequence as set forth in SEQ ID NO: 52. In particular embodiments, an anti-CD90 antibody (naive CD90 5E10 antibody) or fragment thereof includes a variable heavy chain encoded by the sequence as set forth in SEQ ID NO: 1 and a variable light chain encoded by the sequence as set forth in SEQ ID NO: 12.

[0118] In particular embodiments, an anti-CD90 antibody (codon-optimized CD90 5E10 antibody)or fragment thereof includes a variable heavy chain including the sequence as set forth in SEQ ID NO: 40 and a variable light chain including the sequence as set forth in SEQ ID NO: 52. In particular embodiments, an anti-CD90 antibody (codon-optimized CD90 5E10 antibody) or fragment thereof includes a variable heavy chain encoded by the sequence as set forth in SEQ ID NO: 22 and a variable light chain encoded by the sequence as set forth in SEQ ID NO: 31 .

[0119] In particular embodiments, an anti-CD90 antibody (mutated unpaired cysteine CD90 5E10 antibody) or fragment thereof includes a variable heavy chain including the sequence as set forth in SEQ ID NO: 40 and a variable light chain including the sequence as set forth in SEQ ID NO: 145. In particular embodiments, an anti-CD90 antibody (mutated unpaired cysteine CD90 5E10 antibody) or fragment thereof includes a variable heavy chain encoded by the sequence as set forth in SEQ ID NO: 22 and a variable light chain encoded by the sequence as set forth in SEQ ID NO: 144.

[0120] CDRs for these sequences can be found by those skilled in the art, using, for example, ABodyBuilder2.

[0121] In particular embodiments, binding domains disclosed herein can be used to create bi- tri, (or more) specific immune cell engaging molecules (e.g., immune cell engaging antibodies). An example of a multi-specific immune cell engaging molecule includes those which bind both CD90 and an immune cell (e.g., T-cell or NK-cells) activating epitope, with the goal of bringing immune cells to CD90-expressing cells to destroy the CD90-expressing cells. See, for example, US 2008 / 0145362. Such constructs are referred to herein as immune-activating multi-specifics or I- AMS). BiTEs® (Amgen, Thousand Oaks, CA) are one form of l-AMS. Immune cells that can be targeted for localized activation by l-AMS within the current disclosure include, for example, T- cells, natural killer (NK) cells, and macrophages which are discussed in more detail herein.

[0122] T-cell activation can be mediated by two distinct signals: those that initiate antigendependent primary activation and provide a T-cell receptor like signal (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences). l-AMS disclosed herein can target any T-cell activating epitope that upon binding induces T-cell activation. Examples of such T-cell activating epitopes are on T-cell markers including CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, 4-1 BB (CD 137), 0X40, lymphocyte function-associated antigen-1 (LFA-1 ), LIGHT, NKG2C, and B7-H3. Binding domains that bind T-cell markers are known in the art. B cell activation can be initiated by binding of an antigen to the B cell receptor (BCR) (e.g., IgM or IgD). Exemplary immune cell activating factors for NK cells include IL-15 and CD137.

[0123] In particular embodiments, the CD3 binding domain (e.g., scFv) is derived from the OKT3 antibody (the same as the one utilized in blinatumomab). The OKT3 antibody is described in detail in US 5,929,212. In particular embodiments, a CD3 binding domain includes a variable heavy chain including a CDRH1 sequence including KASGYTFTRYTMH (SEQ ID NO: 95), a CDRH2 sequence including INPSRGYTNYNQKFKD (SEQ ID NO: 96), and a CDRH3 sequence including YYDDHYCLDY (SEQ ID NO: 97); and a variable light chain including a CDRL1 sequence including SASSSVSYMN (SEQ ID NO: 92), a CDRL2 sequence including RWIYDTSKLAS (SEQ ID NO: 93), and a CDRL3 sequence including QQWSSNPFT (SEQ ID NO: 94). In particular embodiments, the CD3 T-cell activating epitope binding domain is a human or humanized binding domain (e.g., scFv).

[0124] The following sequence is an scFv derived from OKT3 which retains the capacity to bind CD3:QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYN QKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSSGGG GSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYD TSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINR (SEQ ID NO: 98). It may also be used as a CD3 binding domain.

[0125] In particular embodiments, the CD3 T-cell activating epitope binding domain is a human or humanized binding domain (e.g., scFv) including a variable heavy chain including a CDRH1 sequence including GFTFTKAW (SEQ ID NO: 101 ), a CDRH2 sequence including IKDKSNSYAT (SEQ ID NO: 102), and a CDRH3 sequence including RGVYYALSPFDY (SEQ ID NOU 03); and a variable light chain including a CDRL1 sequence including QSLVHNNGNTY (SEQ ID NO: 99), a CDRL2 sequence including KVS, and a CDRL3 sequence including GQGTQYPFT (SEQ ID NO: 100). These reflect CDR sequences of the 20G6-F3 antibody.

[0126] In particular embodiments, the CD3 T-cell activating epitope binding domain is a human or humanized binding domain (e.g., scFv) including a variable heavy chain including a CDRH1 sequence including GFTFSNAW (SEQ ID NO: 105), a CDRH2 sequence including IKARSNNYAT (SEQ ID NO: 106), and a CDRH3 sequence including RGTYYASKPFDY (SEQ ID NO: 107); and a variable light chain including a CDRL1 sequence including QSLVHDNGNTY (SEQ ID NO: 104), a CDRL2 sequence including KVS, and a CDRL3 sequence including GQGTQYPFT (SEQ ID NO: 100). These reflect CDR sequences of the 4B4-D7 antibody.

[0127] In particular embodiments, the CD3 T-cell activating epitope binding domain is a human or humanized binding domain (e.g., scFv) including a variable heavy chain including a CDRH1 sequence including GFTFSNAW (SEQ ID NO: 105), a CDRH2 sequence including IKDKSNNYAT(SEQ ID NO: 109), and a CDRH3 sequence including RYVHYGIGYAMDA (SEQ ID NO: 166); and a variable light chain including a CDRL1 sequence including QSLEHNNGNTY (SEQ ID NO: 108), a CDRL2 sequence including KVS, and a CDRL3 sequence including GQGTQYPFT (SEQ ID NO: 100). These reflect CDR sequences of the 4E7-C9 antibody.

[0128] In particular embodiments, the CD3 T-cell activating epitope binding domain is a human or humanized binding domain (e.g., scFv) including a variable heavy chain including a CDRH1 sequence including GFTFTNAW (SEQ ID NO: 1 12), a CDRH2 sequence including KDKSNNYAT (SEQ ID NO: 1 13), and a CDRH3 sequence including RYVHYRFAYALDA (SEQ ID NO: 114); and a variable light chain including a CDRL1 sequence including QSLVHTNGNTY (SEQ ID NO: 110), a CDRL2 sequence including KVS, and a CDRL3 sequence including GQGTHYPFT (SEQ ID NO: 1 11). These reflect CDR sequences of the 18F5-H10 antibody.

[0129] Additional examples of anti-CD3 antibodies, binding domains, and CDRs can be found in WO2016 / 116626. TR66 may also be used.

[0130] CD28 is a surface glycoprotein present on 80% of peripheral T-cells in humans and is present on both resting and activated T-cells. CD28 binds to B7-1 (CD80) and B7-2 (CD86) and is the most potent of the known co-stimulatory molecules (June et al., Immunol. Today 15:321 , 1994; Linsley et a / ., Ann. Rev. Immunol. 11 :191 , 1993). In particular embodiments, the CD28 binding domain (e.g., scFv) is derived from CD80, CD86 or the 9D7 antibody. Additional antibodies that bind CD28 include 9.3, KOLT-2, 15E8, 248.23.2, and EX5.3D10. Further, 1YJD provides a crystal structure of human CD28 in complex with the Fab fragment of a mitogenic antibody (5.11 A1 ).

[0131] In particular embodiments, a CD28 binding domain is derived from TGN1412. In particular embodiments, a CD28 binding domain includes avariable heavy chain including the sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNE KFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSS (SEQ ID NO: 1 15) a a variable light chain including the sequence: DIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFS GSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIK (SEQ ID NO: 116).

[0132] In particular embodiments, the CD28 binding domain includes a variable heavy chain including a CDRH1 sequence including GYTFTSYYIH (SEQ ID NO: 120), a CDRH2 sequence including CIYPGNVNTNYNEK (SEQ ID NO: 121 ), and a CDRH3 sequence including SHYGLDWNFDV (SEQ ID NO: 122); and a variable light chain including a CDRL1 sequence including HASQNIYVWLN (SEQ ID NO: 1 17), CDRL2 sequence including KASNLHT (SEQ ID NO: 118), and CDRL3 sequence including QQGQTYPYT (SEQ ID NO: 119).

[0133] In particular embodiments, the CD28 binding domain includes a variable heavy chain including a CDRH1 sequence including SYYIH (SEQ ID NO: 123), a CDRH2 sequence including CIYPGNVNTNYNEKFKD (SEQ ID NO: 124), and a CDRH3 sequence including SHYGLDWNFDV (SEQ ID NO: 122); and a variable light chain including a CDRL1 sequence including HASQNIYVWLN (SEQ ID NO: 117), a CDRL2 sequence including KASNLHT (SEQ ID NO: 118), and a CDRL3 sequence including QQGQTYPYT (SEQ ID NO: 119).

[0134] Activated T-cells express 4-1 BB (CD137). In particular embodiments, the 4-1 BB binding domain includes a variable heavy chain including a CDRH1 sequence including YYWS (SEQ ID NO: 128), a CDRH2 sequence including INH, and a CDRH3 sequence including YGPGNYDWYFDL (SEQ ID NO: 129); and a variable light chain including a CDRL1 sequence including RASQSVS (SEQ ID NO: 125), a CDRL2 sequence including ASNRAT (SEQ ID NO: 126), and a CDRL3 sequence including QRSNWPPALT (SEQ ID NO: 127).

[0135] In particular embodiments, the 4-1 BB binding domain includes a variable heavy chain including a CDRH1 sequence including GYSFSTYWIS (SEQ ID NO: 133), a CDRH2 sequence including KIYPGDSYTNYSPS (SEQ ID NO: 134) and a CDRH3 sequence including GYGIFDY (SEQ ID NO: 135); and a variable light chain including a CDRL1 sequence including SGDNIGDQYAH (SEQ ID NO: 130), a CDRL2 sequence including QDKNRPS (SEQ ID NO: 131 ), and a CDRL3 sequence including ATYTGFGSLAV (SEQ ID NO: 132).

[0136] Particular embodiments disclosed herein including binding domains that bind epitopes on CD8. In particular embodiments, the CD8 binding domain (e.g., scFv) is derived from the OKT8 antibody. For example, in particular embodiments, the CD8 T-cell activating epitope binding domain is a human or humanized binding domain (e.g., scFv) including a variable heavy chain including a CDRH1 sequence including GFNIKD (SEQ ID NO: 139), a CDRH2 sequence including RIDPANDNT (SEQ ID NO: 140), and a CDRH3 sequence including GYGYYVFDH (SEQ ID NO: 141 ); and a variable light chain including a CDRL1 sequence including RTSRSISQYLA (SEQ ID NO: 136), a CDRL2 sequence including SGSTLQS (SEQ ID NO: 137), and a CDRL3 sequence including QQHNENPLT (SEQ ID NO: 138). These reflect CDR sequences of the OKT8 antibody.

[0137] In particular embodiments natural killer cells (also known as NK-cells, K-cells, and killer cells) are targeted for localized activation by l-AMS. NK cells can induce apoptosis or cell lysis by releasing granules that disrupt cellular membranes and can secrete cytokines to recruit other immune cells.

[0138] Examples of activating proteins expressed on the surface of NK cells include NKG2D, CD8, CD16, KIR2DL4, KIR2DS1 , KIR2DS2, KIR3DS1 , NKG2C, NKG2E, and several members of the natural cytotoxicity receptor (NCR) family. Examples of NCRs that activate NK cells uponligand binding include NKp30, NKp44, NKp46, NKp80, and DNAM-1.

[0139] Examples of commercially available antibodies that bind to an NK cell receptor and induce and / or enhance activation of NK cells include: 5C6 and 1 D11 , which bind and activate NKG2D (available from BioLegend® San Diego, CA); mAb 33, which binds and activates KIR2DL4 (available from BioLegend®); P44-8, which binds and activates NKp44 (available from BioLegend®); SK1 , which binds and activates CD8; and 3G8 which binds and activates CD16.

[0140] In particular embodiments, the l-AMS can bind to and block an NK cell inhibitory receptor to enhance NK cell activation. Examples of NK cell inhibitory receptors that can be bound and blocked include KIR2DL1 , KIR2DL2 / 3, KIR3DL1 , NKG2A, and KLRG1. In particular embodiments, a binding domain that binds and blocks the NK cell inhibitory receptors KIR2DL1 and KIR2DL2 / 3 includes a variable heavy chain region of the sequenceQVQLVQSGAEVKKPGSSVKVSCKASGGTFSFYAISWVRQAPGQGLEWMGGFIPIFGAANYAQ KFQGRVTITADESTSTAYMELSSLRSDDTAVYYCARIPSGSYYYDYDMDVWGQGTTVTVSS (SEQ ID NO: 143) and a variable light chain region of the sequence EIVLTQSPVTLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSG SGSGTDFTLTISSLEPEDFAVYYCQQRSNWMYTFGQGTKLEIKRT (SEQ ID NO: 142). Additional NK cell activating antibodies are described in WQ / 2005 / 0003172 and US Patent No. 9,415,104.

[0141] In particular embodiments macrophages are targeted for localized activation by l-AMS. Macrophages are a type of leukocyte (or white blood cell) that can engulf and digest cells, cellular debris, and / or foreign substances in a process known as phagocytosis.

[0142] The l-AMS can be designed to bind to a protein expressed on the surface of macrophages. Examples of activating proteins expressed on the surface of macrophages (and their precursors, monocytes) include CD11 b, CD11 c, CD64, CD68, CD119, CD163, CD206, CD209, F4 / 80, IFGR2 Toll-like receptors (TLRs) 1 -9, IL-4Ra, and MARCO. Commercially available antibodies that bind to proteins expressed on the surface of macrophages include M1 / 70, which binds and activates CD11 b (available from BioLegend®); KP1 , which binds and activates CD68 (available from ABCAM®, Cambridge, United Kingdom); and ab87099, which binds and activates CD163 (available from ABCAM®).

[0143] In particular embodiments, l-AMS can target a pathogen recognition receptor (PRR). PRRs are proteins or protein complexes that recognize a danger signal and activate and / or enhance the innate immune response. Examples of PRRs include the TLR4 / MD-2 complex, which recognizes gram negative bacteria; Dectin-1 and Dectin-2, which recognize mannose moieties on fungus and other pathogens; TLR2 / TLR6 or TLR2 / TLR1 heterodimers, which recognize grampositive bacteria; TLR5, which recognizes flagellin; and TLR9 (CD289), which recognizes CpG motifs in DNA. In particular embodiments, l-AMS can bind and activate TLR4 / MD-2, Dectin-1 , Dectin-2, TRL2 / TLR6, TLR2 / TLR1 , TLR5, and / or TLR9.

[0144] In particular embodiments, l-AMS can target the complement system. The complement system refers to an immune pathway that is induced by antigen-bound antibodies and involves signaling of complement proteins, resulting in immune recognition and clearance of the antibody- coated antigens.

[0145] Cytolytic properties of l-AMS molecules can be confirmed in comparative in vitro assays. Briefly, for cell line experiments, target cells can be incubated in 96-well round bottom plates at 5-10,000 cells / well containing increasing concentrations of the various l-AMS antibodies with / without healthy donor T-cells (used at an E:T cell ratio of 1 :1 and 3:1 ). After 48 hours, cell numbers and drug-induced cytotoxicity, using 4',6-diamidino-2-phenylindole (DAPI) to detect non- viable cells, can be determined by flow cytometry. In experiments where healthy donor T-cells are added, cells can be identified by forward / side scatter properties and negativity for CellVue Burgundy dye. Experiments can include technical duplicates.

[0146] In particular embodiments, T-cell activating epitope binding domains including l-AMS constructs include one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions or non-conservative amino acid substitutions), or a combination of the above-noted changes, when compared with the Va, Vp, Ca, or Cp of a known TCR. An insertion, deletion or substitution may be anywhere in a Va, Vp, Ca, or Cp region, including at the amino- or carboxy-terminus or both ends of these regions, provided that each CDR includes zero changes or at most one, two, or three changes and provided a binding domain including a modified Va, Vp, Ca, or Cp region can still specifically bind its target with an affinity similar to wild type.

[0147] In some embodiments, a multi-domain binding molecule includes a basic immunoglobulin structure such as an IgA domain or an IgM domain. Basic immunoglobulin structures in vertebrate systems are described above and are well understood. (See, e.g., Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. 1988).

[0148] In particular embodiments, multi-domain binding molecules are multimers of an antibody disclosed herein. Multimerization strategies include formation of a fusion protein using protein linkers or use of IgA or IgM constant regions as a multimerization scaffold. In certain aspects, multimerization is achieved by linking antibodies or binding domains of antibodies in a fusion protein with protein linkers. Fusion proteins include different protein domains linked to each otherdirectly or through intervening linker segments such that the function of each included domain is retained.

[0149] Multimerized antibodies and antibody-like molecules such as IgA and IgM antibodies have emerged as promising drug candidates in the fields of, e.g., immuno-oncology and infectious diseases allowing for improved specificity, improved avidity, and the ability to bind to multiple binding targets. See, e.g., U.S. Patent Nos. 9,951 ,134, 10,400,038, and 9,938,347, U.S. Patent Application Publication Nos. US20190100597A1 , US20180118814A1 , US201801 18816A1 , US20190185570A1 , and US20180265596A1 , and PCT Publication Nos. WO 2018 / 017888, WO 2018 / 017763, WO 2018 / 017889, WO 2018 / 017761 , and WO 2019 / 165340.

[0150] Particular embodiments include using IgA and IgM constant region domains to allow the binding portion of molecules provided herein to readily multimerize into dimers, pentamers or hexamers. Basic immunoglobulin structures in vertebrate systems are described above and are well understood. (See, e.g., Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. 1988).

[0151] Immunoglobulin A (IgA), as the major class of antibody present in the mucosal secretions of most mammals, represents a key first line of defense against invasion by inhaled and ingested pathogens. IgA is also found at significant concentrations in the serum of many species, where it functions as a second line of defense mediating elimination of pathogens that have breached the mucosal surface. Receptors specific for the Fc region of IgA, FcaR, are key mediators of IgA effector function. Native IgA is a tetrameric protein including two identical light chains (K or A) and two identical heavy chains. IgA, similarly to IgG, contains three constant domains (CA1 -CA3), with a hinge region between the CA1 and CA2 domains. The main difference between lgA1 and lgA2 resides in the hinge region that lies between the two Fab arms and the Fc region. Ig A1 has an extended hinge region due to the insertion of a duplicated stretch of amino acids, which is absent in lgA2. Both forms of IgA have the capacity to form dimers, in which two monomer units, are arranged in an end-to-end configuration stabilized by disulfide bridges and incorporation of a J-chain. J-chains are also part of IgM pentamers and are discussed in more detail below.

[0152] Both IgA and IgM (discussed further below in relation to pentamers and hexamers) possess an 18-amino acid extension in the C terminus called the "tailpiece" (tp). The IgA and IgM tp is highly conserved among various animal species. The conserved penultimate cysteine residue in the IgA and IgM tp has been demonstrated to be involved in multimerization by forming a disulfide bond between heavy chains to permit formation of a multimer. Both tp contain an N- linked carbohydrate addition site, the presence of which is required for dimer formation in IgA and J-chain incorporation and pentamer formation in IgM. However, the structure and composition ofthe N-linked carbohydrates in the tp differ, suggesting differences in the accessibility of the glycans to processing by glycosyltransferases. Particularly, the IgA (atp) and IgM (ptp) tp differ at seven amino acid positions.

[0153] The human lgA1 constant region typically includes the amino acid sequence: ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDASGDL YTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPR LSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVL PGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLAR GFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMV GHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY (SEQ ID NO: 198). Referring to this SEQ ID NO: 198, the human CA1 domain extends from amino acid 6 to amino acid 98; the human lgA1 hinge region extends from amino acid 102 to amino acid 124, the human CA2 domain extends from amino acid 125 to amino acid 219, the human CA3 domain extends from amino acid 228 to amino acid 330, and the tp extends from amino acid 331 to amino acid 352.

[0154] The human lgA2 constant region typically includes the amino acid sequence ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESGQNVTARNFPPSQDASGD LYTTSSQLTLPATQCPDGKSVTCHVKHYTNPSQDVTVPCPVPPPPPCCHPRLSLHRPALEDLL LGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAQPWNHG ETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRW LQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQK TIDRLAGKPTHVNVSVVMAEVDGTCY (SEQ ID NO: 199). Referring to this SEQ ID NO: 199, the human CA1 domain extends from amino acid 6 to amino acid 98, the human lgA2 hinge region extends from amino acid 102 to amino acid 111 , the human CA2 domain extends from amino acid 113 to amino acid 206, the human CA3 domain extends from amino acid 215 to amino acid 317, and the tp extends from amino acid 318 to amino acid 340.

[0155] As indicated, two IgA binding units can form a complex with two additional polypeptide chains, the J chain (e.g., SEQ ID NO: 32, the mature human J chain) and the secretory component to form a bivalent secretory IgA (slgA)-derived binding molecule. An exemplary precursor secretory component includes the sequence MLLFVLTCLLAVFPAISTKSPIFGPEEVNSVEGNSVSITCYYPPTSVNRHTRKYWCRQGARGGC ITLISSEGYVSSKYAGRANLTNFPENGTFVVNIAQLSQDDSGRYKCGLGINSRGLSFDVSLEVS QGPGLLNDTKVYTVDLGRTVTINCPFKTENAQKRKSLYKQIGLYPVLVIDSSGYVNPNYTGRIRL DIQGTGQLLFSVVINQLRLSDAGQYLCQAGDDSNSNKKNADLQVLKPEPELVYEDLRGSVTFH CALGPEVANVAKFLCRQSSGENCDVVVNTLGKRAPAFEGRILLNPQDKDGSFSVVITGLRKEDAGRYLCGAHSDGQLQEGSPIQAWQLFVNEESTIPRSPTVVKGVAGGSVAVLCPYNRKESKSIK YWCLWEGAQNGRCPLLVDSEGWVKAQYEGRLSLLEEPGNGTFTVILNQLTSRDAGFYWCLTN GDTLWRTTVEIKIIEGEPNLKVPGNVTAVLGETLKVPCHFPCKFSSYEKYWCKWNNTGCQALP SQDEGPSKAFVNCDENSRLVSLTLNLVTRADEGWYWCGVKQGHFYGETAAVYVAVEERKAA GSRDVSLAKADAAPDEKVLDSGFREIENKAIQDPRLFAEEKAVADTRDQADGSRASVDSGSSE EQGGSSRALVSTLVPLGLVLAVGAVAVGVARARHRKNVDRVSIRSYRTDISMSDFENSREFGA NDNMGASSITQETSLGGKEEFVATTESTTETKEPKKAKRSSKEEAEMAYKDFLLQSSTVAAEA QDGPQEA (SEQ ID NO: 200). An exemplary mature secretory component includes KSPIFGPEEVNSVEGNSVSITCYYPPTSVNRHTRKYWCRQGARGGCITLISSEGYVSSKYAGR ANLTNFPENGTFVVNIAQLSQDDSGRYKCGLGINSRGLSFDVSLEVSQGPGLLNDTKVYTVDL GRTVTINCPFKTENAQKRKSLYKQIGLYPVLVIDSSGYVNPNYTGRIRLDIQGTGQLLFSVVINQL RLSDAGQYLCQAGDDSNSNKKNADLQVLKPEPELVYEDLRGSVTFHCALGPEVANVAKFLCR QSSGENCDVVVNTLGKRAPAFEGRILLNPQDKDGSFSVVITGLRKEDAGRYLCGAHSDGQLQE GSPIQAWQLFVNEESTIPRSPTVVKGVAGGSVAVLCPYNRKESKSIKYWCLWEGAQNGRCPLL VDSEGWVKAQYEGRLSLLEEPGNGTFTVILNQLTSRDAGFYWCLTNGDTLWRTTVEIKIIEGEP NLKVPGNVTAVLGETLKVPCHFPCKFSSYEKYWCKWNNTGCQALPSQDEGPSKAFVNCDEN SRLVSLTLNLVTRADEGWYWCGVKQGHFYGETAAVYVAVEERKAAGSRDVSLAKADAAPDEK VLDSGFREIENKAIQDPR (SEQ ID NO: 201 ). While not wishing to be bound by theory, and as indicated above, the assembly of two IgA binding units into a dimeric IgA-derived binding molecule is thought to involve the CA3 and tp domains. See, e.g., Braathen, R., el al., J. Biol. Chem. 277:42755-42762 (2002). Accordingly, a multimerizing dimeric IgA-derived binding molecule provided in this disclosure typically includes IgA constant regions that include at least the CA3 and tp domains.

[0156] An engineered IgA heavy chain constant region can additionally include a CA2 domain or a fragment thereof, an IgA hinge region or fragment thereof, a CA1 domain or a fragment thereof, and / or other IgA (or other immunoglobulin, e.g., IgG) heavy chain domains, including, e.g., an IgG hinge region. In certain embodiments, a binding molecule as provided herein can include a complete IgA heavy chain constant region (e.g., SEQ ID NO: 198 or SEQ ID NO: 199), or a variant, derivative, or analog thereof.

[0157] In particular embodiments, the IgA heavy chain constant regions can include amino acids 125 to 353 of SEQ ID NO: 198 or amino acids 113 to 340 of SEQ ID NO: 199. In particular embodiments, the IgA heavy chain constant regions can each further include an IgA or IgG hinge region situated N-terminal to the IgA CA2 domains. For example, the IgA heavy chain constant regions can include amino acids 102 to 353 of SEQ ID NO: 198 or amino acids 102 to 340 of SEQID NO: 199. In particular embodiments, the IgA heavy chain constant regions can each further include an IgA CA1 domain situated N-terminal to the IgA hinge region.

[0158] Each of the strategies discussed above can be used to create IgA antibody-based dimers.

[0159] Particular embodiments include IgM immunoglobulin constant region domains that allow the binding portion of molecules provided herein to readily multimerize into pentamers or hexamers.

[0160] Particular embodiments include IgM constant regions (or variants thereof). These embodiments have the ability to form hexamers, or in association with a J-chain, form pentamers. Embodiments with an IgM constant region typically include at least the Cp4-tp domains of the IgM constant region but can include heavy chain constant region domains from other antibody isotypes, e.g., IgG, from the same species or from a different species. In particular embodiments, one or more constant region domains can be deleted so long as the IgM antibody is capable of forming hexamers and / or pentamers. Thus, an IgM antibody can be, e.g., a hybrid IgM / IgG antibody or can be a “multimerizing fragment” of an IgM-derived binding molecule.

[0161] The assembly of five or six IgM binding units into a pentameric or hexameric IgM antibody is thought to involve the Cp4 and tp domains. See, e.g., Braathen, R., et al., J Biol. Chem. 277:42755-42762 (2002). Accordingly, a pentameric or hexameric IgM antibody described in this disclosure typically includes at least the Cp4 and / or tp domains (also referred to herein collectively as Cp4-tp). A “multimerizing fragment” of an IgM heavy chain constant region thus includes at least the Cp4-tp domains. An IgM heavy chain constant region can additionally include a Cp3 domain or a fragment thereof, a Cp2 domain or a fragment thereof, a Cp1 domain or a fragment thereof, and / or other IgM heavy chain domains.

[0162] Five IgM monomers form a complex with a J-chain to form a native IgM molecule. The J- chain is considered to facilitate polymerization of p chains before IgM is secreted from antibodyproducing cells. Sequences for the human IGJ gene are known in the art, for example, (IGMT Accession: J00256, X86355, M25625, AJ879487). The J chain establishes the disulfide bridges between IgM antibodies to form multimeric structures such as pentamers. See, for example, Sorensen et al. International Immunology, (2000), pages 19-27. While crystallization of IgM has proved to be notoriously challenging, Czajkowsky and Shao (PNAS 106(35): 14960-14965, 2009) published a homology-based structural model of IgM, based on the structure of the IgE Fc domain and the known disulfide pairings. The authors report that the human IgM pentamer is a mushroomshaped molecule with a flexural bias. The IgM heavy (p) chain contains five N-linked glycosylation sites: Asn-171 , Asn-332, Asn-395, Asn-402 and Asn-563. In an IgM antibody where each binding unit is bivalent, the binding molecule itself can have 10 or 12 valencies.

[0163] The Kabat numbering system for the human IgM constant domain can be found in Kabat, et. al. “Tabulation and Analysis of Amino acid and nucleic acid Sequences of Precursors, V- Regions, C-Regions, J-Chain, T-Cell Receptors for Antigen, T-Cell Surface Antigens, b-2 Microglobulins, Major Histocompatibility Antigens, Thy-I, Complement, C-Reactive Protein, Thymopoietin, Integrins, Post-gamma Globulin, a-2 Macroglobulins, and Other Related Proteins,” U.S. Dept of Health and Human Services (1991 ). IgM constant regions can be numbered sequentially (i.e., amino acid #1 starting with the first amino acid of the constant region) or by using the Kabat numbering scheme.

[0164] A “full length IgM antibody heavy chain” is a polypeptide that includes, in N- terminal to C- terminal direction, an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CM1 or Cp1 ), an antibody heavy chain constant domain 2 (CM2 or Cp2), an antibody heavy chain constant domain 3 (CM3 or Cp3), and an antibody heavy chain constant domain 4 (CM4 or Cp4) that can include a tp, as indicated above.

[0165] In particular embodiments, each binding unit of a multimeric binding molecule as provided herein includes two IgM heavy chain constant regions or multimerizing fragments or variants thereof, each including at least an IgM Cp4 domain and an IgM tp domain. In certain embodiments the IgM heavy chain constant regions can each further include an IgM C|i3 domain situated N- terminal to the IgM Cp4 and IgM tp domains.

[0166] In particular embodiments, the IgM heavy chain constant regions can each further include an IgM Cp2 domain situated N-terminal to the IgM Cp3 domain. Exemplary multimeric binding molecules provided herein include human IgM constant regions that include the wild-type human C|i2, C|i3, and Cp4-tp domains as follows: VIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAE AKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSF ASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWN SGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADV FVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNR VTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 202).

[0167] In certain IgM-derived multimeric binding molecules as provided herein each IgM constant region can include, instead of, or in addition to an IgM Cp2 domain, an IgG hinge region or functional variant thereof situated N-terminal to the IgM Cp3 domain. An exemplary variant human lgG1 hinge region amino acid sequence in which the cysteine at position 6 is substituted with serine is VEPKSSDKTHTCPPCPAP (SEQ ID NO: 212). An exemplary IgM constant region of this type includes the variant human IgG 1 hinge region fused to a multimerizing fragment of the humanIgM constant region including the C i3, Cp4, and tp domains, and includes the amino acid sequence: VEPKSSDKTHTCPPCPAPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNG EAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHR PDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPG RYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 203).

[0168] Human IgM constant regions, and also certain non-human primate IgM constant regions, as provided herein typically include five (5) naturally-occurring asparagine (N)-linked glycosylation motifs or sites. As used herein “an N-linked glycosylation motif” includes the amino acid sequence N-X1 -S / T, wherein N is asparagine, X1 is any amino acid except proline (P), and S / T is serine (S) or threonine (T). The glycan is attached to the nitrogen atom of the asparagine residue. See, e.g., Drickamer K, Taylor ME (2006), Introduction to Glycobiology (2nd ed.). Oxford University Press, USA. N-linked glycosylation motifs occur in the human IgM heavy chain constant regions of SEQ ID NO: 204 or SEQ ID NO: 205 starting at positions 46 (“N1”), 209 (“N2”), 272 (“N3”), 279 (“N4”), and 440 (“N5”). These five motifs are conserved in non-human primate IgM heavy chain constant regions, and four of the five are conserved in the mouse IgM heavy chain constant region. Each of these sites in the human IgM heavy chain constant region, except for N4, can be mutated to prevent glycosylation at that site, while still allowing IgM expression and assembly into a hexamer or pentamer.

[0169] The human IgM heavy chain constant region typically includes the amino acid sequence GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGK YAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNP RKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWL SQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSV TISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTIS RPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAP MPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVM SDTAGTCY (SEQ ID NO: 204; identical to, e.g., GenBank Accession Nos. pir||S37768, CAA47708.1 , and CAA47714.1 ). Referring to this SEQ ID NO: 204, the human Cp1 region ranges from amino acid 5 to amino acid 102; the human Cp2 region ranges from amino acid 1 14 to amino acid 205, the human Cp3 region ranges from amino acid 224 to amino acid 319, the Cp4 region ranges from amino acid 329 to amino acid 430, and the tp ranges from amino acid 431 to amino acid 453.

[0170] In particular embodiments, an IgM heavy chain constant region includes the sequence: GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGK YAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNP RKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWL GQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDS VTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTI SRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSA PMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLV MSDTAGTCY (SEQ ID NO: 205; (UniProt ID P01871 ) — allele IGHM*04). This sequence differs from SEQ ID NO: 204 by one amino acid at position 191 .

[0171] Other forms of the human IgM constant region with minor sequence variations exist, including GenBank Accession Nos. P01871.4, CAB37838.1 , and pir||MHHU. The amino acid substitutions, insertions, and / or deletions at positions corresponding to SEQ ID NO: 204 described herein can likewise be incorporated into alternate human IgM sequences, as well as into IgM constant region amino acid sequences of other species, e.g., those shown in FIG. 1 of PCT / US2019 / 020374.

[0172] In certain aspects, a variant human IgM constant region includes an amino acid substitution corresponding to the wild-type human IgM constant region at position P311 , P313, R344, E345, S401 , E402, and / or E403 of SEQ ID NO: 204. These positions correspond to the Kabat numbering system as follows: S401 of SEQ ID NO: 204 corresponds to S524 of Kabat; E402 of SEQ ID NO: 204 corresponds to E525 of Kabat; E403 of SEQ ID NO: 204 corresponds to E526 of Kabat; R344 of SEQ ID NO: 204 corresponds to R467 of Kabat; and E345 of SEQ ID NO: 204 corresponds to E468 of Kabat.

[0173] In particular embodiments, “corresponds to” means the designated position of SEQ ID NO: 204 and the amino acid in the sequence of the IgM constant region of any species which is homologous to the specified position. See FIG. 1 of PCT / US2019 / 020374.

[0174] In particular embodiments, P311 of SEQ ID NO: 204 can be substituted, e.g., with alanine (P31 1A), serine (P31 1S), or glycine (P311G) and / or P313 of SEQ ID NO: 204 can be substituted, e.g., with alanine (P313A), serine (P313S), or glycine (P313G). P311 and P313 of SEQ ID NO: 204 can be substituted with alanine (P311 A) and serine (P313S), respectively as shown in the following sequence: (mutations in bold underline) GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGK YAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNP RKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSV TISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLASSLKQTIS RPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVM SDTAGTCY (SEQ ID NO: 206).

[0175] In certain aspects, S401 of SEQ ID NO: 204 can be substituted with any amino acid. In certain aspects, S401 of SEQ ID NO: 204 can be substituted with alanine (A) as follows (alanine substitution indicated by bold underline):GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGK YAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNP RKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWL SQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSV TISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTIS RPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAP MPEPQAPGRYFAHSILTVAEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVM SDTAGTCY (SEQ ID NO: 207).

[0176] In certain aspects, E402 of SEQ ID NO: 204 can be substituted with any amino acid. In certain aspects, E402 of SEQ ID NO: 204 can be substituted with alanine (A) as follows (alanine substitution indicated by bold underline):GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGK YAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNP RKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWL SQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTIS RPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAP MPEPQAPGRYFAHSILTVSAEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVM SDTAGTCY (SEQ ID NO: 208).

[0177] In certain aspects, E403 of SEQ ID NO: 204 can be substituted with any amino acid. In certain aspects, E403 of SEQ ID NO: 204 can be substituted with alanine (A) as follows (alanine substitution indicated by bold underline):GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGK YAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNP RKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWL SQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTIS RPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAP MPEPQAPGRYFAHSILTVSEAEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVM SDTAGTCY (SEQ ID NO: 209).

[0178] In certain aspects, R344 of SEQ ID NO: 204 can be substituted with any amino acid. In certain aspects, R344 of SEQ ID NO: 204 can be substituted with alanine (A) as follows (alanine substitution indicated by bold underline):GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGK YAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNP RKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWL SQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSV TISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTIS RPKGVALHRPDVYLLPPAREQLNLAESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAP MPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVM SDTAGTCY (SEQ ID NO: 210).

[0179] In certain aspects, E345 of SEQ ID NO: 204 can be substituted with any amino acid. In certain aspects, E345 of SEQ ID NO: 204 can be substituted with alanine (A) as follows (alanine substitution indicated by bold underline):GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGK YAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNP RKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWL SQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSV TISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTIS RPKGVALHRPDVYLLPPAREQLNLRASATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAP MPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVM SDTAGTCY (SEQ ID NO: 211 ).

[0180] As indicated, five IgM binding units can form a complex with a J-chain to form a pentameric IgM antibody. The precursor form of the human J-chain includes:MKNHLLFWGVLAVFIKAVHVKAQEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLN NRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKC YTAVVPLVYGGETKMVETALTPDACYPD (SEQ ID NO: 39). The signal peptide extends from amino acid 1 to amino acid 22 of SEQ ID NO: 39 and the mature human J-chain extends from amino acid 23 to amino acid 159 of SEQ ID NO: 39.

[0181] The mature human J-chain includes the amino acid sequenceQEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDL CKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTPD ACYPD (SEQ ID NO: 32).

[0182] The term “J-chain” as used herein refers to the J-chain of native sequence IgM or IgA antibodies of any animal species. When specified, it can also refer to any functional fragment thereof, derivative thereof, and / or variant thereof, including a mature human J-chain amino acid sequence provided herein as SEQ ID NO: 32. A functional fragment, derivative, and / or variant of a J-chain has at least 90% sequence identity to the reference J-chain and retains the multimerizing function of the reference J-chain.

[0183] In certain aspects, the J-chain of the IgM antibody as provided herein includes an amino acid substitution at the amino acid position corresponding to amino acid Y102, T103, N49 or S51 of SEQ ID NO: 32.

[0184] By “an amino acid corresponding to” a position of SEQ ID NO: 32 is meant the amino acid in the sequence of the J-chain of any species which is homologous to the referenced residue in the human J-chain. For example, the position corresponding to Y102 in SEQ ID NO: 32 is conserved in the J-chain amino acid sequences of at least 43 other species. The position corresponding to T103 in SEQ ID NO: 32 is conserved in the J-chain amino acid sequences of at least 37 other species. The positions corresponding to N49 and S51 in SEQ ID NO: 32 are conserved in the J-chain amino acid sequences of at least 43 other species. See FIG. 4 of U.S. Patent No. 9,951 ,134 and FIG. 2 of PCT / US2019 / 020374.

[0185] In certain aspects, the amino acid corresponding to Y102 of SEQ ID NO: 32 can be substituted with any amino acid. In certain aspects, the amino acid corresponding to Y102 of SEQ ID NO: 32 can be substituted with alanine (alanine substitution indicated by bold underline): QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDL CKKCDPTEVELDNQIVTATQSNICDEDSATETCATYDRNKCYTAVVPLVYGGETKMVETALTPD ACYPD (SEQ ID NO: 33),With serine (serine substitution indicated by bold underline):QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDL CKKCDPTEVELDNQIVTATQSNICDEDSATETCSTYDRNKCYTAVVPLVYGGETKMVETALTPD ACYPD (SEQ ID NO: 34),Or with arginine (arginine substitution indicated by bold underline):QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDL CKKCDPTEVELDNQIVTATQSNICDEDSATETCRTYDRNKCYTAVVPLVYGGETKMVETALTPD ACYPD (SEQ ID NO: 35).

[0186] In certain aspects, the amino acid corresponding to T103 of SEQ ID NO: 32 can be substituted with any amino acid. In a particular aspect, the amino acid corresponding to T103 of SEQ ID NO: 32 can be substituted with alanine as follows (alanine substitution indicated by bold underline):QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDL CKKCDPTEVELDNQIVTATQSNICDEDSATETCYAYDRNKCYTAVVPLVYGGETKMVETALTP DACYPD (SEQ ID NO: 36).

[0187] In certain aspects, the variant J-chain or functional fragment thereof of the IgM antibody as provided herein includes an amino acid substitution at the amino acid position corresponding to amino acid N49 or amino acid S51 of SEQ ID NO: 32, provided that S51 is not substituted with threonine (T), or wherein the J-chain includes amino acid substitutions at the amino acid positions corresponding to both amino acids N49 and S51 of SEQ ID NO: 32.

[0188] The amino acids corresponding to N49 and S51 of SEQ ID NO: 32 along with the amino acid corresponding to 150 of SEQ ID NO: 32 include an N-linked glycosylation motif in the J- chain. Accordingly, mutations at N49 and / or S51 (with the exception of a single threonine substitution at S51) can prevent glycosylation at this motif. In certain aspects, the asparagine at the position corresponding to N49 of SEQ ID NO: 32 can be substituted with any amino acid. In certain aspects, the asparagine at the position corresponding to N49 of SEQ ID NO: 32 can be substituted with alanine (A), glycine (G), threonine (T), serine (S) or aspartic acid (D). In a particular aspect the position corresponding to N49 of SEQ ID NO: 32 can be substituted with alanine (A). In a particular aspect the J-chain is a variant human J-chain and includes the amino acid sequence:QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNREAISDPTSPLRTRFVYHLSDL CKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTPD ACYPD (SEQ ID NO: 37).

[0189] In certain aspects, the serine at the position corresponding to S51 of SEQ ID NO: 32 can be substituted with any amino acid except threonine. In certain aspects, the serine at the position corresponding to S51 of SEQ ID NO: 32 can be substituted with alanine (A) or glycine (G). In a particular aspect the position corresponding to S51 of SEQ ID NO: 32 can be substituted with alanine (A). In a particular aspect the variant J-chain or functional fragment thereof is a variant human J-chain and includes the amino acid sequence: EDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENIADPTSPLRTRFVYHLSDLC KKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTPDA CYPD (SEQ ID NO: 38).

[0190] Particular embodiments include a heterologous polypeptide (e.g., a single-domain antibody binding domain) fused to the J-chain or functional fragment thereof via a peptide linker, e.g., a peptide linker including at least 5 amino acids, but no more than 25 amino acids. In certain aspects, the peptide linker includes (GGGGS)n (SEQ ID NO: 79) wherein n is 1 -5.

[0191] A single-domain antibody binding domain can be introduced into the J-chain at any location that allows the binding of the binding domain to its binding target without interfering with J-chain function or the function of an associated IgA, IgM, or hybrid IgG antibody. Insertion locations include at or near the C- terminus, at or near the N-terminus or at an internal location that, based on the three-dimensional structure of the J-chain, is accessible. In certain aspects, the antigen-binding domain can be introduced into the mature human J-chain of SEQ ID NO: 32 between cysteine residues 92 and 101 of SEQ ID NO: 32. In a further aspect, the antigen-binding domain can be introduced into the human J-chain of SEQ ID NO: 32 at or near a glycosylation site. In a further aspect, the antigen-binding domain can be introduced into the human J-chain of SEQ ID NO: 32 within 10 amino acid residues from the C- terminus, or within 10 amino acids from the N-terminus.

[0192] In particular embodiments, the single-domain antibody is introduced into the native human J-chain sequence of SEQ ID NO: 32 by chemical or chemo-enzymatic derivatization. In particular embodiments, the single-domain antibody is introduced into the native human J-chain sequence of SEQ ID NO: 32 by a chemical linker. In some embodiments, the chemical linker is a cleavable or non-cleavable linker. In particular embodiments, the cleavable linker is a chemically labile linker or an enzyme-labile linker. In some embodiments, the linker is selected from the group including N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-l-carboxylate (SMCC), N-succinimidyl-4-(2-pyridylthio) pentanoate (SPP), iminothiolane (IT), afunctional derivatives of imidoesters, active esters, aldehydes, bis-azido compounds, bis-diazonium derivatives, diisocyanates, and bis-active fluorine compounds. In particular embodiments, the modified J-chain is modified by insertion of an enzyme recognition site, and by post-translationally attaching a binding moiety at the enzyme recognition site through a peptide or non-peptide linker.

[0193] In certain aspects the modified J-chain can include the formula X[Ln]J or J[Ln]X, where J includes a mature native J-chain or functional fragment thereof, X includes a heterologous binding domain, and [Ln] is a linker sequence including n amino acids, where n is a positive integer from 1 to 100, 1 to 50, or 1 to 25. In certain aspects N is 5, 10, 15, or 20.

[0194] J-chains from the following species can also be used in certain embodiments: Pan troglodytes, Pongo abelii, Callithrix jacchus, Macaca mulatta, Papio Anubis, Saimiri boliviensis,Tupaia chinensis, Tursiops truncatus, Orcinus orca, Loxodonta Africana, Leptonychotes weddellii, Ceratotherium simum, Felis catus, Canis familiaris, Ailuropoda melanoleuca, Mustela furo, Equus caballus, Cavia porcellus, Camelus ferus, Capra hircus, Chinchilla lanigera, Mesocricetus auratus, Ovis aries, Myotis lucifugus, Pantholops hodgsonii, Bos taurus, Mus musculus, Rattus norvegicus, Echinops telfairi, Oryctolagus cuniculus, Monodelphis domestica, Alligator mississippiensis, Chrysemys picta, Sarcophilus harrisii, Ornithorhynchus anatinus, Melopsittacus undulates, Anas platyrhynchos, Gallus gallus, Meleagris gallopavo, Falco peregrinus, Zonotrichia albicollis, and Pteropus alecto.

[0195] In particular embodiments, the antibodies can multimerize by optionally including a multimerization domain. A “multimerization domain” is a domain that causes two or more proteins (monomers) to interact with each other through covalent and / or non-covalent association(s). Multimerization domains present in proteins can result in protein interactions that form dimers, trimers, tetramers, pentamers, hexamers, heptamers, etc., depending on the number of units / monomers incorporated into the multimer.

[0196] In particular embodiments, the multimerization domain is a dimerization domain that allows binding of two complementary monomers to form a dimer. In particular embodiments, a dimerization and docking domain (DDD) can be derived from the cAMP-dependent protein kinase (PKA) regulatory subunits and can be paired with an anchoring domain (AD). The AD can be derived from a specific region found in various A-kinase anchoring proteins (AKAPs) that mediates association with the R subunits of PKA. Additional DDDs and ADs include: the 4-helix bundle type DDD (Newlon, etal. EMBO J. 2001 ; 20: 1651 -1662; Newlon, etal. Nature Struct Biol. 1999; 3: 222-227) domains obtained from p53, DCoH (pterin 4 a carbinolamine dehydratase / dimerization cofactor of hepatocyte nuclear factor 1 a (TCF1 )) and HNF-1 (hepatocyte nuclear factor 1) (Rose, et al. Nature Struct Biol. 2000; 7: 744-748). Other AD sequences of potential use may be found in US 2003 / 0232420A1 .

[0197] In particular embodiments, complementary binding domains can dimerize. In particular embodiments, the binding domain is a transmembrane polypeptide derived from a FCERI chain. In particular embodiments, an antibody or fragment thereof can include a part of a FCERI a chain and another antibody or fragment thereof can include a part of an FCERI p chain such that said FCERI chains spontaneously dimerize together to form a dimeric antibody (e.g., bispecific antibody). In particular embodiments, an antibody or fragment thereof can include a part of a FCERI a chain and another antibody or fragment thereof part of a FCERI y chain such that said FCERI chains spontaneously trimerize together to form a trimeric antibody, and in another embodiment the multi-domain binding molecule can include a part of FCERI a chain, a part ofFCERI p chain and a part of FCERI Y chain such that said FCERI chains spontaneously tetramerize together to form a tetrameric multi-domain binding molecule.

[0198] Leucine zippers are described in US 5932448; SH2 and SH3 are described in Vidal et al., Biochemistry, 43:7336- 44, 2004); PTB is described in Zhou et al., Nature, 378:584- 592, 1995); WW is described in Sudol Prog Biochys MoL Bio, 65:113-132, 1996; PDZ is described in Kim et al., Nature, 378: 85-88, 1995 and Komau et al., Science, 269:1737-1740, 1995; and WD40 is described in Hu et al., J Biol Chem., 273:33489- 33494, 1998.

[0199] Additional multimerization domains and systems are described in, for example, Hodneland, et al. Proc Natl Acd Sci USA. 2002; 99: 5048-5052; Arakawa et al., J Biol. Chem., 269:27833-27839, 1994; Radziejewski et al., Biochem, 32: 1350, 1993; WO2012001647 A2; US 5821333; GenBank Accession no. AAF73912.1 (Nishi et al., Mol Cell Biol, 25: 2607-2621 , 2005), the SH3 domain of IB1 from GenBank Accession no. AAD22543.1 (Kristensen el al., EMBO J., 25: 785-797, 2006), the PTB domain of human DOK-7 from GenBank Accession no. NP 005535.1 (Wagner et al., Cold Spring Harb Perspect Biol. 5: a008987, 2013), the PDZ-like domain of SATB1 from UniProt Accession No. Q01826 (Galande et al., Mol Cell Biol. Aug; 21 : 5591-5604, 2001 ), the WD40 repeats of APAF from UniProt Accession No. 014727 (Jorgensen et al., 2009. PLOS One. 4(12):e8463), the PAS motif of the dioxin receptor from UniProt Accession No. I6L9E7 (Pongratz etal., Mol Cell Biol, 18:4079-4088, 1998) and the EF hand motif of parvalbumin from UniProt Accession No. P20472 (Jamalian et al., Int J Proteomics, 2014: 153712, 2014). C4b, dextrameric, and ferritin-based multimerization can be used.

[0200] In particular embodiments, complementary binding domains can be induced using a third molecule or chemical inducer. This method of dimerization requires that one antibody or fragment thereof include a chemical inducer of dimerization binding domain 1 (CBD1 ) and the second antibody or fragment thereof include the second chemical inducer of dimerization binding domain (CBD2), wherein CBD1 and CBD2 are capable of simultaneously binding to a chemical inducer of dimerization (CID). CBD1 may include a rapamycin binding domain of FK-binding protein 12 (FKBP12) and CBD2 may include a FKBP12-Rapamycin Binding (FRB) domain of mTOR.

[0201] (IV) Expression of Recombinant Antibodies. Antibodies disclosed herein can be produced by recombinant expression. Recombinant polynucleotide constructs typically include an expression control sequence operably linked to the coding sequences of antibody chains, including naturally-associated or heterologous promoter regions. Preferably, the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotidesequences, and the collection and purification of the cross-reacting antibodies.

[0202] In particular embodiments, mammalian cells are used as a host for expressing nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes to Clones, (VCH Publishers, NY, 1987). A number of suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art, and include CHO cell lines (e.g., DG44), various COS cell lines, HeLa cells, HEK293 cells, L cells, and non- antibody-producing myelomas including Sp2 / 0 and NSO. Preferably, the cells are nonhuman. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and information processing sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. In particular embodiments, expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, and bovine papillomavirus (see Co et al., J. Immunol. 1992, 148:1149).

[0203] Once expressed, antibodies can be purified according to standard procedures of the art, including high-performance liquid chromatography (HPLC) purification, column chromatography, gel electrophoresis and the like (see generally, Scopes, Protein Purification (Springer- Verlag, NY, 1982)).

[0204] In particular embodiments, antibodies are formed using the Daedalus expression system as described in Pechman et al. (Am J Physiol 294: R1234-R1239, 2008). The Daedalus system utilizes inclusion of minimized ubiquitous chromatin opening elements in transduction vectors to reduce or prevent genomic silencing and to help maintain the stability of decigram levels of expression. This system can bypass tedious and time-consuming steps of other protein production methods by employing the secretion pathway of serum-free adapted human suspension cell lines, such as 293 Freestyle. Using optimized lentiviral vectors, yields of 20-100 mg / l of correctly folded and post-translationally modified, endotoxin-free protein of up to 70 kDa in size, can be achieved in conventional, small-scale (100 ml) culture. At these yields, most proteins can be purified using a single size-exclusion chromatography step, immediately appropriate for use in structural, biophysical or therapeutic applications. Bandaranayake et al., Nucleic Acids Res., 39(21 ) 2011 . In some instances, purification by chromatography may not be needed due to the purity of manufacture according to the methods described herein.

[0205] (V) Anti-CD90 Antibody Conjugates. An antibody conjugate refers to an antibody or binding fragment thereof disclosed herein linked to another entity other than additional binding domain. The other entity can be, for example, a toxin, a drug, a detectable label, a radioisotope, a particle, or a bead. In particular examples, an antibody conjugate includes an immunotoxin, anantibody-drug conjugate (ADC), an antibody-detectable label conjugate, an antibody-radioisotope conjugate, an antibody-particle conjugate, or an antibody-bead conjugate.

[0206] In particular embodiments, antibodies are formed as immunotoxins. Anti-CD90 immunotoxins include an anti-CD90 antibody or binding fragment thereof disclosed herein conjugated to one or more cytotoxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof). A toxin can be any agent that is detrimental to cells. Frequently used plant toxins are divided into two classes: (1 ) holotoxins (or class II ribosome inactivating proteins), such as ricin, abrin, mistletoe lectin, and modeccin, and (2) hemitoxins (class I ribosome inactivating proteins), such as pokeweed antiviral protein (PAP), saporin, Bryodin 1 , bouganin, and gelonin. Commonly used bacterial toxins include diphtheria toxin (DT) and Pseudomonas exotoxin (PE). Kreitman, Current Pharmaceutical Biotechnology 2:313-325 (2001). The toxin may be obtained from essentially any source and can be a synthetic or a natural product.

[0207] Immunotoxins with multiple (e.g., four) cytotoxins per binding domain can be prepared by partial reduction of the binding domain with an excess of a reducing reagent such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP) at 37°C for 30 min, then the buffer can be exchanged by elution through SEPHADEX G-25 resin with 1 mM DTPA (diethylene triamine penta-acetic acid) in Dulbecco’s phosphate-buffered saline (DPBS). The eluent can be diluted with further DPBS, and the thiol concentration of the binding domain can be measured using 5,5'- dithiobis(2-nitrobenzoic acid) [Ellman's reagent]. An excess, for example 5-fold, of a linker- cytotoxin conjugate can be added at 4°C. for 1 hr, and the conjugation reaction can be quenched by addition of a substantial excess, for example 20-fold, of cysteine. The resulting immunotoxin mixture can be purified on SEPHADEX G-25 equilibrated in PBS to remove unreacted linker- cytotoxin conjugate, desalted if desired, and purified by size-exclusion chromatography. The resulting immunotoxin can then be sterile filtered, for example, through a 0.2 pm filter, and can be lyophilized if desired for storage.

[0208] In particular embodiments, immunotoxins can include anti-CD90 antibodies or binding fragments thereof conjugated to toxins for targeted CD90+cell killing.

[0209] In particular embodiments, immunotoxins can be conjugated to anti-CD90 reagents for targeted CD90+ apoptosis.

[0210] Antibody-drug conjugates (ADC) allow for the targeted delivery of a drug moiety to a CD90- expressing cell. In particular embodiments, the drug moiety can include a cytotoxic drug or a therapeutic drug or agent.

[0211] In particular embodiments, ADC refer to targeted molecules which combine properties ofboth antibodies and cytotoxic drugs (e.g., chemotherapeutic drugs) by targeting potent cytotoxic drugs to antigen-expressing cells (Teicher, B. A. (2009) Current Cancer Drug Targets 9:982- 1004), thereby enhancing the therapeutic index by maximizing efficacy and minimizing off-target toxicity (Carter, P. J. and Senter P. D. (2008) The Cancer Jour. 14(3):154-169; Chari, R. V. (2008) Acc. Chem. Res. 41 :98-107). See also Kamath & Iyer (Pharm Res. 32(11 ): 3470-3479, 2015), which describes considerations for the development of ADCs.

[0212] The drug moiety of the ADC may include any compound, moiety or group that has a cytotoxic or cytostatic effect. Cytotoxic drug moieties may impart their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding or intercalation, and inhibition of RNA polymerase, protein synthesis, and / or topoisomerase. Exemplary drugs include actinomycin D, anthracycline, auristatin, calicheamicin, camptothecin, CC1065, colchicin, cytochalasin B, daunorubicin, 1 -dehydrotestosterone, dihydroxy anthracinedione, dolastatin, doxorubicin, duocarmycin, elinafide, emetine, ethidium bromide, etoposide, gramicidin D, glucocorticoids, lidocaine, maytansinoid (including monomethyl auristatin E [MMAE]; vedotin), mithramycin, mitomycin, mitoxantrone, nemorubicin, PNU-159682, procaine, propranolol, puromycin, pyrrolobenzodiazepine (PBD), taxane, taxol, tenoposide, tetracaine, trichothecene, vinblastine, vinca alkaloid, vincristine, and stereoisomers, isosteres, analogs, and derivatives thereof that have cytotoxic activity.

[0213] ADC compounds of the disclosure include those with anti-CD90 activity. In particular embodiments, the ADC compounds include an antibody conjugated, i.e., covalently attached, to the drug moiety. In particular embodiments, the antibody is covalently attached to the drug moiety through a linker. A linker can include any chemical moiety that is capable of linking an antibody, antibody fragment (e.g., antigen binding fragments which are also referred to as binding fragments) or functional equivalent to another moiety, such as a drug moiety. Linkers can be susceptible to cleavage (cleavable linker), such as, acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, at conditions under which the compound or the antibody remains active. Alternatively, linkers can be substantially resistant to cleavage (e.g., stable linker or noncleavable linker). In some aspects, the linker is a procharged linker, a hydrophilic linker, or a dicarboxylic acid-based linker. The ADCs selectively deliver an effective dose of a drug to cells expressing CD90 (e.g., cancer cells) whereby greater selectivity, i.e. a lower efficacious dose, may be achieved while increasing the therapeutic index (“therapeutic window”).

[0214] To prepare ADCs, linker-drug conjugates can be made by conventional methods analogous to those described by Doronina et al. (Bioconjugate Chem. 17: 1 14-124, 2006).Antibody-drug conjugates with multiple (e.g., four) drugs per antibody can be prepared by partial reduction of the antibody with an excess of a reducing reagent such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP) at 37°C for 30 min, then the buffer can be exchanged by elution through SEPHADEX G-25 resin with 1 mM DTPA in Dulbecco’s phosphate-buffered saline (DPBS). The eluent can be diluted with further DPBS, and the thiol concentration of the antibody can be measured using 5,5'-dithiobis(2-nitrobenzoic acid) [Ellman's reagent]. An excess, for example 5-fold, of a linker-drug conjugate can be added at 4°C. for 1 hr, and the conjugation reaction can be quenched by addition of a substantial excess, for example 20-fold, of cysteine. The resulting ADC mixture can be purified on SEPHADEX G-25 equilibrated in PBS to remove unreacted linker-drug conjugate, desalted if desired, and purified by size-exclusion chromatography. The resulting ADC can then be sterile filtered, for example, through a 0.2 pm filter, and can be lyophilized if desired for storage.

[0215] In particular embodiments, the antibody or binding fragment thereof can be conjugated to a detectable label to form an antibody-detectable label conjugate. A detectable label is a compound or composition that is conjugated directly or indirectly to another molecule, such as an antibody, to facilitate detection of a molecule. Detectable labels can include any suitable label or detectable group detectable by, for example, optical, spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Such detectable labels include fluorescent labels, enzyme labels, chemiluminescence labels, spectral colorimetric labels, radiolabels, radioacoustic labels, gold beads, magnetic beads (e.g., Dynabeads™), and affinity tags.

[0216] Fluorescent labels can be particularly useful in cell staining, identification, and isolation uses. Exemplary fluorescent labels include blue fluorescent proteins e.g. eBFP, eBFP2, Azurite, mKalamal , GFPuv, Sapphire, T-sapphire); cyan fluorescent proteins (e.g. eCFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan, mTurquoise); green fluorescent proteins (e.g. GFP, GFP-2, tagGFP, turboGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green (mAzamigreen)), CopGFP, AceGFP, avGFP, ZsGreenl, Oregon GreenTM(Thermo Fisher Scientific)); Luciferase; orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato); red fluorescent proteins (mKate, mKate2, mPlum, DsRed monomer, mCherry, mRuby, mRFP1 , DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611 , mRaspberry, mStrawberry, Jred, Texas Red™ (Thermo Fisher Scientific)); far red fluorescent proteins (e.g., mPlum and mNeptune); yellow fluorescent proteins (e.g., YFP, eYFP, Citrine, SYFP2, Venus, YPet, PhiYFP, ZsYellowl); and tandem conjugates.

[0217] Exemplary fluorescence labels include rhodamine, phycoerythrin, and fluorescein.

[0218] Exemplary enzyme labels include horseradish peroxidase, hydrolases, and alkaline phosphatase.

[0219] Chemiluminescent labels can include lucigenin, luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, or oxalate ester.

[0220] Spectral colorimetric labels can include colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, and latex) beads.

[0221] Affinity tags can include, for example, His tag (HHHHHH (SEQ ID NO: 23)), Flag tag (DYKDDDD (SEQ ID NO: 24), Xpress tag (DLYDDDDK (SEQ ID NO: 25)), Avi tag (GLNDIFEAQKIEWHE (SEQ ID NO: 26)), Calmodulin binding peptide (CBP) tag (KRRWKKNFIAVSAANRFKKISSSGAL (SEQ ID NO: 27)), Polyglutamate tag (EEEEEE (SEQ ID NO: 28)), HA tag (YPYDVPDYA (SEQ ID NO: 29)), Myc tag (EQKLISEEDL (SEQ ID NO: 30)), Strep tag (WRHPQFGG (SEQ ID NO: 1 1 )), STREP® tag II (WSHPQFEK (SEQ ID NO: 167); IBA Institut fur Bioanalytik, Germany; see, e.g., US 7,981 ,632), Softag 1 (SLAELLNAGLGGS (SEQ ID NO: 168)), Softag 3 (TQDPSRVG (SEQ ID NO: 169)), and V5 tag (GKPIPNPLLGLDST (SEQ ID NO: 170)).

[0222] Anti-CD90-radioisotope conjugates (or antibody-radioisotope conjugates) include a CD90 antibody or binding fragment thereof linked to a radioisotope (i.e., radioactive isotope) for use in nuclear medicine. Nuclear medicine refers to the diagnosis and / or treatment of conditions by administering radioactive isotopes (radioisotopes or radionuclides) to a subject. Therapeutic nuclear medicine is often referred to as radiation therapy or radioimmunotherapy (R IT) .

[0223] Examples of radioactive isotopes that can be conjugated to antibodies or binding fragments thereof of the present disclosure include actinium-225, iodine-131 , arsenic-21 1 , iodine- 131 , indium-1 1 1 , yttrium-90, and lutetium-177, as well as alpha-emitting radionuclides such as astatine-21 1 or bismuth-212 or bismuth-213. Methods for preparing radioimmunoconjugates are established in the art. Examples of radioimmunoconjugates are commercially available, including Zevalin™ (DEC Pharmaceuticals), and similar methods can be used to prepare radioimmunoconjugates using the antibodies of the disclosure.

[0224] Examples of radionuclides that are useful for radiation therapy include225Ac and227Th.225Ac is a radionuclide with the half-life of ten days. As225Ac decays the daughter isotopes221Fr,213Bi, and209Pb are formed.227Th has a half-life of 19 days and forms the daughter isotope223Ra.

[0225] Additional examples of useful radioisotopes include228Ac,111Ag,124Am,74As,211As,209At,132La,262Lr,169Lu,174mLu,176mLu,257Md,260Md,28Mg,52Mn,90Mo,24Na,95Nb,138Nd,57Ni,66Ni,234Np,150,1820s,189mOs,l910s,32P,201Pb,101Pd,143Pr,191Pt,243Pu,225Ra,81Rb,188Re,105Rh,211Rn,103Ru,35S,44Sc,72Se,153Sm,125Sn,91Sr,173Ta,154Tb,127Te,234Th,45Ti,166Tm,230U,237U,240ll,48V,178W,181W,188W,125Xe,127Xe,133Xe,133mXe,135Xe,85mY,86Y,90Y,93Y,169Yb,175Yb,65Zn,71mZn,86Zr,95Zr, and / or97Zr. Radioisotopes can be used as a type of detectable label called a radiolabel. In particular embodiments, a radioisotope includes 131 1, 90Y, and / or 21 1 At. In particular embodiments, a radioisotope is selected that does not emit daughter radionuclides that cause organ toxicity.

[0226] In particular embodiments, the antibody conjugate includes antibody-particle conjugates. Antibody-particle conjugates can function in the targeted delivery of a payload (e.g., small molecules or genetic engineering components) to a CD90+ cell ex vivo or in vivo. For example, scFv or other binding fragments can be linked to the surface of particles to guide delivery to CD90+ cells. The linkage can be through, for example, covalent attachment. In particular embodiments, a particle is a nanoparticle or a microparticle.

[0227] Examples of particles include metal particles. Metal particles can be made of gold, platinum, or silver. Proteins and / or DNA can be conjugated to the surface of metal particles using surface chemistry. In particular embodiments, the particle can include gold particles.

[0228] Examples of particles include liposomes. Liposomes are microscopic vesicles including at least one concentric lipid bilayer. Vesicle-forming lipids are selected to achieve a specified degree of fluidity or rigidity of the final complex. In particular embodiments, liposomes provide a lipid composition that surrounds an aqueous core. In certain examples, the structure of a liposome can be used to encapsulate a particle within its core (i.e., a liposomal nanoparticle). Lipid nanoparticles (LNPs) are liposome-like structures that lack the continuous lipid bilayer characteristic of liposomes. Solid lipid nanoparticles (SLNs) are LNPs that are solid at room and body temperatures.

[0229] Liposomes and similar structures described in the preceding paragraph can be neutral (cholesterol) or bipolar and include phospholipids, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and sphingomyelin (SM) and other type of bipolar lipids including dioleoylphosphatidylethanolamine (DOPE), with a hydrocarbon chain length in the range of 14-22, and saturated or with one or more double C=C bonds. Examples of lipids capable of producing a stable liposome, alone, or in combination with other lipid components are phospholipids, such as hydrogenated soy phosphatidylcholine (HSPC), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebro sides,distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE) and dioleoylphosphatidylethanolamine 4-(N- maleimido-methyl)cyclohexane-1 -carboxylate (DOPE-mal). Additional non-phosphorous containing lipids that can become incorporated into liposomes include stearylamine, dodecylamine, hexadecylamine, isopropyl myristate, triethanolamine-lauryl sulfate, alkyl-aryl sulfate, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate, amphoteric acrylic polymers, polyethyloxylated fatty acid amides, DDAB, dioctadecyl dimethyl ammonium chloride (DODAC), 1 ,2-dimyristoyl-3-trimethylammonium propane (DMTAP), DOTAP, DOTMA, DC-Chol, phosphatidic acid (PA), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylglycerol, DOPG, and dicetylphosphate. In particular embodiments, lipids used to create liposomes disclosed herein include cholesterol, hydrogenated soy phosphatidylcholine (HSPC) and, the derivatized vesicle-forming lipid PEG-DSPE.

[0230] Methods of forming liposomes are described in, for example, US Patent Nos. 4,229,360; 4,224,179; 4,241 ,046; 4,737,323; 4,078,052; 4,235,871 ; 4,501 ,728; and 4,837,028, as well as in Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980) and Hope et al., Chem. Phys. Lip. 40:89 (1986). For additional information regarding particles or nanoparticles, see Yetisgin et al., Molecules 2020, 25, 2193.

[0231] Particles can also be formed from polymer and lipid blends. Blends of lipids and polymers in any concentration and in any ratio can also be used. Blending different polymer types in different ratios using various grades can result in characteristics that borrow from each of the contributing polymers. Various terminal group chemistries can also be adopted.

[0232] The polymer may be a biodegradable polymer. Examples of polymers that can be used within particles include polyglutamic acid (PGA); poly(lactic-co-glycolic acid) (PLGA); Polylactic acid (PLA); poly-D-lactic acid (PDLA); PLGA-dimethacrylate; polyamines; polyorganic amines (e.g., polyethyleneimine (PEI), polyethyleneimine celluloses); poly(amidoamines) (PAMAM); polyamino acids (e.g., polylysine (PLL), polyarginine); polysaccharides (e.g, cellulose, dextran, DEAE dextran, starch); spermine, spermidine, poly(vinylbenzyl trialkyl ammonium), poly(4-vinyl- N-alkyl-pyridiumiun), poly(acryloyl-trialkyl ammonium), and Tat proteins. In particular embodiments, the particle can include a polyglutamic acid (PGA) particle with a poly(P-amino ester) (PbAE) core. In particular embodiments, particles are prepared using two steps: 1 ) genetic engineering components are complexed with a positively-charged PBAE polymer to form nanosized complexes and 2) an antibody-functionalized PGA is added to the complexes to shield the positive charge and provide targeting (Moffet et al., Nature Communications. 2017. 8(1):389). Inparticular embodiments, antibody-functionalized PGA includes PGA activated with 1 -ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride and combined with antibodies. In particular embodiments, the antibodies include anti-CD90 antibodies. In particular embodiments, the genetic engineering components include mRNA.

[0233] In particular embodiments, polymers disclosed herein can include "star shaped polymers," which refer to branched polymers in which two or more polymer branches extend from a core. The core is a group of atoms having two or more functional groups from which the branches can be extended by polymerization.

[0234] Examples of positively charged lipids commonly used in particles include esters of phosphatidic acid with an aminoalcohol, such as an ester of dipalmitoyl phosphatidic acid or distearoyl phosphatidic acid with hydroxyethylenediamine. More particular examples of positively charged lipids include 3p-[N-(N',N'-dimethylaminoethyl)carbamoyl) cholesterol (DC-chol); N,N'- dimethyl-N,N'-dioctacyl ammonium bromide (DDAB); N,N'-dimethyl-N,N'-dioctacyl ammonium chloride (DDAC); 1 ,2-dioleoyloxypropyl-3-dimethyl-hydroxyethyl ammonium chloride (DORI); 1 ,2- dioleoyloxy-3-[trimethylammonio]-propane (DOTAP); N-(1-(2,3-dioleyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTMA); dipalmitoylphosphatidylcholine (DPPC); 1 ,2- dioctadecyloxy-3-[trimethylammonio]-propane (DSTAP); and the cationic lipids described in e.g. Martin et al., Current Pharmaceutical Design 2005, 11 , 375-394.

[0235] Particular embodiments disclosed herein can also utilize porous particles constructed from any material capable of forming a porous network. Exemplary materials include metals, transition metals and metalloids. Exemplary metals, transition metals and metalloids include lithium, magnesium, zinc, aluminum and silica. In particular embodiments, the porous particles include silica. The exceptionally high surface area of mesoporous silica (exceeding 1 ,000 m2 / g) enables nucleotide loading at levels exceeding conventional DNA carriers such as liposomes.

[0236] Particles can be formed in a variety of different shapes, including spheroidal, cuboidal, pyramidal, oblong, cylindrical, toroidal, and the like. Particle cargo can be included in the pores of particles in a variety of ways. For example, particle cargo can be encapsulated in the porous particles. In other aspects, particle cargo can be associated (e.g., covalently and / or non- covalently) with the surface or close underlying vicinity of the surface of a porous particle. In particular embodiments, the particle cargo can be incorporated in porous particles e.g., integrated in the material of the porous particles. For example, the particle cargo can be incorporated into a polymer matrix of polymer particles.

[0237] In particular embodiments, the particles can include a coating, particularly when used in vivo. A coating can serve to shield the encapsulated cargo and / or reduce or prevent off-targetbinding. Off-target binding is reduced or prevented by reducing the surface charge of the particles to neutral or negative. Coatings can include neutral or negatively charged polymer- and / or liposome-based coatings. In particular embodiments, the coating is a dense surface coating of hydrophilic and / or neutrally charged hydrophilic polymer sufficient to prevent the encapsulated cargo from being exposed to the environment before release into a CD90+ cell. In particular embodiments, the coating covers at least 80% or at least 90% of the surface of the particle. In particular embodiments, the coating includes polyglutamic acid (PGA) or hyaluronic acid. In particular embodiments, PGA can serve as a linker to attach a CD90 binding domain to a particle.

[0238] Examples of neutrally charged polymers that can be used as a particle coating include polyethylene glycol (PEG); polypropylene glycol); and polyalkylene oxide copolymers, (PLURONIC®, BASF Corp., Mount Olive, NJ).

[0239] Neutrally charged polymers also include zwitterionic polymers. Zwitterionic refers to the property of overall charge neutrality while having both a positive and a negative electrical charge. Zwitterionic polymers can behave like regions of cell membranes that resist cell and protein adhesion.

[0240] Zwitterionic polymers include zwitterionic constitutional units including pendant groups (i.e., groups pendant from the polymer backbone) with zwitterionic groups. Exemplary zwitterionic pendant groups include carboxybetaine groups (e.g., -Ra-N+(Rb)(Rc)-Rd-CO2-, where Ra is a linker group that covalently couples the polymer backbone to the cationic nitrogen center of the carboxybetaine groups, Rb and Rc are nitrogen substituents, and Rd is a linker group that covalently couples the cationic nitrogen center to the carboxy group of the carboxybetaine group).

[0241] Examples of negatively charged polymers include alginic acids; carboxylic acid polysaccharides; carboxymethyl cellulose; carboxymethyl cellulose-cysteine; carrageenan (e.g., GELCARIN® 209, GELCARIN® 379, FMC Corporation, Philadelphia, PA); chondroitin sulfate; glycosaminoglycans; mucopolysaccharides; negatively charged polysaccharides (e.g., dextran sulfate); poly(acrylic acid); poly(D-aspartic acid); poly(L-aspartic acid); poly(L-aspartic acid) sodium salt; poly(D-glutamic acid); poly(L-glutamic acid); poly(L-glutamic acid) sodium salt; poly(methacrylic acid); sodium alginate (e.g., PROTANAL® LF 120M, PROTANAL® LF 200M, PROTANAL® LF 200D, FMC Biopolymer Corp., Drammen, Norway); sodium carboxymethyl cellulose (CMC); sulfated polysaccharides (heparins, agaropectins); pectin, gelatin and hyaluronic acid.

[0242] The size of particles can vary over a wide range and can be measured in different ways. In preferred embodiments, nanoparticles are <130 nm in size. However, nanoparticles can also have a minimum dimension of equal to or less than 500 nm, less than 150 nm, less than 140 nm,less than 120 nm, less than 1 10 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, less than 40 nm, less than 30 nm, less than 20 nm, or less than 10 nm. In particular embodiments, nanoparticles are 90 to 130 nm in size. In particular embodiments, a microparticle is 1 pm -1000pm in diameter.

[0243] Dimensions of the particles can be determined using, e.g., conventional techniques, such as dynamic light scattering and / or electron microscopy.

[0244] In particular embodiments, antibody conjugates include antibody-bead conjugates. Antibody-bead conjugates can be prepared by covalently attaching antibodies to a bead. Antibody-bead conjugates can be used for enrichment of CD90+ cells, for example, HSCs or MSCs. The bead can be made of any material suitable for the separation of cells from a cell population. In particular embodiments, the bead can be a magnetic bead or paramagnetic bead. In particular embodiments, the antibody-bead conjugates can be used for column-based enrichment of CD90+ cells.

[0245] The sample or composition of cells to be separated is incubated with the antibody-bead conjugates such that the antibody is able to bind to a molecule on the surface of the cell. The cell is then separated from the population of cells by negative or positive selection of the beads. In particular embodiments, the bead is a magnetizable or magnetically responsive material and the cells are selected using a magnetic field. The beads can be Dynabeads or magnetic-activated cell sorting (MACS) (Miltenyi Biotec, Auburn, Calif.). There are many well- known magnetically responsive materials used in magnetic separation methods. Suitable magnetic particles include those described in Molday, U.S. Pat. No. 4,452,773, and in European Patent EP 452342. Colloidal sized particles, such as those described in Owen U.S. Pat. No. 4,795,698, and Liberti et al., U.S. Pat. No. 5,200,084 are other examples.

[0246] (VI) Antibody-Enhanced Viral Vector Delivery. Current virus-based vector systems can often have broad cell specificity. The current disclosure provides a system for and method of refining a viral vector’s natural tropism to target CD90+ cells more specifically by incorporating an antibody or binding fragment thereof disclosed herein on the surface of a viral vector. In particular embodiments, the antibody or binding fragment thereof is part of a recombinant protein, expressed together with a viral structural protein. In certain examples, the viral structural protein is a viral attachment and / or viral fusion protein. This approach can be used with any viral vector delivery system.

[0247] In particular embodiments, a viral vector delivery system includes a retrovirus, adeno- associated virus (AAV), or other viruses disclosed herein. In particular embodiments, a retrovirus includes a lentivirus or a foamy virus.

[0248] Retroviruses are a family of RNA viruses that have an enzyme, reverse transcriptase, capable of making a complementary DNA copy of the viral RNA, which then is integrated into a host cell’s DNA. During replication the RNA is converted into DNA. Following infection of the cell a double-stranded molecule of DNA is generated from the two molecules of RNA which are carried in the viral particle by the molecular process known as reverse transcription. The DNA form becomes covalently integrated in the host cell genome as a provirus, from which viral RNAs are expressed with the aid of cellular and / or viral factors. The expressed viral RNAs are packaged into particles and released as infectious virion. Retroviruses have been classified in various ways but the nomenclature has been standardized in the last decade (see ICTVdB — The Universal Virus Database, v 4 on the World Wide Web (www) at ncbi.nlm.nih.gov / ICTVdb / ICTVdB / and the text book “Retroviruses” Eds Coffin, Hughs and Varmus, Cold Spring Harbor Press 1997).

[0249] "Lentivirus" refers to a genus of retroviruses that are capable of infecting dividing and nondividing cells and typically produce high viral titers. Lentiviral vectors have been employed in gene therapy for a number of diseases. For example, hematopoietic gene therapies using lentiviral vectors or gamma retroviral vectors have been used for x-linked adrenoleukodystrophy and beta thalassaemia. See, e.g., Kohn et al., 2010 Clin Immunol. 135:247-254; Cartier et al., 2012 Meth. Enzymol. 507:187-198; and Cavazzana-Calvo et al., 2010. Nature 467:318-322. Several examples of lentiviruses include HIV (human immunodeficiency virus: including HIV type 1 , and HIV type 2); equine infectious anemia virus; feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV).

[0250] Foamy viruses (FVes) are the largest retroviruses known today and are widespread among different mammals, including all non-human primate species, however are absent in humans. FV vectors are suitable for gene therapy applications because they can (1 ) accommodate large transgenes (> 9kb), (2) transduce slowly dividing cells efficiently, and (3) integrate as a provirus into the genome of target cells, thus enabling stable long-term expression of the transgene(s). FV vectors do need cell division for the pre-integration complex to enter the nucleus, however the complex is stable for at least 30 days and still infective. The intracellular half-life of the FV pre-integration complex is comparable to the one of lentiviruses and significantly higher than for gammaretroviruses, therefore FV are also - similar to LV vectors - able to transduce rarely dividing cells. FV vectors are natural self-inactivating vectors and characterized by the fact that they seem to have hardly any potential to activate neighboring genes. In addition, FV vectors can enter any cells known (although the receptor is not identified yet) and infectious vector particles can be concentrated 100-fold without loss of infectivity due to a stable envelope protein. FV vectors achieve high transduction efficiency in pluripotent HSC and have been usedin animal models to correct monogenetic diseases such as leukocyte adhesion deficiency (LAD) in dogs and Fanconi anemia in mice. FV vectors are also used in preclinical studies of [3- thalassemia.

[0251] AAV vectors may also be used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93 / 24641 ; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994). Construction of recombinant AAV vectors is described in a number of publications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251 -3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81 :6466-6470 (1984); and Samulski et aL, J. Virol. 63:03822-3828 (1989).

[0252] Additional examples of viral vectors include those derived from adenoviruses (e.g., adenovirus 5 (Ad5), adenovirus 35 (Ad35), adenovirus 11 (Ad11 ), adenovirus 26 (Ad26), adenovirus 48 (Ad48) or adenovirus 50 (Ad50)), alphaviruses, cytomegaloviruses (CMV), flaviviruses, herpes viruses (e.g., herpes simplex), influenza viruses, papilloma viruses (e.g., human and bovine papilloma virus; see, e.g., U.S. Pat. No. 5,719,054), poxviruses, vaccinia viruses, etc. See Kozarsky and Wilson, 1993; Rosenfeld, et al., 1991 ; Rosenfeld, et al., 1992; Mastrangeli, et al., 1993; Walsh, et al., 1993; and Lundstrom, 1999. Examples include modified vaccinia Ankara (MVA) and NYVAC, or strains derived therefrom; avipox vectors, such as a fowlpox vectors (e.g., FP9) or canarypox vectors (e.g., ALVAC and strains derived therefrom); the measles virus; vesicular stomatitis virus, ebola virus, coronavirus, nipah virus, respiratory syncytial virus, rabies virus, and human immunodeficiency virus (HIV).

[0253] Viral vectors can be pseudotyped according to methods known to those of ordinary skill in the art. A pseudotyped viral vector refers to a viral vector particle bearing a structural protein (e.g., an envelope glycoprotein or capsid) derived from a virus distinct from the viral vector genome. Vector particles can be readily prepared by the skilled person, for example by following the general guidance provided by Sandrin et al. (2002) Blood 100:823-832. Briefly, the vector particles may be generated by co-expressing the packaging elements (i.e., the core and enzyme components), the genome component and the envelope component in a producer cell, e.g., 293T human embryonic kidney cells. Typically, from three to four plasmids may be employed, but the number may be greater depending upon the degree to which the lentiviral components are broken up into separate units. For the generation of a pseudotyped vector, the env gene, originally derived from the same virus as the gag and pol genes is exchanged for the structural protein(s) of a different virus. A method of generating a viral vector includes transfecting producer cells withplasmids. The plasmids include a transfer plasmid, one or more packaging plasmids, and an envelope (or structural) plasmid. After incubating in appropriate media, the packaging cells will produce virus and the virus can be harvested from the supernatant media. In particular embodiments, the transfer plasmid includes a 5’ long terminal repeat (LTR), a promoter, and a coding sequence. The packaging plasmid contains the structural (gag), and replication (pol) genes which code for some of the proteins required to produce the viral vector. The pseudotyped protein is encoded by the envelope plasmid which contains a promoter, a sequence encoding the structural protein, and a polyA tail.

[0254] Herein, structural proteins refer to proteins (including glycoproteins) that make up the outermost layer of most viruses. A structural protein can include envelope proteins, membrane proteins, matrix proteins, attachment proteins, viral entry proteins, or other viral glycoproteins.

[0255] Measles is one of the most contagious viral diseases, and remains a major cause of childhood morbidity and mortality worldwide. The measles virus encodes at least eight structural proteins, which have letter names: F, C, H, L, M, N, P, and V. Of these proteins, H (hemagglutinin) has a role in the attachment of the virus to host cells, and F (fusion) is involved in the spread of the virus from one cell to another.

[0256] Vesicular stomatitis virus (VSV) is transmitted naturally by arthropods to a number of animal species including cattle, horses, and pigs. Vesicular stomatitis virus G (VSV-G) protein (accession number KT429217.1 ) is a type III viral fusion protein that mediates both cell attachment and membrane fusion.

[0257] Influenza viruses are orthomyxoviruses, members of the family Orthomyxoviridae, which include the Influenza virus A, B and C, Thogotovirus, Quaranjavirus, and Isavirus. Influenza is an enveloped virus with structural proteins that can be found associated with the virus envelope, a lipid bilayer derived from the plasma membrane of the host cell. The viral envelope contains three of the viral transmembrane proteins: hemagglutinin (HA), neuraminidase (NA), and the matrix ion channel M2. HA (e.g., accession number AAA43099.1 or CAA40728.1) and NA proteins are the main proteins at the virus surface and HA is more abundant compared to NA.

[0258] Ebolavirus is an enveloped filamentous RNA virus that causes severe hemorrhagic fever. It enters the cell by macropinocytosis and membrane fusion in a late endosomal compartment. Fusion is mediated by the ebolavirus envelope glycoprotein GP, which includes subunits GP1 and GP2.

[0259] Coronaviruses (CoVs) are enveloped viruses with a positive sense, single-stranded RNA genome. The coronaviral genome encodes four major structural proteins: the spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, and the envelope (E) protein, all of which areprovided to produce a structurally complete viral particle.

[0260] Lassa virus is an enveloped, single-stranded RNA virus. The two RNA segments in its genome encode four viral proteins, including zinc-binding protein (Z), RNA polymerase (L), nucleoprotein (NP), and the surface glycoprotein precursor (GP, or spike protein).

[0261] The Nipah virus envelope contains two surface glycoproteins used for cellular entry: the attachment protein (NiV-G) and fusion protein (NiV-F). Niv-G allows for attachment of the virion to cellular receptors. NiV-F mediates the fusion of the virus and cellular membranes.

[0262] Rabies virus is a rod- or bullet-shaped, single-stranded, negative-sense, unsegmented, enveloped RNA virus. The virus genome encodes five proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and polymerase (L).

[0263] Respiratory syncytial virus (RSV) is an enveloped negative-sense single-stranded RNA virus. RSV encodes four envelope-associated proteins, of which three are membrane proteins: the small hydrophobic protein (SH), fusion protein (F), and attachment protein (G). The matrix protein (M) coordinates virion assembly.

[0264] Other structural proteins that could be used include HIV gp120, protein F of paramyxoviruses (accession number AAV54052.1 ), protein GP2 of filoviruses (accession number Q89853.1 or AAV48577.1 ), protein E of flaviviruses (accession number AAR87742.1 ), protein E1 of alphaviruses (e.g., accession number LC064744.1 [Chikungunya virus]), protein S of coronaviruses (accession number AAP33697.1 or BAC81404.1 ), protein gH of herpes simplex virus (accession number P06477), protein gB of herpes simplex virus (accession number P06437), and protein G2 of arenaviruses (accession number BAA00964.2 or P03540). Other structural proteins include Chikungunya (E1 Env and E2 Env), Hendra (F glycoprotein and G glycoprotein), hepatitis B (large (L), middle (M), and small (S)), hepatitis C (glycoprotein E1 and glycoprotein E2), HIV envelope (Env), among many others.

[0265] Viral vectors may be further targeted using an antigen-binding portion of an antibody. The current disclosure provides example scFv sequences that bind CD90 (FIGs. 4A-4C) that can additionally be expressed as a recombinant protein with a structural protein (see, e.g., hemagglutinin, FIGs. 5A and 5B).

[0266] In particular embodiments, an anti-CD90 scFv is conjugated to a structural protein. In particular embodiments, the anti-CD90 scFv includes KB4 (SEQ ID NO: 197), KB1 (SEQ ID NO: 63) , KB2 (SEQ ID NO: 156), or KB3 (SEQ ID NO: 157). In particular embodiments, the anti-CD90 scFv referred to herein as KB4 (SEQ ID NO: 197) is conjugated to a structural protein.

[0267] In particular embodiments, the anti-CD90 scFv referred to herein as KB4 (SEQ ID NO: 197) is conjugated to a Measles virus hemagglutinin.

[0268] In particular embodiments, the anti-CD90 scFv referred to herein as KB4 (SEQ ID NO: 197) is conjugated to a VSV-G.

[0269] The antigen-binding portion of the antibody can be conjugated to the structural protein using any method known to those skilled in the art. Methods of conjugation can include enzymatic conjugation or expressing the recombinant protein as the antigen-binding portion of the antibody linked to the structural protein via a linker. In particular embodiments, the linker includes the sequence as set forth in (GGGGSjs (SEQ ID NO: 64). In particular embodiments, the linker includes the sequence as set forth in GSDSNAGHASAGNTS (SEQ ID NO: 66). Additional linkers are described elsewhere herein.

[0270] The structural protein can undergo various modifications to improve its production or in vivo characteristics. Modifications can include mutations to evade the immune system and mutations to the cytoplasmic tail to improve viral titer. For example, when the hemagglutinin protein of a measles virus is selected, hemagglutinin from different strains of measles and / or with mutations to mimic different strains can be selected to allow evasion of the human immune system while maintaining transfection. For example, Kneissl et al. (PLoS ONE. 2012, 7(10): e46667) describes a mutated hemagglutinin protein (Hmut) derived from the NSe variant of the measles virus (MV) vaccine strain Edmonston B. Mutations in the MV receptor recognition ectodomain region include Y481 A, R533A, S548L and F549S. These modifications in the MV glycoproteins’ ectodomain overcame pre-existing MV-specific immunity due to vaccination or natural infection in humans.

[0271] Modification in the cytoplasmic tails of measles hemagglutinin can also improve viral titer and can be implemented, for example, as described in Funke et al., (Mol Ther. 2008 Aug;16(8):1427-36). Funke et al., describes mutant proteins with cytoplasmic tail truncations of up to 30 residues (leaving, in some instances, only three amino acids).

[0272] Additionally, Anliker et al. (Nature Methods. 2010 Nov;7(11):929-935) describes modifications to hemagglutinin that abolish measles CD46 / SLAM targeting and replaces it with scFvs linked to the end of the hemagglutinin. This approach can further improve cell targeting.

[0273] (VII) Recombinant Receptors. Anti-CD90 antibodies and fragments thereof can be utilized within recombinant receptors such as chimeric antigen receptors (CAR) and / or engineered T cell receptors (eTCR).

[0274] CAR include several distinct subcomponents that allow genetically modified cells (e.g., regulatory T cells) to recognize and kill CD90-expressing cells. The subcomponents include at least an extracellular component and an intracellular component. The extracellular component includes a binding domain that specifically binds a CD90 epitope that is preferentially present onthe surface of cells or in the area thereof. When the binding domain binds such epitopes, the intracellular component activates the cell to destroy the bound cell. CAR additionally include a transmembrane domain that directly or indirectly links the extracellular component to the intracellular component, and other subcomponents that can increase the CAR’s function. For example, the inclusion of a spacer region and / or one or more linker sequences can allow the CAR to have additional conformational flexibility, often increasing the binding domain’s ability to bind the targeted epitope.

[0275] eTCR disclosed herein include an anti-CD90 antibody or fragment thereof linked to the Caand / or Cp chains of a TCR. A TCR is a heterodimeric fusion protein that typically includes an a and p chain. Each chain includes a variable region (Vaand Vp) and a constant region (Caand Cp). In particular embodiments, an eTCR does not include the native TCR variable region but does include the native TCR constant region. In particular embodiments, the eTCR includes an anti- CD90 antibody or fragment thereof as the variable region of the a and p chain. In particular embodiments, eTCR include a Caand / or Cp chain sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to an amino acid sequence of a known or identified TCR Ca or Cp.

[0276] In particular embodiments, the extracellular component of a recombinant receptor includes a binding domain that binds CD90. Particular embodiments of binding domains include an anti- CD90 antibody or fragment thereof.

[0277] Recombinant receptors can additionally include spacer regions, transmembrane domains, intracellular effector domains, transduction markers, and tags.

[0278] Spacer regions are used to create appropriate distances and / or flexibility between subcomponents of a protein. Spacer regions typically include 10 to 250 amino acids, 10 to 200 amino acids, 10 to 150 amino acids, 10 to 100 amino acids, 10 to 50 amino acids, or 10 to 25 amino acids. Exemplary spacer regions include all or a portion of an immunoglobulin hinge region.

[0279] Transmembrane domains typically have a three-dimensional structure that is thermodynamically stable in a cell membrane, and generally ranges in length from 15 to 30 amino acids. The structure of a transmembrane domain can include an a helix, a p barrel, a p sheet, a P helix, or any combination thereof. Transmembrane domains can include at least the transmembrane region(s) of the a, p or chain of a T-cell receptor, CD28, CD27, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22; CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.

[0280] A transmembrane domain can include one or more additional amino acids adjacent to thetransmembrane region, e.g., one or more amino acid within the extracellular region of the expressed protein (e.g., up to 15 amino acids of the extracellular region) and / or one or more additional amino acids within the intracellular region of the expressed protein (e.g., up to 15 amino acids of the intracellular components).

[0281] Intracellular effector domains activate the expressing cell when the binding domain binds the antigen. The term “effector domain” is thus meant to include any portion of the intracellular domain sufficient to transduce an activation signal.

[0282] An effector domain can include one, two, three or more intracellular signaling components (e.g., receptor signaling domains, cytoplasmic signaling sequences), co-stimulatory domains, or combinations thereof. Exemplary effector domains include signaling and stimulatory domains selected from: 4-1 BB (CD137), CD3y, CD35, CD3c, CD3 , CD27, CD28, DAP10, ICOS, LAG3, NKG2D, NOTCH1 , 0X40, ROR2, SLAMF1 , TCRa, TCRp, TRIM, Wnt, Zap70, or any combination thereof. In particular embodiments, exemplary effector domains include signaling and costimulatory domains selected from: CD86, FcyRlla, DAP12, CD30, CD40, PD-1 , lymphocyte function-associated antigen-1 (LFA-1 ), LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1 , GITR, BAFFR, SLAMF7, NKp80 (KLRF1 ), CD127, CD19, CD4, CD8a, CD8P, IL2R[3, IL2Ry, IL7Ra, ITGA4, VLA1 , CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11 d, ITGAE, CD103, ITGAL, CD11 a, ITGAM, CD1 1 b, ITGAX, CD11 c, ITGB1 , CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1 , CRTAM, Ly9 (CD229), PSGL1 , CD100 (SEMA4D), CD69, SLAMF6 (NTB- A, Ly108), SLAM (CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, GADS, PAG / Cbp, NKp44, NKp30, or NKp46.

[0283] Intracellular signaling component sequences that act in a stimulatory manner may include iTAMs. Examples of iTAMs including primary cytoplasmic signaling sequences include those derived from CD3y, CD35, CD3E, CD3 , CD5, CD22, CD66d, CD79a, CD79b, and common FcRy (FCER1 G), FcyRlla, FcR|3 (Fee Rib), DAP10, and DAP12. In particular embodiments, variants of CD3 retain at least one, two, three, or all ITAM regions.

[0284] A co-stimulatory domain is a domain whose activation can be included for an efficient lymphocyte response to cellular marker binding. Some molecules are interchangeable as intracellular signaling components or co-stimulatory domains. Examples of costimulatory domains include CD27, CD28, 4-1 BB (CD137), 0X40, PD-1 , ICOS, lymphocyte function-associated antigen-1 (LFA-1), NKG2C, and a ligand that specifically binds with CD83.

[0285] T ransduction markers may be selected from, for example, at least one of a truncated CD19 (tCD19; see Budde et al., Blood 122: 1660, 2013); a truncated human EGFR (tEGFR; see Wanget al., Blood 118: 1255, 2011 ); an extracellular domain of human CD34; and / or RQR8 which combines target epitopes from CD34 (see Fehse et al, Mol. Therapy 1 (5 Pt 1); 448-456, 2000) and CD20 antigens (see Philip et al, Blood 124: 1277-1278).

[0286] Methods to genetically modify cells to express recombinant receptors are well-known in the art. Recombinant receptors can additionally include tags, such as the affinity tags elsewhere herein.

[0287] (VIII) Ex Vivo Cell Manufacturing & Formulation. Anti-CD90 antibodies and binding fragments thereof in any exemplary format or conjugation form described herein can be used in ex vivo cell manufacturing to isolate CD90+ cells from a sample and / or to target CD90+ cells for genetic engineering component delivery.

[0288] HSCs can be harvested from, for example, the bone marrow (BM) or from peripheral blood of a subject. Mobilization is a process whereby stem cells are stimulated out of the BM niche into the peripheral blood (PB), and likely proliferate in the PB. Mobilization allows for a larger frequency of stem cells within the PB minimizing the number of days of apheresis, reaching target number collection of stem cells, and minimizing discomfort to the donor source. Agents that enhance mobilization can either enhance proliferation in the PB, or enhance migration from the BM to PB, or both. Mobilizing agents include cytotoxic drugs, cytokines, and / or small molecules. A historically used regimen is a combination of cyclophosphamide (Cy) plus granulocyte-colony stimulating factor (G-CSF) (Bonig etal., Stem Cells, 2009, 27(4): 836-837). Additional mobilizing agents include alpha4-integrin blockade with anti-functional antibodies and CXCR4 blockade with the small-molecule inhibitor AMD3100. AMD3100 is a bicyclam molecule that specifically and reversibly blocks SDF-1 binding to CXCR4. Another embodiment is the combined regimen of GM- CSF or GCSF with AMD3100. In certain embodiments, C4, a CXC chemokine ligand for the CXCR2 receptor. GRObeta rapidly mobilizes short- and long-term repopulating cells in mice and / or monkeys and synergistically enhances mobilization responses with G-CSF (Pelus et al., 2006). Furthermore, GRObeta can be combined with antagonists of VLA4 to synergistically increase circulating HSPC numbers. In certain embodiments, the CXC4 inhibitor, plerixafor, is used as a single agent for mobilization of HSPCs. Plerixafor is also known commercially under the trade names Mozobil, REvixil, UMK121 , AMD 3000, AMD 3100, AMD3000, AMD3100, GZ 316455, GZ316455, JM 3100, JM3100, SDZ SID 791 , SDZSID791 .

[0289] MSC are adult, multipotent non-hematopoietic stem cells that have the ability to differentiate into mesodermal lineage (e.g., osteocytes, adipocytes, and chondrocytes), ectodermal lineage (e.g., neurocytes), and endodermal lineage (e.g., hepatocytes). MSC can be isolated from many fetal and adult tissues. Although first being isolated from bone marrow(Pittenger, et al., Science. 1999, 2 84:143-147), MSC can also be isolated from adipose tissue (Wagner, et al., Exp. HematoL 2005, 33:1402-1416; and Zhang, et al., Calcif. Tissue Int. 2006, 79:169-178), amniotic fluid (In ’t Anker, etal., Blood. 2003, 102:1548-1549; and Tsai, etal., Hum. Reprod. 2004, 19:1450-1456), amniotic membrane (Cai, et al., J. Biol. Chem. 2010, 285:11227- 11234), dental tissues (Huang, et al., J. Dent. Res. 2009, 88:792-806; and Seifrtova, et al., Int. Endod. J. 2012, 45:401-412), placenta and fetal membrane (Raynaud, et al., Stem Cells Int. 2012, 2012:658356), sub-amniotic umbilical cord lining membrane (Kita, et al., Stem Cells Dev. 2010, 19:491-502), salivary gland (Rotter, et al., Stem Cells Dev. 2008, 17:509-518), endometrium (Schuring, et al., Fertil. Steril. 2011 , 95:423-426), limb bud (Jiao, et al., Cell Reprogram. 2012, 14:324-333), menstrual blood (Allickson, et al., Open Stem Cell J. 2011 , 3:4- 10), peripheral blood (Ab Kadir, et al., ScientificWorldJournal. 2012, 2012:843843), skin and foreskin (Bartsch, et al., Stem Cells Dev. 2005, 14:337-348; and Riekstina, et al., Cytotechnology 2008, 58:153-162), synovial fluid (Morito, et al., Rheumatology 2008, 47:1137- 1143) and Wharton's jelly (Wang, et al., Stem Cells 2004, 22:330-1337; and Hou, et al., Tissue Eng. Part A. 2009, 15:2325-2334). Cell surface markers expressed by MSC include CD29, CD44, CD73, CD90, CD105. MSC lack expression of CD14, CD34, CD45 and human leucocyte antigen (HLA)-DR (Ullah, et al., Biosci Rep. 2015, 35(2): e00191)

[0290] In certain examples, cells can be processed using reduced or minimal manipulation. Reduced manipulation means that the use of electroporation and viral vectors, such as AAV, are not required. Minimal manipulation means that the use of electroporation, viral vectors, and cell selection and purification processes are not required.

[0291] In particular embodiments, cell populations can be isolated with minimal manipulation. In particular embodiments, minimal manipulation means that the use of electroporation, viral vectors such as AAV, cell selection processes, and cell purification processes are not required. In particular embodiments, minimal manipulation means that a sample containing the selected blood cell type is only washed to remove platelets before expansion, manipulation, modification, formulation for delivery, preparation for administration, or preservation as disclosed herein.

[0292] In additional embodiments, CD90+ cells can be collected and isolated from a sample using any appropriate technique. Appropriate collection and isolation procedures can use an antibody conjugate described herein within a procedure such as magnetic separation; fluorescence activated cell sorting (FACS; Radtke, etal., 2020, Molecular Therapy 18:679-691 ; Williams et al., 1985. J Immunol.135(2): 1004-1011 ; Lu et al., 1986. Blood 68(1 ): 126-133); nanosorting based on fluorophore expression; affinity chromatography; cytotoxic agents joined to a monoclonal antibody or used in conjunction with a monoclonal antibody, e.g., complement and cytotoxins;"panning" with antibody attached to a solid matrix (Broxmeyer et al., 1984. J Clin Invest. 73(4):939-953); selective agglutination using a lectin such as soybean (Reisner et al., 1980. Proc Natl Acad Sci. USA 77(11): 6778-6782); immunomagnetic bead-based sorting or combinations of these techniques, etc. These techniques can also be used to assay for successful engraftment or manipulation of cells in vivo, for example for gene transfer, genetic editing or cell population expansion.

[0293] Particular embodiments isolate cells based on the marker profile CD347CD45RA7CD90+; CD347CD45RA7CD907CD133+; CD347CD45RA7CD907CD133+; CD347CD45RA' / CD907CD117+; CD347CD45RA7CD907CD117+; CD347CD45RA7CD907CD117+;CD347CD45RA7CD907CD117-; or CD347CD45RA7CD907CD1177CD1237 Particular embodiments isolate cells based on the marker profile CD347CD45RA7CD9CF and / or do not utilize CD38 or CD49f.

[0294] In particular embodiments, anti-CD90 antibodies can be used to isolate CD90+ cells. In particular embodiments, compositions described herein can be used to capture CD90+ cells using affinity chromatography. Affinity chromatography refers generally to chromatographic procedures that rely on the specific affinity between a substance to be isolated and a molecule that it can bind to. This is a receptor-ligand type interaction. For example, in particular embodiments, affinity chromatography can be accomplished using columns or beads or other surfaces coated in antibodies or other relevant binding domains. Column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then able to adsorb the substance from the solution. Elution occurs by changing the conditions to those in which binding will not occur (alter pH, ionic strength, temperature, etc.).

[0295] In particular embodiments, anti-CD90 antibodies or binding fragments thereof are conjugated to magnetic beads for enrichment or isolation of CD90+ cells. In particular embodiments, enrichment or isolation of CD90+ cells includes column-based, affinity-based, or magnetic selection. In particular embodiments, additional antibodies can be used to further refine the desired cell population.

[0296] The isolation can include one or more of various cell preparation and separation steps, including separation based on one or more properties, such as size, density, sensitivity or resistance to particular reagents, and / or affinity, e.g., immunoaffinity, to antibodies or other binding partners. In particular embodiments, the isolation is carried out using the same apparatus or equipment sequentially in a single process stream and / or simultaneously. In particular embodiments, the isolation, culture, and / or engineering of the different populations is carried out from the same starting composition or material, such as from the same sample.

[0297] Collected cells (e.g., from a subject) or isolated cells can be washed in an appropriate buffer or media. Washing can be accomplished using a semi-automated "flow-through" centrifuge (for example, the Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. Tangential flow filtration (TFF) can also be performed. In particular embodiments, cells can be resuspended in a variety of biocompatible buffers after washing.

[0298] All collected cells (e.g., stem cells or stem cell sources) can be screened for undesirable components and discarded, treated, or used according to accepted current standards at the time. These stem cell sources can be steady state / naive or primed with mobilizing or growth factor agents. Undesirable components can be removed from a sample using, for example, biochemical and / or mechanical methods to remove the undesired components and / or remaining cell populations. Examples include using detergents, hetastarch, hetastarch with centrifugation, cell washing, cell washing with density gradient, Ficoll-hypaque, Sepx, Optipress, Filters, and other protocols that have been used both in the manufacture of cell and / or gene therapies for research and therapeutic purposes.

[0299] Once cells have been collected and isolated, expansion can be performed. Expansion can occur in the presence of one or more growth factors, such as: angiopoietin-like proteins (Angptls, e.g., Angptl2, Angptl3, Angptl7, Angpt15, and Mfap4); erythropoietin; fibroblast growth factor-1 (FGF-1 ); FLT3-ligand (FLT3-L); granulocyte colony stimulating factor (G-CSF); granulocytemacrophage colony stimulating factor (GM-CSF); insulin growth factor-2 (IFG-2); interleukin-3 (IL- 3); interleukin-6 (IL-6); interleukin-7 (IL-7); interleukin-1 1 (IL-1 1); stem cell factor (SCF; also known as the c-kit ligand or mast cell growth factor); thrombopoietin (TPO); and analogs thereof (wherein the analogs include any structural variants of the growth factors having the biological activity of the naturally occurring growth factor; see, e.g., WO 2007 / 1145227 and U.S. Patent Publication No. 2010 / 0183564). For clarity, growth factor agents can also be used as mobilizing agents. Particular embodiments utilize expansion in stem cell supportive media (e.g. StemSpan) supplemented with either SCF, TPO, and FLT3-L or SCF and IL-3, or other combinations of growth factors.

[0300] In particular embodiments, the type, amount and / or concentration of growth factors suitable for expanding cells is the amount or concentration effective to promote proliferation. Cell populations are preferably expanded until a sufficient number of cells are obtained to provide for at least one infusion into a human subject, typically around 104cells / kg to 109cells / kg.

[0301] The amount or concentration of growth factors suitable for expanding cells depends on the activity of the growth factor preparation, and the species correspondence between the growth factors and HSC / MSC, etc. Generally, when the growth factor(s) and cells are of the samespecies, the total amount of growth factor in the culture medium ranges from 1 ng / ml to 5 ig / ml, from 5 ng / ml to 1 pg / ml, or from 5 ng / ml to 250 ng / ml. In additional embodiments, the amount of growth factors can be in the range of 5-1000 or 50-100 ng / ml.

[0302] Cells can be expanded in a tissue culture dish onto which an extracellular matrix protein such as fibronectin (FN), or a fragment thereof (e.g., CH-296 (Dao et. al., 1998, Blood 92(12):4612-21 )) or RetroNectin® (a recombinant human fibronectin fragment; (Clontech Laboratories, Inc., Madison, Wl) is bound.

[0303] During ex vivo manufacturing cells can be genetically modified. Genetic modification is described in the following section.

[0304] Ex vivo manufactured cells can be formulated for delivery. In particular embodiments, formulations include ex vivo manufactured cells and a pharmaceutically acceptable carrier. Formulations of ex vivo manufactured cells are described elsewhere herein.

[0305] It can be necessary or beneficial to cryopreserve a cell and / or cell-based formulation. The terms "frozen / freezing" and “cryopreserved / cryopreserving” can be used interchangeably. Freezing includes freeze drying. As is understood by one of ordinary skill in the art, the freezing of cells can be destructive (see Mazur, P., 1977, Cryobiology 14:251-272) but there are numerous procedures available to prevent such damage. For example, damage can be avoided by (a) use of a cryoprotective agent, (b) control of the freezing rate, and / or (c) storage at a temperature sufficiently low to minimize degradative reactions. Exemplary cryoprotective agents include dimethyl sulfoxide (DMSO) (Lovelock and Bishop, 1959, Nature 183:1394-1395; Ashwood-Smith, 1961 , Nature 190:1204-1205), glycerol, polyvinylpyrrolidine (Rinfret, 1960, Ann. N.Y. Acad. Sci. 85:576), polyethylene glycol (Sloviter and Ravdin, 1962, Nature 196:548), albumin, dextran, sucrose, ethylene glycol, i-erythritol, D-ribitol, D-mannitol (Rowe etal., 1962), D-sorbitol, i-inositol, D-lactose, choline chloride (Bender etal.., 1960), amino acids (Phan The Tran and Bender, 1960), methanol, acetamide, glycerol monoacetate (Lovelock, 1954), and inorganic salts (Phan The Tran and Bender, 1960; Phan The Tran and Bender, 1961). In particular embodiments, DMSO can be used. Addition of plasma (e.g., to a concentration of 20-25%) can augment the protective effects of DMSO. After addition of DMSO, cells can be kept at 0° C until freezing, because DMSO concentrations of 1% can be toxic at temperatures above 4°C.

[0306] Further considerations and procedures for the manipulation, cryopreservation, and longterm storage of cells, can be found in the following exemplary references: U.S. Patent Nos. 4,199,022; 3,753,357; and 4,559,298; Gorin, 1986; Bone-Marrow Conservation, Culture and Transplantation, Proceedings of a Panel, Moscow, July 22-26, 1968, International Atomic Energy Agency, Vienna, pp. 107-186; Livesey and Linner, 1987; Linner et al., 1986; Simione, 1992).

[0307] Following cryopreservation, frozen cells can be thawed for use in accordance with methods known to those of ordinary skill in the art. Frozen cells are preferably thawed quickly and chilled immediately upon thawing. In particular embodiments, the vial containing the frozen cells can be immersed up to its neck in a warm water bath; gentle rotation will ensure mixing of the cell suspension as it thaws and increase heat transfer from the warm water to the internal ice mass. As soon as the ice has completely melted, the vial can be immediately placed on ice.

[0308] As is understood by one of ordinary skill in the art, if a cryoprotective agent that is toxic to humans is used, it should be removed prior to therapeutic use. DMSO has no serious toxicity.

[0309] (IX) In Vivo or Ex Vivo Genetic Modification of Cells. In certain examples, cells will be genetically modified in vivo or ex vivo. For example, CD90+cells can be genetically modified using a viral vector or particle to deliver nucleic acids of interest. In particular embodiments, particle conjugates include anti-CD90 antibodies or binding fragments thereof conjugated to a particle encapsulating a payload (e.g., a nucleic acid, protein, or small molecule drug) for targeted delivery of genetic engineering components to a CD90+ cell in vivo or ex vivo. In particular embodiments, anti-CD90+ antibodies or binding fragments thereof can be used to isolate CD90+ cells for genetic modification ex vivo. In particular embodiments, anti-CD90+ antibodies or binding fragments thereof can be used to target CD90+ cells in vivo for genetic modification.

[0310] In particular embodiments, cells can be genetically modified to express an antibody or fragment thereof (e.g., as in a CAR).

[0311] In particular embodiments, genetic modification by gene transfer can be accomplished using any number of DNA or RNA viral vector based or non-viral vector based gene transfer technologies. As indicated previously, viral-mediated gene transfer can be conducted using retroviral vectors (e.g., lentiviral vectors, foamy viral vectors), adenoviral vectors, or adeno- associated viral vectors. In particular embodiments, viral-mediated gene transfer is conducted using pseudotyped viral vectors. Other nucleic acid gene delivery methods include transposon- mediated delivery, plasmid DNA, particle delivery, or mRNA delivery using transfection, electroporation or nucleofection.

[0312] In certain examples, targeted gene editing systems, such as base editors, clustered regularly interspaced short palindromic repeats (CRISPR), zinc fingers (ZFNs), tai effector nucleases (TALENs), meganucleases or meganuclease-TALEN fusions (MegaTALEs) can be used.

[0313] CRISPR systems and components thereof are described in for example, US8697359, US8771945, US8795965, US8865406, US8871445, US8889356, US8889418, US8895308, US8906616, US8932814, US8945839, US8993233 and US8999641 and applications relatedthereto; and WO2014 / 018423, WO2014 / 093595, WO2014 / 093622, WO2014 / 093635,WO2014 / 093655, WO2014 / 093661 , WO2014 / 093694, WO2014 / 093701 , WO2014 / 093709,WO2014 / 093712, WO2014 / 093718, WO2014 / 145599, WO2014 / 204723, WO2014 / 204724,WO2014 / 204725, WO2014 / 204726, WO2014 / 204727, WO2014 / 204728, WO2014 / 204729,WO2015 / 065964, WO2015 / 089351 , WO2015 / 089354, WO2015 / 089364, WO2015 / 089419,WO2015 / 089427, WO2015 / 089462, WO2015 / 089465, WO2015 / 089473 and WO2015 / 089486, WO2016205711 , WO2017 / 106657, WO2017 / 127807 and applications related thereto.

[0314] For information regarding ZFNs and ZFNs useful within the teachings of the current disclosure, see, e.g., U.S. Patent Nos. 6,534,261 ; 6,607,882; 6,746,838; 6,794,136; 6,824,978; 6,866,997; 6,933, 113; 6,979,539; 7,013,219; 7,030,215; 7,220,719; 7,241 ,573; 7,241 ,574; 7,585,849; 7,595,376; 6,903,185; 6,479,626; and U.S. Application Publication Nos. 2003 / 0232410 and 2009 / 0203140 as well as Gaj et al., Nat Methods, 2012, 9(8):805-7; Ramirez etal., Nucl Acids Res, 2012, 40(12):5560-8; Kim etal., Genome Res, 2012, 22(7): 1327-33; Urnov et al., Nature Reviews Genetics, 2010, 1 1 :636-646; Miller, et al. Nature biotechnology 25, 778- 785 (2007); Bibikova, et al. Science 300, 764 (2003); Bibikova, et al. Genetics 161 , 1169-1175 (2002); Wolfe, et al. Annual review of biophysics and biomolecular structure 29, 183-212 (2000); Kim, et al. Proceedings of the National Academy of Sciences of the United States of America 93, 1156-1 160 (1996); and Miller, et al. The EMBO journal 4, 1609-1614 (1985).

[0315] TALENs, are described in, for example, U.S. Patent Nos. 8,440,431 ; 8,440,432; 8,450,471 ; 8,586,363; and 8,697,853; as well as Joung and Sander, Nat Rev Mol Cell Biol, 2013, 14(l):49-55; Beurdeley et al., Nat Common, 2013, 4: 1762; Scharenberg et al., Curr Gene Ther, 2013, 13(4):291 -303; Gaj etal., Nat Methods, 2012, 9(8) :805-7; Miller, etal. Nature biotechnology 29, 143-148 (2011 ); Christian, et al. Genetics 186, 757-761 (2010); Boch, et al. Science 326, 1509-1512 (2009); and Moscou, & Bogdanove, Science 326, 1501 (2009).

[0316] Base editors directly convert one base or base pair into another, enabling the efficient installation of point mutations in non-dividing cells without generating excess undesired editing by-products, such as insertions and deletions (indels). With base editors, point mutations can be inserted into DNA or RNA without making double-stranded breaks. DNA base editors include a catalytically disabled nuclease fused to a nucleobase deaminase enzyme and, in some cases, a DNA glycosylase inhibitor. RNA base editors achieve analogous changes using components that base modify RNA. Any nuclease of the CRISPR system can be disabled and used within a base editing system (e.g., dCas9). The nucleobase deaminase enzyme can include a cytidine deaminase domain, an adenine deaminase domain. Further, particular embodiments utilize a uracil glycosylase inhibitor (UGI) as a glycosylase inhibitor.

[0317] In particular embodiments, a deaminase domain (cytidine and / or adenine) is fused to the N-terminus of the catalytically disabled nuclease. In these embodiments, a glycosylase inhibitor (e.g., UGI domain) can be fused to the C-terminus of the catalytically disabled nuclease. When multiple glycosylase inhibitors are used, each can be fused to the C-terminus of the catalytically disabled nuclease. Components of base editors can be fused directly (e.g., by direct covalent bond) or via linkers. For additional information regarding base editors, see Seo & Kim, Nature Medicine, 24, 1493-1495 (2018) and Rees & Liu, Nature Reviews Genetics, 19, pages770-788 (2018).

[0318] Particular embodiments can use transposon-based systems as gene editing agents to mediate the integration of a genetic construct into cells. Generally, such methods will involve introducing into cells (i) a first vector encoding a transposase (or a transposase polypeptide) and (ii) a second vector encoding a desired genetic element that is flanked by transposon repeats. Transposons or transposable elements include a (short) nucleic acid sequence with terminal repeat sequences upstream and downstream thereof and encode enzymes that facilitate the excision and insertion of the nucleic acid into target DNA sequences.

[0319] Several transposon / transposase systems have been adapted for genetic insertions of heterologous DNA sequences. Examples of such transposases include sleeping beauty (“SB”, e.g., derived from the genome of salmonid fish); piggyback {e.g., derived from lepidopteran cells and / or the Myotis lucifugus)-, mariner e.g., derived from Drosophila); frog prince {e.g., derived from Rana pipiens); Toll ; Tol2 {e.g., derived from medaka fish); TcBuster {e.g., derived from the red flour beetle Tribolium castaneum), Helraiser, Himarl , Passport, Minos, Ac / Ds, PIF, Harbinger, Harbinger3-DR, HSmarl , and spinON. Transposases and transposon systems are further described in U.S. Pat. Nos. 6,489,458; 7,148,203; 8,227,432; and 9,228,180.

[0320] RNA is used in many targeted genetic engineering systems and can form specific interactions with RNA or DNA. While this property is exploited in many such systems, it also comes with the risk of promiscuous interactions in a nucleic acid-rich cellular environment. One solution to this problem is the formation of ribonucleoprotein particles or complexes (RNPs), in which RNA (e.g., guide RNA) is pre-complexed with a nuclease. Another benefit of RNPs is that they protect the RNA from degradation. To form an RNP, RNA and the nuclease can generally be combined in a 1 :1 molar ratio.

[0321] In particular embodiments, the genetic engineering components include a coding sequence (naked DNA or naked mRNA), guide RNA, homology arms, a base editor, a clustered regularly interspaced short palindromic repeats (CRISPR) nuclease, a zinc finger (ZFN), a tai effector nuclease (TALEN), a meganucleases or a meganuclease-TALEN fusion (MegaTALE).

[0322] In certain examples, CD90+ cells are genetically modified to express a coding sequence encoding a therapeutic molecule (e.g., nucleotide sequence or protein). A “coding sequence” refers to a sequence of nucleotides including genes and regulatory regions such as promoters, enhancers, insulators, and / or post-regulatory elements, such as termination regions. The term further can include all introns and other DNA sequences spliced from the mRNA transcript, along with variants resulting from alternative splice sites. The sequences can also include degenerate codons of a reference sequence or sequences that may be introduced to provide codon preference in a specific organism or cell type.

[0323] Exemplary coding sequences include ABCA3, ABCD1 , Akt, amyloid beta precursor protein (APP), angiopoietin 1 (Ang1 ), angiotensin-converting enzyme 2 (ACE2), antibodies to CD4, antibodies to CD5, antibodies to CD7, antibodies to CD52, etc., antibodies to IL1 , antibodies to IL2, antibodies to IL6, antibodies to TCR specifically present on autoreactive T cells, antibodies to TNF, arylsulfatase A, Bcl-2, brain derived neurotrophic factor (BDNF), cerebral dopamine neurotrophic factor (CDNF), C1q / tumor necrosis factor-related protein-3 (CTRP3), C9ORF72, C- C Motif Chemokine Receptor 1 (CCR1 ), CCR2, chemokine receptor 2 (CXCR2), CXCR4, CXCR7, ciliary neurotrophic factor (CNTF), CLN3, connexin 43 (Cx43), Csx / Nkx 2.5, CTLA, cystic fibrosis transmembrane conductance regulator (CFTR), Cytochrome b5 reductase 3 (CYB5R3), DRB1*1501 / DQBr0602, Dyskerin Pseudouridine Synthase (DKC1), dystrophin, E2F4, endothelial nitric oxide synthase (eNOS), erythropoietin (EPO), extracellular regulating kinase 1 / 2, F8 (coagulation factor VIII), F9 (coagulation factor IX), Fanconi anaemia complementation group (FANC) family coding sequences, Fas L, fibroblast growth factor-2 (FGF-2), fibroblast growth factor 4 (FGF4), Follistatin-like 1 , forkhead box protein (Foxa2), fused in sarcoma (FUS), GATA1 , GATA-4, glial cell line-derived neurotrophic factor (GDNF), globin family coding sequences, granulocyte colony-stimulating factor (G-CSF), HBB, heme oxygenase-1 (HO-1 ), hepatocyte growth factor (HGF), hepatocyte nuclear factor 4a (HNF4a), hypoxia-inducible factor 1 a (HIF-1 a), insulin-like growth factor (IGF)-1 , Intercellular Adhesion Molecule (ICAM)-1 , interferon-beta (IFN-p), integrin a4, interleukin-1 receptor antagonist (IL-1 Ra), interleukin 4 (IL4), IL10, IL12, IL13, IL-33, Islet-1 , Klotho, let-7d, leucine-rich repeat kinase 2 (LRRK2), lipocalin 2 (Len2), Mashl , microRNA-1 (miR-1 ), miR-16-5p, miR-21 , miR-25, miR-30b-3p, miR-34, miR-101 - 3p, miR-124, mlR-126, miR-133, miR-133b, mlR-181 a, miR-199a, miR-199a-3p, miR-211 , miR- 705, nerve growth factor (NGF), neuregulin 4 (Nrg4), neurotrophin-3 (NT3), NLX2.1 , Notch ligand Delta-like-4, Notchl receptor, nuclear factor erythroid-derived 2-like 2 (Nrf2), orphan receptor tyrosine kinase 2 (ROR2), P53, p130, Parkinson disease 2 (PARK2), PARK7, PARKIN, phox, platelet-derived growth factor (PDGF), presenilin 1 (PSEN1 ), PSEN2, protein tyrosinephosphatase, non-receptor type 22 (PTPN22), PTEN-induced kinase 1 (PINK1 ), pyruvate kinase, ribosomal protein coding sequences, secreted Klotho protein (SKL), sirtuin 1 , soluble CD40, soluble interleukin 1 receptor II (sILI RII), sILI RI, soluble TNF alpha receptor I (sTNFRI), sTNFRII, somatostatin 2 (sST2), sonic hedgehog (Shh), superoxide dismutase 1 (SOD1 ), surfactant protein B (SFTPB), SFTPC, synuclein alpha (SNCA), TAR DNA-binding protein 43 (TDP43), telomerase reverse transcriptase (TERT), telomerase RNA component (TERC), TERF interacting nuclear factor 2 (TINF2), TNF-related apoptosis-inducing ligand (TRAIL), transforming growth factor [31 (TGF-|31 ), type 2 angiotensin II receptor (AT2R), tyrosine kinase receptor type 3 (TrkC), ubiquilin 2, vascular endothelial growth factor (VEGF), WASP Actin nucleation promoting factor (WAS), or Wnt / |3-catenin.

[0324] Genetic constructs disclosed herein can be introduced into cells by any method known in the art, including transfection, electroporation, microinjection, lipofection, calcium phosphate mediated transfection, infection with a viral or bacteriophage vector including the gene sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, in vivo particle-mediated delivery, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see e.g., Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen, et al., 1993, Meth. Enzymol. 217:618-644; Cline, 1985, Pharmac. The 29:69-92) and may be used, provided that the necessary developmental and physiological functions of the recipient cells are not unduly disrupted. The technique can provide for the stable transfer of the gene to the cell, so that the gene is expressible by the cell and, in certain instances, preferably heritable and expressible by its cell progeny.

[0325] Electroporation can be useful, for example, to deliver targeting elements and / or cutting elements because the membrane of the cell does not normally allow such foreign molecules into the cell. Electroporation sends an electric shock to the cells that temporarily allows such foreign molecules to pass through the cell membrane.

[0326] (X) Compositions and Formulations for Administration. Any of the antibodies, binding fragments thereof described herein in any exemplary format or conjugation form can be formulated alone or in combination into compositions for administration to subjects. Additionally, nucleic acids encoding the antibodies or fragments thereof can also be formulated into compositions for administration (e.g., nucleic acids encapsulated within particles (e.g., liposomes or polymer-based nanoparticles) and / or as part of a vector delivery system (e.g., a viral vector or plasmid). Binding domains (e.g., antibodies, multi-domain binding molecules, antibody conjugates, recombinant proteins, recombinant receptors) and / or nucleic acids encoding binding domains are collectively referred to herein as “active ingredients”. Certain examples may includeformulations. Formulations include cells genetically modified to express a binding domain disclosed herein (e.g., recombinant receptor) within a pharmaceutically-acceptable carrier.

[0327] Salts and / or pro-drugs of the active ingredients can also be used.

[0328] A pharmaceutically acceptable salt includes any salt that retains the activity of the active ingredient and is acceptable for pharmaceutical use. A pharmaceutically acceptable salt also refers to any salt which may form in vivo as a result of administration of an acid, another salt, or a prodrug which is converted into an acid or salt.

[0329] Suitable pharmaceutically acceptable acid addition salts can be prepared from an inorganic acid or an organic acid. Examples of such inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid. Appropriate organic acids can be selected from aliphatic, cycloaliphatic, aromatic, arylaliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids.

[0330] Suitable pharmaceutically acceptable base addition salts include metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from N,N'-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N- methylglucamine, lysine, arginine and procaine.

[0331] A prodrug includes an active ingredient which is converted to a therapeutically active compound after administration, such as by cleavage or by hydrolysis of a biologically labile group.

[0332] Exemplary generally used pharmaceutically acceptable carriers include any and all absorption delaying agents, antioxidants, binders, buffering agents, bulking agents or fillers, chelating agents, coatings, disintegration agents, dispersion media, gels, isotonic agents, lubricants, preservatives, salts, solvents or co-solvents, stabilizers, surfactants, and / or delivery vehicles. Exemplary carriers include saline, buffered saline, physiological saline, water, Hanks' solution, Ringer's solution, Normosol-R (Abbott Labs), Plasma-Lyte A® (Baxter Laboratories, Inc., Morton Grove, IL), glycerol, ethanol, and combinations thereof.

[0333] Exemplary antioxidants include ascorbic acid, methionine, and vitamin E.

[0334] Exemplary buffering agents include citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers, and / or trimethylamine salts.

[0335] An exemplary chelating agent is EDTA (ethylene-diamine-tetra-acetic acid).

[0336] Exemplary isotonic agents include polyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol.

[0337] Exemplary preservatives include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides,hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol.

[0338] Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which solubilizes the active ingredient or helps to prevent denaturation or adherence to the container wall. Typical stabilizers can include polyhydric sugar alcohols; amino acids, such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, and cyclitols, such as inositol; PEG; amino acid polymers; sulfur-containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, a- monothioglycerol, and sodium thiosulfate; low molecular weight polypeptides (i.e., <10 residues); proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose and glucose; disaccharides such as lactose, maltose and sucrose; trisaccharides such as raffinose, and polysaccharides such as dextran. Stabilizers are typically present in the range of from 0.1 to 10,000 parts by weight based on therapeutic weight.

[0339] The compositions and / or formulations disclosed herein can be formulated for administration by, for example, injection, inhalation, infusion, perfusion, lavage, or ingestion. The compositions and / or formulations disclosed herein can further be formulated for intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular, oral, sublingual, and / or subcutaneous administration.

[0340] The compositions and / or formulations disclosed herein can be pipetted.

[0341] For injection, compositions and / or formulations can be formulated as aqueous solutions, such as in buffers including Hanks' solution, Ringer's solution, or physiological saline. The aqueous solutions can include formulatory agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the composition can be in lyophilized and / or powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0342] Compositions can be formulated as an aerosol. In particular embodiments, the aerosol is provided as part of an anhydrous, liquid or dry powder inhaler. Aerosol sprays from pressurized packs or nebulizers can also be used with a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.

[0343] Additionally, compositions and / or formulationscan be formulated as sustained-release systems utilizing semipermeable matrices of solid polymers including at least one type of activeingredient.

[0344] In particular embodiments, the compositions include active ingredients of at least 0.1% w / v or w / w of the composition; at least 1% w / v or w / w of composition; at least 10% w / v or w / w of composition; at least 20% w / v or w / w of composition; at least 30% w / v or w / w of composition; at least 40% w / v or w / w of composition; at least 50% w / v or w / w of composition; at least 60% w / v or w / w of composition; at least 70% w / v or w / w of composition; at least 80% w / v or w / w of composition; at least 90% w / v or w / w of composition; at least 95% w / v or w / w of composition; or at least 99% w / v or w / w of composition.

[0345] In certain examples, cells are genetically modified to express an antibody or fragment thereof, for example, as part of a recombinant receptor (e.g., a CAR or eTCR). In these embodiments, genetically modified cells can be prepared as formulations for delivery in buffers such as Hanks' solution, Ringer's solution, or physiological saline. Cells can be genetically modified using methods known in the art. Exemplary targeted genetic engineering approaches include the use of CRISPR / Cas nuclease systems, zinc finger nucleases (ZFNs), and / or transcription activator like effector nucleases (TALENs). In particular embodiments, the cells are B cells genetically modified to express an antibody. Methods to genetically modify a B cell to express an antibody are described in PCT / US2018 / 056789.

[0346] Therapeutically effective amounts of cells within formulations can be greater than 102cells, greater than 103cells, greater than 104cells, greater than 105cells, greater than 106cells, greater than 107cells, greater than 108cells, greater than 109cells, greater than 1010cells, or greater than 1011cells.

[0347] In particular embodiments, cells are in a formulation volume of a liter or less, 500 ml or less, 250 ml or less, or 100 ml or less. Hence, the density of administered cells is typically greater than 104cells / ml, 105cells / ml, 106cells / ml, 107cells / ml, or 108cells / ml.

[0348] Any composition or formulation disclosed herein can advantageously include any other pharmaceutically acceptable carriers which include those that do not produce significantly adverse, allergic, or other untoward reactions that outweigh the benefit of administration. Exemplary pharmaceutically acceptable carriers are disclosed in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990. Moreover, compositions or formulations can be prepared to meet sterility, pyrogenicity, general safety, and purity standards as required by U.S. FDA Office of Biological Standards and / or other relevant foreign regulatory agencies.

[0349] (XI) Kits. Also provided herein are kits including at least one antibody or binding fragment thereof or sequences encoding at least one antibody or binding fragment thereof disclosed herein. Kits may be formed with components to practice, for example, the methods described herein. Inparticular embodiments, the kit includes an anti-CD90 antibody, a multi-domain binding molecule, an antibody conjugate, a recombinant protein, or a recombinant receptor, or sequences encoding an antibody, a multi-domain binding molecule, an anti-CD90 antibody conjugate, a recombinant protein, or a recombinant receptor, as described herein. In particular embodiments, the kit includes cells expressing an antibody, binding fragment, or engineered format thereof (e.g., recombinant protein or recombinant receptor), or composition to modify cells to express an antibody, binding fragment, or engineered format thereof. The kit may include material(s), which may be desirable from a user standpoint, such as a buffer(s), a diluent(s), a standard(s), and / or other material useful in sample processing, washing, or conducting any other step of the method described herein.

[0350] The kit according to the present disclosure may also include instructions for carrying out the method. Instructions included in the kit of the present disclosure may be affixed to packaging material or may be included as a package insert. While instructions are typically written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term “instructions” can include the address of an internet site which provides instructions.

[0351] (XII) Methods of Use. The compositions or formulations disclosed herein can be used for treating subjects (humans, veterinary animals (dogs, cats, reptiles, birds, etc.), livestock (horses, cattle, goats, pigs, chickens, etc.), and research animals (monkeys, rats, mice, fish, etc.). Treating subjects includes delivering therapeutically effective amounts. Therapeutically effective amounts include those that provide effective amounts, prophylactic treatments, and / or therapeutic treatments.

[0352] An “effective amount” is the amount of a composition or formulation necessary to result in a desired physiological change in a subject. Effective amounts are often administered for research purposes. Effective amounts disclosed herein can cause a statistically-significant effect in an in vitro assay, an animal model, clinical study, or clinical study relevant to the assessment of an condition’s development, progression, and / or resolution, as well as the effects of the condition.

[0353] A "prophylactic treatment" includes a treatment administered to a subject who does not display signs or symptoms of a condition to be treated or displays only early signs or symptoms of the condition to be treated such that treatment is administered for the purpose of diminishing, preventing, or decreasing the risk of developing the condition. Thus, a prophylactic treatment functions as a preventative treatment against a condition.

[0354] A "therapeutic treatment" includes a treatment administered to a subject who displays symptoms or signs of a condition and is administered to the subject for the purpose of reducing the severity or progression of the condition.

[0355] Function as an effective amount, prophylactic treatment or therapeutic treatment are not mutually exclusive, and in particular embodiments, administered dosages may accomplish more than one treatment type.

[0356] In particular embodiments, a composition disclosed herein can be used to target or isolate cells for genetic modification. In particular embodiments, the targeted or isolated cells can be genetically modified to express a coding sequence. As one example, a coding sequence can be selected to provide a therapeutically effective response against a condition that, in particular embodiments, is inherited. In particular embodiments, the condition can be Grave's Disease, rheumatoid arthritis, pernicious anemia, Multiple Sclerosis (MS), inflammatory bowel disease, systemic lupus erythematosus (SLE), adenosine deaminase deficiency (ADA-SCID) or severe combined immunodeficiency disease (SCID) stemming from deficiency in gamma chain (X-linked SCID), JAK 3 kinase deficiency, purine nucleoside phsosphorylase deficiency, adenosine deaminase (ADA) deficiency, MHO Class II deficiency or recombinase activating gene (RAG) deficiency, Wiskott-Aldrich syndrome (WAS), chronic granulomatous disease (CGD), Fanconi anemia (FA), Battens disease, adrenoleukodystrophy (ALD) or metachromatic leukodystrophy (MLD), muscular dystrophy, pulmonary aveolar proteinosis (PAP), pyruvate kinase deficiency, Shwachmann-Diamond-Blackfan anemia, dyskeratosis congenita, xeroderma pigmentosa, cystic fibrosis, Parkinson’s disease, Alzheimer’s disease, or amyotrophic lateral sclerosis (Lou Gehrig’s disease). In particular embodiments, depending on the condition, the therapeutic coding sequence may be a coding sequence that encodes a protein and / or a coding sequence whose function has been interrupted. Exemplary therapeutic coding sequences or products thereof include: soluble CD40; CTLA; Fas L; antibodies to CD4, CD5, CD7, CD52, etc.; antibodies to IL1 , IL2, IL6; an antibody to TOR specifically present on autoreactive T cells; IL4; IL10; IL12; IL13; IL1 Ra, sILI RI, sILI RII; sTNFRI; sTNFRII; antibodies to TNF; P53, PTPN22, and DRB1 *1501 / DQB1 *0602; globin family genes; WAS; phox; FANC family genes; dystrophin; pyruvate kinase; CLN3; ABCD1 ; arylsulfatase A; SFTPB; SFTPC; NLX2.1 ; ABCA3; GATA1 ; ribosomal protein genes; TERT; TERC; DKC1 ; TINF2; CFTR; LRRK2; PARK2; PARK7; PINK1 ; SNCA; PSEN1 ; PSEN2; APP; SOD1 ; TDP43; FLIS; ubiquilin 2; and / or C9ORF72. Therapeutically effective amounts may provide function to immune and other blood cells and / or microglial cells or may alternatively - depending on the treated condition - inhibit lymphocyte activation, induce apoptosis in lymphocytes, eliminate various subsets of lymphocytes, inhibit T cell activation,eliminate or inhibit autoreactive T cells, inhibit Th-2 or Th-1 lymphocyte activity, antagonize IL1 or TNF, reduce inflammation, induce selective tolerance to an inciting agent, reduce or eliminate an immune-mediated condition; and / or reduce or eliminate a symptom of the immune-mediated condition. Therapeutic effective amounts may also provide functional DNA repair mechanisms; surface protein expression; telomere maintenance; lysosomal function; breakdown of lipids or other proteins such as amyloids; permit ribosomal function; and / or permit development of mature blood cell lineages which would otherwise not develop such as macrophages other white blood cell types.

[0357] In particular embodiments, HSCs can be genetically modified to provide a therapeutically effective response against diseases related to red blood cells and clotting. In particular embodiments, a coding sequence can be selected to provide a therapeutically effective response against diseases related to red blood cells and clotting. In particular embodiments, the disease is a hemoglobinopathy like thalassemia, or a sickle cell disease / trait. The therapeutic coding sequence may be, for example, a coding sequence that induces or increases production of hemoglobin; induces or increases production of beta-globin, or alpha-globin; or increases the availability of oxygen to cells in the body. The therapeutic coding sequence may be, for example, HBB or CYB5R3. Exemplary effective treatments may, for example, increase blood cell counts, improve blood cell function, or increase oxygenation of cells in patients. In another particular embodiment, the disease is hemophilia. The therapeutic coding sequence may be, for example, a coding sequence that increases the production of coagulation / clotting factor VIII or coagulation / clotting factor IX, causes the production of normal versions of coagulation factor VIII or coagulation factor IX, a coding sequence that reduces the production of antibodies to coagulation / clotting factor VIII or coagulation / clotting factor IX, or a coding sequence that causes the proper formation of blood clots. Exemplary therapeutic coding sequences include factor VIII and factor IX. Exemplary effective treatments may, for example, increase or induce the production of coagulation / clotting factors VIII and IX; improve the functioning of coagulation / clotting factors VIII and IX, or reduce clotting time in subjects.

[0358] Other uses with supporting detail are found in PCT / US2016 / 014378.

[0359] MSC are widely used in the field of regenerative medicine and have shown therapeutic potential in diseases such as cancer, cardiovascular diseases, lung diseases, gastrointestinal and liver diseases, kidney diseases, and neurological disorders. Several genetic engineering techniques have been used in recent years with the aim of enhancing the therapeutic potential of MSC in order to improve the outcomes after transplantation, increase survival and proliferation, improve pro-regenerative capacity, and induce expression of desired factors (Damascene et al.,2020. Front. Cell Dev Biol. 8: 737).

[0360] MSC can be used to deliver anticancer immunostimulatory agents, such as chemokines and cytokines, to cancer site. In particular embodiments, MSC can be genetically engineered to increase the expression of anticancer agents including: chemokine receptor 4 (CXCR4) (Hmadcha et al., 2020. Front. Bioeng. Biotechnol. 8:43; and Bobis-Wozowicz et al., 2011. Exp. Hematol. 39, 686-696), CXC receptor 7 (CXCR7) (Liu et al., 2018. Sci. Pep. 8, 1-10), cytokine interferon-beta (IFN-|3) (Han et al., 2015. Anticancer. Res. 35, 4749-4756), TNF-related apoptosis-inducing ligand (TRAIL) (Spano et al., 2019. Sci. Rep. 9, 1-14), sirtuin 1 (Yu et al., 2016. 7, 71112-71122), hepatocyte nuclear factor 4a (HNF4a) (Wu et al., 2016. Cancer Biol. Ther. 17, 558-565), and microRNAs such as miR-16-5p (Xu et al., 2019. J. Cell. Physiol. 234, 21380-21394), miR-34 (Wang et al., 2019. Stem Cell Res. 41 :101605), miR-101-3p and miR- 199a (Xie et a!., 2019. Mol. Cell. Biochem. 458, 11-26).

[0361] MSC can be used to treat cardiovascular disease because of their ability to promote neovascularization, cytoprotection, cardiac regeneration, modulation of inflammatory and fibrogenic processes, cardiac contractility and cardiac metabolism (Gnecchi and Cervio. 2013. Mesenchymal Stem Cell Therapy, eds L. G. Chase and M. C. Vemuri (Totowa, NJ: Humana Press), 241-270). In particular embodiments, MSC can be used in the treatment of cardiovascular diseases by genetic modification to overexpress factors including vascular endothelial growth factor (VEGF) (Locatelli, et al., 2015. Gene Ther. 22, 449-457), hepatocyte growth factor (HGF) (Zhao et al., 2016. Exp. Cell Res. 344, 30-39), insulin-like growth factor (IGF)-1 (Haider et al., 2008. Circ. Res. 103, 11 ,1300-1308), C1q / tumor necrosis factor-related protein-3 (CTRP3), CCR1 (Zhang etal., 2019. Stem Cell Res. Ther. 10:74), granulocyte colony-stimulating factor (G- CSF) (Silva et al., 2018. Front. Immunol. 9:1449), Csx / Nkx 2.5, GATA-4 (Gao et al., 2011. Circ. J. 75, 1 1 ,2683-2691), Akt (Gnecchi et al., 2009. Stem Cells 27, 971-979), CXCR4 (Huang et al., 2012. Cell Biochem. and Funct. 30, 139-144), IL-10 (Meng et al., 2019. J. Biol. Eng. 13:49), IL- 33 (Schmitz etal., 2005. Immunity 23, 479-490), endothelial nitric oxide synthase (eNOS) (Chen L. et al., 2017. Cardiovasc. Drugs. Ther. 31 , 9-18), heme oxygenase-1 (HO-1) (Liu and Qian, 2015. Int. J. Clin. Exp. Med. 8, 19867-19873), Follistatin-like 1 (Shen etal., 2019. Stem Cell Res. Ther. 10, 1-14), Islet-1 (Xiang et al., 2018. Stem Cell Res. Ther. 9:51 ), Notch ligand Delta-like-4, Bcl-2, Cx43 and the overexpression of micro RNAs such as mlR-126 (Huang et al., 2013. Int. J. Mol. Med. 3 , 484-492), microRNA-1 (Huang et al., 2013. Cardiology 125, 18-30), mlR-181 a (Wei etal., 2019. Life Sci. 232:1 16632), miR-21 (Zeng etal., 2017. Oncotarget. 8, 29161-29173), miR-133 (Chen Y. et al., 2017. Stem Cell Res. Ther. 8:268), and miR-211 (Hu et al., 2016. Stem Cells 34, 1846-1858).

[0362] In particular embodiments, MSC can be used and genetically modified to express different factors to promote a patients’ recovery from lung disease. In particular embodiments, these factors include HGF, G-CSF (Guo et al., 2013. Heart Vessels 29, 520-531), secreted Klotho protein (SKL) (Varshney et al., 2016. Hypertension 68, 1255-1263), angiopoietin 1 (Ang1 ) (Mei et al., 2007. PLoS Med. 4:e269), Notchl receptor (Cheng et al., 2017. Int. J. Chron. Obstruct. Pulmon. Dis. 12, 3133-3147), Wnt / p-catenin (Cai et al. 2015. Stem Cell Res. The 6:65), IL-10, CXCR4, CXCR7 (Wang et al., 2018. DNA Cell Biol. 37, 53-61 ; Jerkic et al., 2019. J. Clin. Med. 8:847; Shao etal., 2019. Biomed. Pharmacother. 109, 1233-1239; Zhang etal., 2019. Stem Cells Int. 2019:2457082), angiotensin-converting enzyme 2 (ACE2) (He et al., 2015. Cell Transplant. 24, 1699-1715), soluble IL-1 receptor type 1 (sST2) (Martinez-Gonzalez etal., 2014. Stem Cells Dev. 23, 2352-2363), heme-oxygenase-1 (HO-1 ) (Chen et al., 2018. J. Cell. Physiol. 234, 7301-7319), type 2 angiotensin II receptor (AT2R) (Xu et al., 2018. Stem Cells Transl. Med. 7, 721-730), orphan receptor tyrosine kinase 2 (ROR2) (Cai et al., 2016. Cell Transplant. 25, 1561-1574), nuclear factor erythroid-derived 2-like 2 (Nrf2) (Zhang et al., 2017. J. Cell. Biochem. 119, 1627-1636), and microRNAs such as miR-30b-3p (Yi et al., 2019. Exp. Cell Res. 383:1 11454), let-7d (Huleihel et al., 2017. Am. J. Physiol. Lung Cell. Mol. Physiol. 313, L92-L103), and p130 and E2F4 (Zhang et al., 2019. Stem Cell Res. Ther. 10:74).

[0363] In particular embodiments, MSC can be used and genetically modified to express different factors in the treatment of gastrointestinal and liver diseases. In particular embodiments, these factors include: HGF (Zhang et al., 2018. Cell Death Dis. 9, 1-12), fibroblast growth factor 4 (FGF4) (Wang et al., 2015. Int. J. Clin. Exp. Med. 8, 12774-12782), IGF-1 (Fiore et al., 2015. Stem Cell. Dev. 24, 791-801 ), neuregulin 4 (Nrg4) (Wang W. et al., 2019. Exp. Biol. Med. 244, 565-578), interleukin-1 receptor antagonist (IL-1 Ra) (Zheng et al., 2012. PLoS One 7:e41392), IL-10 (Choi et al., 2019. Biomater. Sci. 7, 1078-1087), CXCR2 (Shen et al., 2018. Cell Death Dis. 9, 1-14), Intercellular Adhesion Molecule (ICAM)-1 (Li et al., 2019. Stem Cell Res. Ther. 10, 1-11 ), HNF4a (Ye et al., 2019), and forkhead box protein (Foxa2) (Chae et al., 2019).

[0364] In particular embodiments, MSC can be used and genetically modified to express different factors in the treatment of kidney diseases. In particular embodiments, these factors include: glial cell line-derived neurotrophic factor (GDNF) (Huang et al., 2012. J. Control. Release 162, 464- 473; Wang, et al., 2019. Stem Cell Res. 41 :101605), IGF-1 , erythropoietin (EPO) (Kucic et al., 2008. Am. J. Physiol. Renal Physiol. 295, F488-F496), TGF-[31 (Cai etal., 2019. Cytotherapy 21 , 535-545), lipocalin 2 (Len2) (Roudkenar et al., 2011. Free Radio. Res. 45, 810-819), CXCR4 (Liu et al., 2013. Am. J. Physiol. Renal Physiol. 305, F1064-F1073), Klotho gene (Kuro-o et al., 1997. Nature 390, 45-51 ), and miR-199a-3p (Zhu et al., 2019. J. Cell. Physiol. 234, 23736-23749).

[0365] In particular embodiments, MSC can be used and genetically modified to express different factors in the treatment of several neurological disorders. In particular embodiments, these factors include brain derived neurotrophic factor (BDNF), Glial cell-derived neurotrophic factor (GDNF), fibroblast growth factor-2 (FGF-2), HGF, IGF-1 , nerve growth factor (NGF) and platelet-derived growth factor (PDGF) (Joyce et al., 2010. Regen. Med. 5, 933-946), ciliary neurotrophic factor (CNTF) (Abbaszadeh et al., 2015. Cytotherapy 17 , 912-921 ), sonic hedgehog (Shh) (Jia et al., 2014. Neurosci. Lett. 573, 46-51 ), brain dopamine factor (CDNF) (Zhao et al., 2014. Mol. Neurobiol. 53, 187-199), neurotrophin-3 (NT3) (Moradian et al., 2017. Mater. Sci. Eng. C Mater. Biol. Appl. 76, 934-943), VEGF (Man et al., 2016. Cell. Mol. Bioeng. 9, 96-106), IL-4 (Payne et al., 2012. Cell Adh. Migr. 6, 179-189), IL-10 (Nakajima et al., 2017. Mol. Ther. Methods Clin. Dev. 6, 102— 1 11 ), C-C Motif Chemokine Receptor 2 (CCR2) (Huang etal., 2018. Theranostics 8, 5929-5944), PARKIN (Bonilla-Porras et al., 2018. Cytotherapy 20, 45-61 ), extracellular regulating kinase 1 / 2 or integrin a4 (Gao etal., 2019. Brain Res. Bull. 149, 42-52), miR-705 (Ji et al., 2017. Mol. Med. Rep. 16, 8323-8328), miR-25 (Huang et al., 2018. Exp. Cell Res. 371 , 269- 277; Zhao et al., 2018. J. Thorac. Cardiovasc. Surg. 157, 508-517), miR-21 , miR-124, or miR- 133b (Zou et al., 2014. Neural Regen. Res. 9, 1241-1248; and Xin et al., 2017. Cell Transplant. 26, 243-257; Zhang H. etal., 2018. Front. Neurol. 9:931 ), hypoxia-inducible factor 1 a (HIF-1 a) (Yang et al., 2014. Int. J. Mol. Med. 34, 1622-1628), Mashl (Wang et al., 2013. Brain Res. Bull. 99, 84-94), and tyrosine kinase receptor type 3 (TrkC) (Ding et al., 2015. Sci. Rep. 5, 1-14).

[0366] In particular embodiments, compositions and / or formulations can be used to treat cancers expressing CD90. In particular embodiments, a method of treating a CD90-expressing cancer in a subject in need thereof includes administering a composition and / or formulation described herein to the subject in need thereof. Examples of CD90-expressing cancer cells include cancer stem cells in liver cancer, gastric cancer, lung cancer, or gliomas. In particular embodiments, CD90+ cancer cells include cancer stem cells from liver cancer, gastric cancer, lung cancer, brain cancer (e.g., glioma), bone marrow cancer (e.g., acute myeloid leukemia), skin cancer, esophageal cancer, or prostate cancer.

[0367] For administration, therapeutically effective amounts (also referred to herein as doses) can be initially estimated based on results from in vitro assays and / or animal model studies. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes an IC50 as determined in cell culture against a particular target. Such information can be used to more accurately determine useful doses in subjects of interest.

[0368] The actual dose amount administered to a particular subject can be determined by a physician, veterinarian or researcher taking into account parameters such as physical and physiological factors including target, body weight, severity of condition, type of disease, previous or concurrent therapeutic interventions, idiopathy of the subject and route of administration.

[0369] Useful doses of active ingredients within compositions range from, for example, 0.1 to 5 pg / kg or from 0.5 to 1 pg / kg. In other examples, a dose can include 1 ng / kg, 15 pg / kg, 30 pg / kg, 50 pg / kg, 55 pg / kg, 70 pg / kg, 90 pg / kg, 150 pg / kg, 350 pg / kg, 500 pg / kg, 750 pg / kg, 1000 pg / kg, 0.1 to 5 mg / kg or from 0.5 to 1 mg / kg. In other examples, a dose can include 1 mg / kg, 10 mg / kg, 30 mg / kg, 50 mg / kg, 70 mg / kg, 100 mg / kg, 300 mg / kg, 500 mg / kg, 700 mg / kg, 1000 mg / kg, 2000 mg / kg, 3000 mg / kg, 4000 mg / kg, 5000 mg / kg or more.

[0370] Exemplary doses of cell-based formulations can include 104to 109cells / kg body weight, or 103to 1011cells / kg body weight. Therapeutically effective amounts to administer can include greater than 102cells, greater than 103cells, greater than 104cells, greater than 105cells, greater than 106cells, greater than 107cells, greater than 108cells, greater than 109cells, greater than 1010cells, or greater than 1011cells.

[0371] In particular embodiments, cell-based formulations are generally in a volume of a liter or less, 500 mis or less, 250 mis or less or 100 mis or less. Hence the density of administered cells is typically greater than 104cells / ml , 107cells / m I, or 108cells / ml.

[0372] Therapeutically effective amounts can be achieved by administering single or multiple doses during the course of a treatment regimen (e.g., daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months or yearly. In particular embodiments, therapeutically effective amounts can be achieved by administering repeated doses during the course of a treatment regimen. In particular embodiments, the treatment protocol may be dictated by a clinical trial protocol or an FDA-approved treatment protocol.

[0373] The compositions and formulations described herein can be administered by, for example, injection, inhalation, infusion, perfusion, lavage, or ingestion. Routes of administration can include intravenous, intradermal, intraarterial, intranodal, intravesicular, intrathecal, intraperitoneal, intraparenteral, intranasal, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, intrathecal, intratumoral, intramuscular, oral, subcutaneous, and / or sublingual administration.

[0374] The Exemplary Embodiments and Example below are included to demonstrate particular embodiments of the disclosure. Those of ordinary skill in the art should recognize in light of thepresent disclosure that many changes can be made to the specific embodiments disclosed herein and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

[0375] (XIII) Exemplary Embodiments.1 . An antibody or binding fragment thereof that binds CD90 including a variable heavy chain including a complementarity determining region (CDR) heavy (H) 1 , a CDRH2, and a CDRH3; and a variable light chain including a CDR light (L)1 , a CDRL2, and CDRL3, wherein: the CDRH1 includes a sequence as set forth in SEQ ID NO: 41 , the CDRH2 includes a sequence as set forth in SEQ ID NO: 42, the CDRH3 includes a sequence as set forth in SEQ ID NO: 43, CDRL1 includes a sequence as set forth in SEQ ID NO: 53, the CDRL2 includes the sequence YTS, and the CDRL3 includes a sequence as set forth in SEQ ID NO: 54, according to IMGT; the CDRH1 includes a sequence as set forth in SEQ ID NO: 44, the CDRH2 includes a sequence as set forth in SEQ ID NO: 45, the CDRH3 includes a sequence as set forth in SEQ ID NO: 46, the CDRL1 includes a sequence as set forth in SEQ ID NO: 55, the CDRL2 includes the sequence as set forth in SEQ ID NO: 195, and the CDRL3 includes a sequence as set forth in SEQ ID NO: 54, according to Kabat; the CDRH1 includes a sequence as set forth in SEQ ID NO: 47, the CDRH2 includes a sequence as set forth in SEQ ID NO: 48, the CDRH3 includes a sequence as set forth in SEQ ID NO: 46, the CDRL1 includes a sequence as set forth in SEQ ID NO: 55, the CDRL2 includes the sequence as set forth in SEQ ID NO: 195, and the CDRL3 includes a sequence as set forth in SEQ ID NO: 54, according to Chothia; the CDRH1 includes a sequence as set forth in SEQ ID NO: 49, the CDRH2 includes a sequence as set forth in SEQ ID NO: 50, the CDRH3 includes a sequence as set forth in SEQ ID NO: 43, the CDRL1 includes a sequence as set forth in SEQ ID NO: 55, the CDRL2 includes the sequence as set forth in SEQ ID NO: 195, and the CDRL3 includes a sequence as set forth in SEQ ID NO: 54, according to North; or the CDRH1 includes a sequence as set forth in SEQ ID NO: 56, the CDRH2 includes a sequence as set forth in SEQ ID NO: 57, the CDRH3 includes a sequence as set forth in SEQ ID NO: 58, the CDRL1 includes a sequence as set forth in SEQ ID NO: 67, the CDRL2 includes the sequence as set forth in SEQ ID NO: 68, and the CDRL3 includes a sequence as set forth in SEQ ID NO: 69, according to Contact.2. The antibody or binding fragment thereof of embodiment 1 , wherein the variable heavy chain has at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 40 and the variable light chain has at least 90% sequence identity to the sequence as set forth in SEQID NO: 193. The antibody or binding fragment thereof of embodiments 1 or 2, wherein the variable heavy chain has the sequence as set forth in SEQ ID NO: 40 and the variable light chain has the sequence as set forth in SEQ ID NO: 193. The antibody or binding fragment thereof of any of embodiments 1 -3, wherein the variable heavy chain is encoded by a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 22 and the variable light chain is encoded by a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 190. The antibody or binding fragment thereof of any of embodiments 1 -4, wherein the variable heavy chain is encoded by a sequence as set forth in SEQ ID NO: 20 and the variable light chain is encoded by a sequence as set forth in SEQ ID NO: 190. The antibody or binding fragment thereof of any of embodiments 1 -5, wherein the antibody or binding fragment thereof further includes a human constant region. The antibody or binding fragment thereof of embodiment 6, wherein the human constant region includes a human heavy chain constant region and / or a human light chain constant region. The antibody or binding fragment thereof of embodiment 7, wherein the human heavy chain constant region includes an IgG, IgM, IgD, IgA, or IgE heavy chain constant region. The antibody or binding fragment thereof of embodiments 7 or 8, wherein the human heavy chain constant region includes an IgG heavy chain constant region. The antibody or binding fragment thereof of embodiment 9, wherein the IgG heavy chain constant region includes an IgG 1 heavy chain constant region, an lgG2 heavy chain constant region, an lgG3 heavy chain constant region, and an lgG4 heavy chain constant region. The antibody or binding fragment thereof of embodiments 9 or 10, wherein the IgG heavy chain constant region includes an IgG 1 heavy chain constant region. The antibody or binding fragment thereof of embodiment 9, wherein the IgG heavy chain constant region includes an lgG4 heavy chain constant region with an S228P mutation. The antibody or binding fragment thereof of any of embodiments 9-12, wherein the IgG heavy chain constant region includes the sequence as set forth in SEQ ID NOs: 15-19. The antibody or binding fragment thereof of any of embodiments 7-13, wherein the human light chain constant region includes a human IgK light chain constant region or a human IgA light chain constant region. The antibody or binding fragment thereof of embodiment 14, wherein the human IgK light chain constant region includes the sequence as set forth in SEQ ID NO: 13.The antibody or binding fragment thereof of embodiment 14, wherein the human IgA light chain constant region includes the sequence as set forth in SEQ ID NO: 14. The antibody or binding fragment thereof of any of embodiments 1 -16, wherein the antibody further includes an Fc modification. The antibody or binding fragment thereof of any of embodiments 1 -17, wherein the antibody or binding fragment thereof is humanized. The antibody or binding fragment thereof of any of embodiments 1 -18, wherein the antibody or binding fragment thereof is PEGylated. The antibody or binding fragment thereof of any of embodiments 1 -19, wherein the antibody or binding fragment thereof is conjugated to polyglutamic acid (PGA). The antibody or binding fragment thereof of embodiment 20, wherein the PGA shields a positive charge of a polymeric nanoparticle. The antibody or binding fragment thereof of any of embodiments 1 -21 , including M428L / N434S mutations. The antibody or binding fragment thereof of any of embodiments 1 -22, including G236A / S239D / A330L / I332E mutations. The antibody or binding fragment thereof of any of embodiments 1 -23, wherein the antibody or binding fragment thereof includes a heavy chain and a light chain, wherein the heavy chain includes the sequence as set forth in SEQ ID NO: 3 and the light chain includes the sequence as set forth in SEQ ID NO: 7, or the heavy chain includes a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 3 and the light chain includes a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 7. The antibody or binding fragment thereof of any of embodiments 1 -24, wherein the antibody or binding fragment thereof includes a heavy chain and a light chain, wherein the heavy chain includes the sequence as set forth in SEQ ID NO: 5 and the light chain includes the sequence as set forth in SEQ ID NO: 7, or the heavy chain includes a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 5 and the light chain includes a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 7. The antibody or binding fragment thereof of any of embodiments 1 -25, wherein the antibody or binding fragment thereof includes a signal peptide. The antibody or binding fragment thereof of embodiment 26, wherein the signal peptide includes a sequence selected from SEQ ID NO: 191 , SEQ ID NO: 192, SEQ ID NO: 8, or SEQID NO: 9. A multi-domain binding molecule including at least two binding domains, wherein at least one binding domain of the at least two binding domains includes the antibody or binding fragment thereof of any of embodiments 1 -27. The multi-domain binding molecule of embodiment 28, wherein the multi-domain binding molecule includes a bispecific antibody, a trispecific antibody, or a tetraspecific antibody. The multi-domain binding molecule of embodiments 28 or 29, wherein the multi-domain binding molecule includes an immune cell engaging molecule. The multi-domain binding molecule of embodiment 30, wherein the immune cell engaging molecule activates a T cell, natural killer (NK) cell, B cell, or macrophage. The multi-domain binding molecule of embodiment 31 , wherein the T cell is a CD3 T cell, a CD4 T cell, a CD8 T cell, a central memory T cell, an effector memory T cell, and / or a naive T cell. The multi-domain binding molecule of embodiments 30 or 31 , wherein a binding domain of the immune cell engaging molecule binds CD3, CD28, CD8, NKG2D, CD8, CD16, KIR2DL4, KIR2DS1 , KIR2DS2, KIR3DS1 , NKG2C, NKG2E, NKG2D, NKp30, NKp44, NKp46, NKp80, DNAM-1 , CD11 b, CD11c, CD64, CD68, CD119, CD163, CD206, CD209, F4 / 80, IFGR2, Tolllike receptors 1 -9, IL-4Ra, or MARCO. The multi-domain binding molecule of any of embodiments 30-33, wherein a binding domain of the immune cell engaging molecule binds CD3, CD28, 4-1 BB, or CD27. The multi-domain binding molecule of any of embodiments 30-33, wherein a binding domain of the immune cell engaging molecule binds CD8, CD16, NKG2A, NKG2D, KIR2DL1 , KIR2DL2 / 3, KIR2DL4, KIR3DL1 , NKp44, or KLRG1. The multi-domain binding molecule of any of embodiments 28-35, wherein the at least two binding domains include at least two copies of the antibody or binding fragment thereof of any of embodiments 1 -27. The multi-domain binding molecule of embodiment 36, wherein the at least two copies are joined by a linker. The multi-domain binding molecule of embodiment 37, wherein the linker is a Gly-Ser linker. The multi-domain binding molecule of embodiment 38, wherein the Gly-Ser linker is (GlyxSery)nwherein x and y are independently an integer from 0 to 10 provided that x and y are not both 0 and wherein n is an integer of 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10. The multi-domain binding molecule of any of embodiments 28-39, including 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the antibody or binding fragment thereof of any of embodiments 1 -27.The multi-domain binding molecule of any of embodiments 28-40, wherein the multi-domain binding molecule is a dimer, trimer, tetramer, pentamer, hexamer, or heptamer. The multi-domain binding molecule of any of embodiments 36-41 , wherein the at least two copies are linked to an Fc region of an antibody. The multi-domain binding molecule of embodiment 42, wherein the Fc region is an IgA Fc region or an IgM Fc region. The multi-domain binding molecule of embodiments 42 or 43, wherein the Fc region is an IgA Fc region having the sequence as set forth in SEQ ID NOs: 198 and 199. The multi-domain binding molecule of embodiments 42 or 43, wherein the Fc region is an IgM Fc region having the sequence as set forth in SEQ ID NOs: 202-211 . The multi-domain binding molecule of any of embodiments 42-45, wherein the Fc region includes a multimerizing fragment of an IgA Fc region or a multimerizing fragment of an IgM Fc region. The multi-domain binding molecule of embodiment 46, wherein the multimerizing fragment of the IgA Fc region includes an IgA tailpiece. The multi-domain binding molecule of embodiment 47, wherein the IgA tailpiece has a sequence of residues 331 -352 as set forth in SEQ ID NO: 198 or the sequence of residues 318-340 as set forth in SEQ ID NO: 199. The multi-domain binding molecule of any of embodiments 46-48, wherein the multimerizing fragment of the IgA Fc region includes an IgA CA3 domain and an IgA tailpiece. The multi-domain binding molecule of any of embodiments 46-49, wherein the multimerizing fragment of the IgA Fc region includes an IgA CA2 domain, an IgA CA3 domain, and an IgA tailpiece. The multi-domain binding molecule of any of embodiments 46-50, wherein the multimerizing fragment of the IgA Fc region includes an IgA CA1 domain, an IgA CA2 domain, an IgA CA3 domain, and an IgA tailpiece. The multi-domain binding molecule of embodiment 46, wherein the multimerizing fragment of the IgM Fc region includes an IgM tailpiece. The multi-domain binding molecule of embodiments 46 or 52, wherein the multimerizing fragment of the IgM Fc region includes a Cp4 domain and an IgM tailpiece. The multi-domain binding molecule of any of embodiments 46-53, wherein the multimerizing fragment of the IgM Fc region includes a Cp3 domain, a Cp4 domain, and an IgM tailpiece. The multi-domain binding molecule of any of embodiments 46-54, wherein the multimerizing fragment of the IgM Fc region includes a C|i2 domain, a Cp3 domain, a Cp4 domain, and anIgM tailpiece. The multi-domain binding molecule of any of embodiments 46-55, wherein the multimerizing fragment of the IgM Fc region includes a Cp1 domain, a Cp2 domain, a Cp3 domain, a Cp4 domain, and an IgM tailpiece. The multi-domain binding molecule of any of embodiments 46-56, wherein the multimerizing fragment of the IgM Fc region has the sequence as set forth in SEQ ID NO: 204. A single-chain variable fragment (scFv) including an antibody binding fragment of any of embodiments 1 -27. The scFv of embodiment 58, further including a linker. The scFv of embodiment 59, wherein the linker includes a sequence as set forth in SEQ ID NO: 66. The scFv of embodiments 59 or 60, wherein the linker includes a Gly-Ser linker. The scFv of embodiment 61 , wherein the Gly-Ser linker includes a sequence as set forth in SEQ ID NO: 64, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81 , SEQ ID NO: 82. SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, or SEQ ID NO: 91 . The scFv of embodiment 61 , wherein the Gly-Ser linker includes a sequence as set forth in SEQ ID NO: 66. The scFv of any of embodiments 58-63, having the sequence as set forth in SEQ ID NO: 194. A conjugate including the antibody or binding fragment thereof of any of embodiments 1 -27 linked to a toxin, a drug, a detectable label, a radioisotope, a particle, or a bead. The conjugate of embodiment 65, wherein the toxin includes a holotoxin or a hemitoxin. The conjugate of embodiment 65, wherein the drug includes actinomycin D, anthracycline, auristatin, calicheamicin, camptothecin, CC1065, colchicin, cytochalasin B, daunorubicin, 1 - dehydrotestosterone, dihydroxy anthracinedione, dolastatin, doxorubicin, duocarmycin, elinafide, emetine, ethidium bromide, etoposide, gramicidin D, glucocorticoids, lidocaine, maytansinoid, mithramycin, mitomycin, mitoxantrone, nemorubicin, PNU-159682, procaine, propranolol, puromycin, pyrrolobenzodiazepine, taxane, taxol, tenoposide, tetracaine, trichothecene, vinblastine, vinca alkaloid, or vincristine. The conjugate of embodiment 65, wherein the detectable label includes a fluorescent label, an enzyme label, chemiluminescent label, a spectral colorimetric label, or an affinity tag. The conjugate of embodiment 68, wherein the fluorescent label includes blue fluorescent protein, cyan fluorescent protein, green fluorescent proteins, luciferase, orange fluorescent protein, red fluorescent protein, far red fluorescent protein, or yellow fluorescent protein.The conjugate of embodiment 68, wherein the enzyme label includes horseradish peroxidase, hydrolase, or alkaline phosphatase. The conjugate of embodiment 68, wherein the chemiluminescent label includes lucigenin, luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, or oxalate ester. The conjugate of embodiment 68, wherein the spectral colorimetric label includes colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, and latex) beads. The conjugate of embodiment 68, wherein the affinity tag includes a sequence as set forth in SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 1 1 , SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, or SEQ ID NO: 170. The conjugate of embodiment 65, wherein the radioisotope includes actinium-225, iodine- 131 , arsenic-21 1 , iodine-131 , indium-1 1 1 , yttrium-90, lutetium-177, astatine-21 1 , bismuth-212, or bismuth-213. The conjugate of embodiment 65, wherein the radioisotope includes228Ac,111Ag,124Am,74As,211At,209At,194Au,128Ba,7Be,206Bi,245Bk,246Bk,76Br,11C,14C,47Ca,254Cf,242Cm,51Cr,67Cu,153Dy,157Dy,159Dy,165Dy,166Dy,171Er,250Es,254Es,147Eu,157Eu,52Fe,59Fe,251Fm,252Fm,253Fm,66Ga,72Ga,146Gd,153Gd,68Ge,3H,17°Hf,171Hf,193Hg,193mHg,16°mHo,130l,1311,135l,114mln,185lr,42K,43K,76Kr,79Kr,81mKr,132La,262Lr,169Lu,174mLu,176ml_u,257Md,260Md,28Mg,52Mn,90Mo,24Na,95Nb,138Nd,57Ni,66Ni,234Np,15O,1820s,189mOs,191Os,32P,201Pb,101Pd,143Pr,191Pt,243Pu,225Ra,81Rb,188Re,105Rh,211Rn,103Ru,35S,44Sc,72Se,153Sm,125Sn,91Sr,173Ta,154Tb,127Te,234Th,45Ti,166Tm,230U,237U,240U,48V,178W,181W,188W,125Xe,127Xe,133Xe,133mXe,135Xe,85mY,86Y,90Y,93Y,169Yb,175Yb,65Zn,71mZn,86Zr,95Zr, or97Zr. The conjugate of any of embodiments 65-75, wherein the radioisotope does not emit daughter radionuclides. The conjugate of embodiment 65, wherein the particle includes a metal nanoparticle, liposome, a polymer nanoparticle, or a lipid nanoparticle. The conjugate of embodiment 65, wherein the bead includes a magnetic or a paramagnetic bead. A recombinant protein including the antibody or binding fragment thereof of any of embodiments 1 -27 linked to a viral structural protein. The recombinant protein of embodiment 79, wherein the viral structural protein includes measles virus hemagglutinin (H), measles virus fusion (F), vesicular stomatis virus G (VSV- G), influenza hemagglutinin, ebolavirus glycoprotein (GP)1 or GP2, coronavirus spike, lassavirus glycoprotein precursor (GP) or spike, nipah virus NiV-G or NiV-F, rabies virus glycoprotein (G), respiratory syncytial virus fusion protein (F) or attachment protein (G), human immunodeficiency virus (HIV) gp41 , HIV gp120, paramyxovirus F protein, filovirus GP2 protein, flavivirus E protein, alphavirus E1 protein, herpes simplex virus gH or gB protein, or arenavirus G2 protein. The recombinant protein of embodiments 79 or 80, wherein the viral structural protein includes measles virus hemagglutinin. The recombinant protein of embodiments 79 or 80, wherein the viral structural protein includes VSV-G. The recombinant protein of embodiment 81 , wherein the measles virus hemagglutinin includes a cytoplasmic tail truncation of up to 30 residues. The recombinant protein of embodiments 81 or 83, wherein the measles virus hemagglutinin includes mutations Y481A, R533A, S548L, and F549S. The recombinant protein of any of embodiments 79-84, wherein the antibody or binding fragment thereof includes an scFv. A pseudotyped viral vector including an antibody or binding fragment thereof of any of embodiments 1 -27 linked to a viral structural protein. The pseudotyped viral vector of embodiment 86, wherein the viral structural protein includes measles virus hemagglutinin (H), measles virus fusion (F), vesicular stomatis virus G (VSV- G), influenza hemagglutinin, ebolavirus glycoprotein (GP)1 or GP2, coronavirus spike, lassa virus glycoprotein precursor (GP) or spike, nipah virus NiV-G or NiV-F, rabies virus glycoprotein (G), respiratory syncytial virus fusion protein (F) or attachment protein (G), human immunodeficiency virus (HIV) gp41 , HIV gp120, paramyxovirus F protein, filovirus GP2 protein, flavivirus E protein, alphavirus E1 protein, herpes simplex virus gH or gB protein, or arenavirus G2 protein. The pseudotyped viral vector of embodiments 86 or 87, wherein the viral structural protein includes a measles virus hemagglutinin. The pseudotyped viral vector of embodiments 86 or 87, wherein the viral structural protein includes VSV-G. The pseudotyped viral vector of any of embodiments 86-89, wherein the pseudotyped viral vector is a retroviral vector, an adenoviral vector, or an adeno-associated viral (AAV) vector. The pseudotyped viral vector of embodiment 90, wherein the retroviral vector includes a lentiviral vector or a foamy virus vector. The pseudotyped viral vector of any of embodiments 86-91 , wherein the antibody or bindingfragment thereof is linked to the viral structural protein with a linker. The pseudotyped viral vector of embodiment 92, wherein the linker is a Gly-Ser linker. A pseudotyped viral vector including a viral vector expressing the recombinant protein of any of embodiments 79-85. The pseudotyped viral vector of embodiment 94, wherein the viral vector includes a retroviral vector, an adenoviral vector, or adeno-associated viral (AAV) vector. A recombinant receptor that, when expressed by a cell, includes an extracellular component including the scFv of any of embodiments 58-64. The recombinant receptor of embodiment 96, wherein the recombinant receptor includes a chimeric antigen receptor (CAR) or engineered T cell receptor (eTCR). The recombinant receptor of embodiment 97, wherein the CAR includes an intracellular component linked to the extracellular component by a transmembrane domain. The recombinant receptor of embodiment 98, wherein the intracellular component includes an effector domain including: 4-1 BB (CD137), CD3y, CD35, CD3c, CD3 , CD27, CD28, DAP10, ICOS, LAG3, NKG2D, NOTCH1 , 0X40, ROR2, SLAMF1 , TCRa, TCR|3, TRIM, Wnt, Zap70, or a combination thereof. . The recombinant receptor of embodiments 98 or 99, wherein the transmembrane domain includes a transmembrane region of: an a, p or £ chain of a T-cell receptor; CD28; CD27; CD3; CD45; CD4; CD5; CD8; CD9; CD16; CD22; CD33; CD37; CD64; CD80; CD86; CD134; CD137; CD154; or a combination thereof. . The recombinant receptor of any of embodiments 97-100, wherein the CAR further includes a spacer region. . The recombinant receptor of any of embodiments 97-100, wherein the eTCR includes a constant alpha domain (Ca) and a constant beta domain (Cp). . The recombinant receptor of any of embodiments 102, wherein the scFv of any of embodiments 58-64 is linked to the Cadomain and / or the Cp domain. . A nucleic acid encoding the antibody or binding fragment thereof of any of embodiments 1 -27. . The nucleic acid of embodiment 104, wherein the nucleic acid is codon-optimized. . The nucleic acid of embodiments 104 or 105, wherein nucleic acid includes a variable heavy chain coding sequence and a variable light chain coding sequence, wherein the variable heavy chain coding sequence includes the sequence as set forth in SEQ ID NO: 22 and the variable light chain coding sequence includes the sequence as set forth in SEQ ID NO: 190, or the variable heavy chain coding sequence includes a sequence having at least90% sequence identity to the sequence as set forth in SEQ ID NO: 22 and the variable light chain coding sequence includes a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 190. . The nucleic acid of any of embodiments 104-106, wherein the nucleic acid includes a heavy chain coding sequence and a light chain coding sequence, wherein the heavy chain coding sequence includes the sequence as set forth in SEQ ID NO: 2 and the light chain coding sequence includes the sequence as set forth in SEQ ID NO: 6, or the heavy chain coding sequence includes a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 2 and the light chain coding sequence includes a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 6. . The nucleic acid of any of embodiments 104-107, wherein the nucleic acid includes a heavy chain coding sequence and a light chain coding sequence, wherein the heavy chain coding sequence includes the sequence as set forth in SEQ ID NO: 4 and the light chain coding sequence includes the sequence as set forth in SEQ ID NO: 6, or the heavy chain coding sequence includes a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 4 and the light chain coding sequence includes a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 6. . A genetic construct including the nucleic acid of any of embodiments 104-108 operably linked to a sequence encoding a viral structural protein. . The genetic construct of embodiment 109, wherein the viral structural protein includes measles virus hemagglutinin (H), measles virus fusion (F), vesicular stomatis virus G (VSV- G), influenza hemagglutinin, ebolavirus glycoprotein (GP)1 or GP2, coronavirus spike, lassa virus glycoprotein precursor (GP) or spike, nipah virus NiV-G or NiV-F, rabies virus glycoprotein (G), respiratory syncytial virus fusion protein (F) or attachment protein (G), human immunodeficiency virus (HIV) gp41 , HIV gp120, paramyxovirus F protein, filovirus GP2 protein, flavivirus E protein, alphavirus E1 protein, herpes simplex virus gH or gB protein, or arenavirus G2 protein.. . The genetic construct of embodiments 109 or 110, wherein the viral structural protein includes measles virus hemagglutinin. . The genetic construct of embodiments 109 or 110, wherein the viral structural protein includes VSV-G. . The genetic construct of any of embodiments 109-1 12, wherein the nucleic acid is operably linked to the sequence encoding the viral structural protein by a sequence encoding linker.. The genetic construct of embodiment 113, wherein the sequence encoding the linker includes the sequence as set forth in SEQ ID NO: 78, SEQ ID NO: 164, or SEQ ID NO: 165 or a sequence having at least 95% sequence identity to a sequence as set forth in SEQ ID NO: 78, SEQ ID NO: 164, or SEQ ID NO: 165. . The genetic construct of embodiments 113 or 1 14, wherein the linker includes the sequence as set forth in SEQ ID NO: 64 or SEQ ID NO: 66 or a sequence having at least 95% sequence identity to a sequence as set forth in SEQ ID NO: 64 or SEQ ID NO: 66. . A cell genetically modified to express any of the preceding embodiments. . The cell of embodiment 116, wherein the cell is an immune cell. . The cell of embodiment 117, wherein the immune cell is a T cell, B cell, natural killer cell, or macrophage. . A composition including any of the preceding embodiments; and a pharmaceutically- acceptable carrier. . A formulation including the cell of any of embodiments 1 16-118 and a pharmaceutically- acceptable carrier. . A kit including the antibody or binding fragment thereof of any of embodiments 1 -27, the multi-domain binding molecule of any of embodiments 28-57, the scFv of any of embodiments 58-64, the conjugate of any of embodiments 65-78, the pseudotyped viral vector of any of embodiments 86-93 or 94-95, the nucleic acid of any of embodiments 104-108, the genetic construct of any of embodiments 109-1 15, or the cell of any of embodiments 116-118. . A method of delivering genetic engineering components to a CD90+ cell including administering the pseudotyped viral vector of any of embodiments 86-93 or 94-95 or the conjugate of any of embodiments 65-78. . The method of embodiment 122, wherein the conjugate includes the antibody or binding fragment thereof linked to a particle. . The method of embodiments 122 or 123, wherein the genetic engineering components provide a therapeutic coding sequence to the CD90+ cell. . The method of embodiment 124, wherein the therapeutic coding sequence inserts or alters a coding sequence selected from ABCA3, ABCD1 , Akt, amyloid beta precursor protein (APP), angiopoietin 1 (Ang1 ), angiotensin-converting enzyme 2 (ACE2), antibodies to CD4, antibodies to CD5, antibodies to CD7, antibodies to CD52, etc., antibodies to IL1 , antibodies to IL2, antibodies to IL6, antibodies to TCR specifically present on autoreactive T cells, antibodies to TNF, arylsulfatase A, Bcl-2, brain derived neurotrophic factor (BDNF), cerebral dopamine neurotrophic factor (CDNF), C1q / tumor necrosis factor- related protein-3 (CTRP3),C9ORF72, C-C Motif Chemokine Receptor 1 (CCR1 ), CCR2, chemokine receptor 2 (CXCR2), CXCR4, CXCR7, ciliary neurotrophic factor (CNTF), CLN3, connexin 43 (Cx43), Csx / Nkx 2.5, CTLA, cystic fibrosis transmembrane conductance regulator (CFTR), Cytochrome b5 reductase 3 (CYB5R3), DRB1 *1501 / DQB1 *0602, Dyskerin Pseudouridine Synthase (DKC1 ), dystrophin, E2F4, endothelial nitric oxide synthase (eNOS), erythropoietin (EPO), extracellular regulating kinase 1 / 2, F8 (coagulation factor VIII), F9 (coagulation factor IX), Fanconi anaemia complementation group (FANC) family genes, Fas L, fibroblast growth factor-2 (FGF-2), fibroblast growth factor 4 (FGF4), Follistatin-like 1 , forkhead box protein (Foxa2), fused in sarcoma (FUS), GATA1 , GATA-4, glial cell line-derived neurotrophic factor (GDNF), globin family genes, granulocyte colony-stimulating factor (G-CSF), HBB, heme oxygenase-1 (HO-1 ), hepatocyte growth factor (HGF), hepatocyte nuclear factor 4a (HNF4a), hypoxia-inducible factor 1 a (HIF-1 a), insulin-like growth factor (IGF)-1 , Intercellular Adhesion Molecule (ICAM)-1 , interferon -beta (IFN-[3), integrin a4, interleukin-1 receptor antagonist (IL-1 Ra), interleukin 4 (IL4), IL10, IL12, IL13, IL-33, Islet-1 , Klotho gene, let-7d, leucine-rich repeat kinase 2 (LRRK2), lipocalin 2 (Len2), Mashl , microRNA-1 (miR-1 ), miR-16-5p, miR-21 , miR- 25, miR-30b-3p, miR-34, miR-101 -3p, miR-124, mlR-126, miR-133, miR-133b, mlR-181a, miR-199a, miR-199a-3p, miR-211 , miR-705, nerve growth factor (NGF), neuregulin 4 (Nrg4), neurotrophin-3 (NT3), NLX2.1 , Notch ligand Delta-like-4, Notchl receptor, nuclear factor erythroid-derived 2-like 2 (Nrf2), orphan receptor tyrosine kinase 2 (ROR2), P53, p130, Parkinson disease 2 (PARK2), PARK7, PARKIN, phox, platelet-derived growth factor (PDGF), presenilin 1 (PSEN1 ), PSEN2, protein tyrosine phosphatase, non-receptor type 22 (PTPN22), PTEN-induced kinase 1 (PINK1 ), pyruvate kinase, ribosomal protein genes, secreted Klotho protein (SKL), sirtuin 1 , soluble CD40, soluble interleukin 1 receptor II (sILI RII), sILI RI, soluble TNF alpha receptor I (sTNFRI), sTNFRII, somatostatin 2 (sST2), sonic hedgehog (Shh), superoxide dismutase 1 (SOD1 ), surfactant protein B (SFTPB), SFTPC, synuclein alpha (SNCA), TAR DNA-binding protein 43 (TDP43), telomerase reverse transcriptase (TERT), telomerase RNA component (TERO), TERF interacting nuclear factor 2 (TINF2), TNF-related apoptosis-inducing ligand (TRAIL), transforming growth factor pi (TGF- 1 ), type2 angiotensin II receptor (AT2R), tyrosine kinase receptor type 3 (TrkC), ubiquilin 2, vascular endothelial growth factor (VEGF), WASP Actin nucleation promoting factor (WAS), or Wnt / p- catenin. . The method of any of embodiments 122-125, wherein the CD90+ cells are hematopoietic stem cells or mesenchymal stem / stromal cells. . The method of any of embodiments 122-126, wherein the genetic engineeringcomponents include coding sequence, guide RNA, homology arms, a base editor, a clustered regularly interspaced short palindromic repeats (CRISPR) nuclease, a zinc finger (ZFN), a tai effector nuclease (TALEN), a meganucleases or a meganuclease-TALEN fusion (MegaTALE). . The method of any of embodiments 122-127, wherein the administering is in vivo or ex vivo. . The method of any of embodiments 122-128, wherein the administering includes intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, intrathecal, intrauterine, intraplacental, intramuscular, intravesicular, and / or subcutaneous administration. . The method of any of embodiments 122-129, wherein the administering includes pipetting.. The method of any of embodiments 122-130, further including isolating CD90+ cells.. A method of isolating CD90+ cells from a sample including contacting the sample with the conjugate of any of embodiments 65-78, thereby creating a conjugate-target complex; and separating the conjugate-target complex from the sample, thereby isolating CD90+ cells.. The method of embodiment 132, wherein the sample includes bone marrow, peripheral blood, adipose tissue, amniotic fluid, amniotic membrane, dental tissue, placental and fetal membrane, sub-amniotic umbilical cord lining membrane, salivary gland, endometrium, limb bud, menstrual blood, skin, synovial fluid, or Wharton's jelly. . The method of embodiments 132 or 133, wherein the sample includes hematopoietic stem cells or mesenchymal stem / stromal cells. . The method of any of embodiments 132-134, wherein the separating includes affinity chromatography. . The method of any of embodiments 132-134, wherein the separating includes magnetic separation. . The method of any of embodiments 132-134, wherein the separating includes fluorescence activated cell sorting. . The method of any of embodiments 132-137, wherein the CD90+ cells are isolated with reduced or minimal manipulation. . The method of embodiment 138, wherein the CD90+ cells are isolated without electroporation, viral vectors, cell selection processes, or cell purification processes. . The method of embodiments 138 or 139 wherein the CD90+ cells are isolated solely by washing cells to remove platelets. . The method of any of embodiments 132-140, further including washing the sample.142. The method of any of embodiments 132-141 , further including washing the isolated cells.143. The method of any of embodiments 132-142, further including expanding isolated cells.144. The method of any of embodiments 132-143, further including culturing isolated cells.145. The method of any of embodiments 132-144, further including genetically modifying isolated cells.146. The method of any of embodiments 132-145, further including storing isolated cells.147. The method of any of embodiments 132-146, further including cryopreserving isolated cells.148. The method of any of embodiments 132-147, further including formulating isolated cells.149. A method of treating a subject in need thereof including administering a therapeutically effective amount of the composition of embodiment 119 or the formulation of embodiment 120 to the subject thereby treating the subject in need thereof.150. The method of embodiment 149, wherein the subject has a CD90-positive cancer.151 . The method of embodiments 149 or 150, wherein the administering includes intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, intrathecal, intrauterine, intraplacental, intramuscular, intravesicular, and / or subcutaneous administration.

[0376] (XIV) Experimental Example. CD90-targeted measles-pseudotyped lentiviral vectors for HSC gene therapy.

[0377] Abstract. Hematopoietic stem cell (HSC) gene therapy is currently performed on CD34+hematopoietic stem and progenitor cells (HSPCs) containing less than 1% true HSCs and requiring a highly specialized infrastructure for cell manufacturing and transplantation. The CD34+CD90+subset has previously been identified to be exclusively responsible for short- and long-term engraftment. However, purification and enrichment of this subset is laborious and expensive. HSC-specific delivery agents for the direct modification of rare HSCs are currently lacking. Here, targeted viral vectors were developed to specifically transduce CD90-expressing HSCs. Anti-CD90 single chain variable fragments (scFvs) were engineered onto measles- and VSV-G-pseudotyped lentiviral vectors that were knocked out for native targeting. A custom hydrodynamic titration methodology was developed to assess the loading of surface-engineered capsids, measure antigen recognition of the scFv, and predict the performance on cells. Engineered vectors formed with minimal impairment in the functional titer, maintained their ability to fuse with the target cells, and showed highly specific recognition of CD90 on cells ex vivo. Most importantly, targeted vectors selectively transduced human HSCs with secondary colony-forming potential. The disclosed HSC-targeted viral vectors significantly enhance the feasibility of ex vivogene therapy and pave the way for future in vivo applications.

[0378] Introduction. Currently existing gene therapy approaches modify CD34+hematopoietic stem and progenitor cells (HSPCs), a heterogeneous population that contains less than 1 % HSCs with long-term multilineage engraftment potential (Majeti, et al., 2007, Cell Stem Cell 1 , 635-645). Consequently, massive use of expensive reagents, potential off-target effects in non-HSCs, and low on-target efficiency currently impact the feasibility of HSC gene therapy. In addition, current gene therapy approaches rely on the modification of HSCs outside the patient's body (ex vivo) and require highly specialized facilities similar to bone marrow transplantation, severely limiting the accessibility of this treatment option. Injection of gene therapy agents and modification of HSCs directly in the patient (in vivo) would overcome all these limitations. However, a major obstacle to perform gene therapy in vivo is the lack of HSC-targeted gene therapy agents to highly specifically deliver the therapeutic cargo and avoid off-target effects.

[0379] This targeted delivery depends on the uptake of the vector after attaching to the cell / antigen, which has not been achieved with CD34-targeted HSC gene therapy vectors (Benedict, et al., 1999, Hum Gene Ther 10, 545-557). As a result, alternative markers of HSCs such as CD117 and CD133 have been considered (Radtke, et al., 2020, Mol Ther Methods Clin Dev 18, 679-691 ; and Brendel, et al., 2015, Mol Ther 23, 63-70). However, CD117+ / CD133+cells contain various erythro-myeloid and lympho-myeloid progenitors (Gorgens, et aL, 2013, Cell Rep 3, 1539-1552; and Radtke, et al., 2015, Br J Haematol 169, 868-878) that bear the risk for unwanted off-target effects. Further, CD133 as well as CD117 are readily expressed on cells in the lung, liver, and reproductive organs (proteinatlas.org), likely leading to rapid clearance of CD133 / CD1 17-targeted vectors as well as unknown side-effects when applied in vivo.

[0380] Previous work has shown that the CD90+subset of CD34+HSPCs is exclusively responsible for rapid recovery onset, robust long-term multilineage engraftment, and an entire reconstitution of the bone marrow (BM) stem cell compartment (Radtke, et aL, 2020, Mol Ther Methods Clin Dev 18, 679-691 ; Radtke, et aL, 2017, Sci Transl Med 9. 10.1126 / scitranslmed.aan1145; and Humbert, et aL, 2019, Sci Transl Med 1 1 , eaaw3768. 10.1126 / scitranslmed.aaw3768). Furthermore, CD34+CD90+cells are almost entirely depleted for phenotypically, transcriptionally, and functionally committed progenitor cells (Radtke, et aL, 2020, Mol Ther Methods Clin Dev 18, 679-691 ; Masiuk, et aL, 2017, Mol Ther 25, 2163-2175; Zonari, et aL, 2017, Stem Cell Reports 8, 977-990; and Gordon, et aL, 2003, Bone Marrow Transplant 31 , 17-22). Isolation and direct targeting of this HSC-enriched subset from mobilized human apheresis products ex vivo significantly increased lentivirus-mediated gene transfer without impacting the cells long-term multilineage engraftment potential (Benedict, et aL, 1999, HumGene Ther 10, 545-557).

[0381] To enable highly-specific targeting of CD90+HSCs, CD90-targeted viral vectors engineering an anti-CD90 single chain variable fragment (scFv) onto measles- and VSV-G- pseudotyped lentiviral vectors were herein designed. CD90-targeted viral vectors were further knocked out for their native targeting to optimize on-target specificity. Lastly, a hydrodynamicsbased methodology was developed to easily and reliably titrate and validate the disclosed surface-engineered viral vectors.

[0382] Results. CD90 scFv Design, Modification, and Validation. In order to generate HSC- targeted viral vectors for gene therapy, an scFv recognizing CD90 was designed and computationally validated. Sequences for the scFv design were extracted from the hybridoma cell line producing the CD90 antibody clone 5E10 (Craig, et al., 1993, J Exp Med 177, 1331 -1342). Briefly, mRNA was extracted from the cell line, reverse transcribed, and primers designed to sequence the cDNA fragments encoding for the variant light and heavy regions (FIG. 11 A). To determine the framework and complementary defining regions of the antibody, sequences were annotated using IMGT (Lefranc, et al., 2015, Nucleic Acids Res 43, D413-D422; and Lefranc, 2014, Front Immunol 5, 22). Next, scFvs were designed linking the light- and heavy-chain sequences using a 3x(GGGGS) (SEQ ID NO: 64) linker (FIG. 11 B). Finally, to predict the binding ability of the anti-CD90 scFv with the extracellular domain of CD90, interactions were modeled using SwissDock (Grosdidier, et al., 2011 , Nucleic Acids Res 39, W270-W277) (FIG. 11 C). Although bioinformatics predicted successful binding, no expression of the anti-CD90 scFv could be achieved (data not shown). Comprehensive comparison of the sequences with the database revealed two cysteine residues (position 31 and 59) that were not found in any other reference and were not predicted to form disulfate bonds running SAbPred modeling (Dunbar, et al., 2016, Nucleic Acids Res 44, W474-W478). Cysteines were therefore exchanged (position 31 Tyrosine [Y], position 59 Arginine [R]) to match the reference sequences. In addition, the linker was exchanged to an alternate linker sequence (GSDSNAGHASAGNTS) (SEQ ID NO: 66) (Anliker, et al., 2010, Nature Methods 7, 929-935) to enable easier production and sequencing of variants.

[0383] Design, Production, and Hydrodynamic Titration of CD90-Targeted Measles-Pseudotyped Viral Vectors. Measles-pseudotyped lentiviral vectors efficiently transduce a wide variety of cell types that recognize the broadly expressed cell surface antigens CD46 and CD150 (Levy, et al., 2017, Blood Adv 1 , 2088-2104; and Frecha, et al., 2008, Blood 112, 4843-4852). Here the measles envelope was stepwise developed to remove the native targeting and instead recognize CD90 as previously described for other target cells (Anliker, et aL, 2010, Nature Methods 7, 929- 935). In addition to the wildtype envelope (Variant H1 : WT, FIG. 12A), three variants of themeasles envelope were generated (FIG. 12B). To remove its ability to target CD46 / 150 and thus create a virus that should not be able to elicit fusion and transduce cells (Funke, et aL, 2008, Mol Ther 16, 1427-1436), four modifications were introduced to the amino acid sequence of the measles glycoprotein (Variant H2: knockout (KO), FIG. 12C). A variant was created that is capable to detect CD46, CD150, and CD90, inserting the CD90 scFv at the end of the glycoprotein (Variant H3: WT / CD90, FIG. 12D). Finally, the CD46 / CD150 KO and CD90 recognition was combined to lead to a variant that should only bind and fuse on CD90-expressing cells (Variant H4: KO / CD90, FIG. 12E).

[0384] Due to the inability of the KO variants to be titrated on standard cell lines such as HT 1080 (FIG. 12F), a methodology was developed using hydrodynamics combined with fluorescence to determine the number of total as well as RNA-filled viral particles. Size and number of viral particles were determined using the ZetaView® (Particle Metrix), a Nanoparticle Tracking Analysis (NTA) instrument for measuring particle size, zeta potential, concentration, and fluorescence (FIG. 12G). Regardless of the modifications and addition of the CD90 scFv, all four envelope variants equally produced viral particles (FIG. 12H). Successful loading of viral capsids and loading efficiency was determined staining the particles with SYBR II. The number of RNA- loaded viral particles was highly variable across batches (FIG. 121). However, the loading efficiency of viral particles (frequency of RNA-loaded particles within total particles) was very consistent, averaging at 10% to 20% with no obvious differences due to the modification made (FIG. 12J).

[0385] CD90 scFv on Viral Vectors Bind CD90 Protein. To ensure the ability of CD90-targeted viral vectors to recognize CD90 antigen, a custom assay was designed using NTA. The extracellular domain of CD90 was fused with the fluorochrome mCherry (CD90-mCh) and the fusion protein produced with the Daedalus system (Bandaranayake, et aL, 2011 , Nucleic Acids Res 39, e143). The quality of the CD90-mCh fusion protein was confirmed via its interaction with anti-CD90 Fabs from the hybridoma cell line measured through size exclusion chromatography (SEC) using an A200 column run in PBS buffer. Each protein exhibited a single peak at the expected molecular weight (MW) (FIG. 13A). Fab binding to CD90-mCh was confirmed by the shift of the elution peak to a larger MW species and by SDS-PAGE of the peak showing presence of both proteins.

[0386] Next, the size of the vector variants were measured in the presence and absence of the CD90-mCh protein (FIG. 13B). All four vectors were very similar in size (range 1 10-130 nm) regardless of the modifications made measuring the scattered light (FIG. 13C, scatter, top row). Adding SYBR II to stain only RNA-loaded particles and observing the fluorescence in the 500 nmfilter, particles appeared slightly larger across all four variants and a broader size range due to variability in RNA loading (FIG. 13C, + SYBR II, middle row). Finally, vectors incubated with CD90- mCh protein were measured (FIG. 13C, + SYBR II + CD90-mCh, bottom row). Due to the background noise of unbound protein in the 600 nm filter, measurements were performed in the 500 nm filter focusing on RNA-loaded particles. CD90-decorated viral variants, H3 and H4 with CD90-mCh, formed new peaks at 300 nm, indicating formation of a complex. Only a minor change in size was seen for variant H1 and H2, which lacked the CD90 scFv, due to additional background noise signal from the CD90-mCh protein.

[0387] In summary, an anti-CD90 scFv was successfully engineered onto the measles envelope without impairing the vector production and maintaining viral titers. Most importantly, anti-CD90 scFvs on the viral capsid are fully functional and able to recognize CD90 antigen in the custom hydrodynamic readout confirming CD90 specificity of the scFv.

[0388] Targeted Viral Vectors Transduce CD90-Expressing Jurkat Cells and Human HSCs. The measles envelope has been extensively modified and previous attempts to use similar systems to specifically transduce CD34+cells failed due to the lack of fusion with the target cells (Benedict, et al., 1999, Hum Gene Ther 10, 545-557). Consequently, the CD90-targeted viral vectors were tested for fusion and transduction ability. For the initial tests, CD90-expressing Jurkat cells were used and incubated with all four viral variants. Jurkat cells were kept in culture for 5-7 days to guarantee stable integration of the transgene and wash out transiently delivered fluorescent protein sticking on the viral particles.

[0389] Determined by flow-cytometry, transduction of Jurkat cells were observed with variant H1 (FIG. 14A). Relative to variant H1 , knock-out of CD46 and CD150 recognition reduced the ability of variant H2 to transduce Jurkat cells by 81 .6±13.4% (FIG. 14A). Addition of the CD90 scFv on the H WT virus (variant H3) had no impact on the transduction ability in comparison to the WT variant H1 . Finally, variant H4 (KO / CD90) regained transduction capability over variant H2 (KO), reaching on average 44.8±17.9% transduction efficiency. The reliability was further evaluated of hydrodynamic titers determined with NTA for variant H1 (FIG. 14B). A very high correlation (R2=0.9315) was found in between the number of mRNA-loaded particles and the number of transduced Jurkat cells.

[0390] Next, human GCSF-mobilized CD34+cells were transduced with variant H1 and H4 vectors to analyze the targeting efficiency of the CD34+CD90+subset (FIG. 14C). Due to the internalization / blocking and resulting inability to detect CD90 surface expression after exposure to the variant 4 virus by flow-cytometry, mScarlet expressing CD34+cells from both conditions were FACS-purified and introduced into colony-forming cell (CFG) assays to functionallydetermine differences in the targeting of both viruses. No differences in the total colony-formation were seen comparing the WT and CD90-targeted virus in primary CFCs, whereas more colonies were found in secondary CFCs when the targeted virus was used, indicating selective transduction of more primitive human HSPCs with enhanced proliferation potential with variant 4. Colonies in secondary CFC assays from variant H4-transduced cells contained more total cells as well as a greater number of stably mScarlet-expressing cells, confirming greater proliferation and expansion potential of cells transduced with the CD90-targeted virus.

[0391] These data confirm that CD90-targeted viral vectors are capable of recognizing CD90 antigen on the surface of Jurkat cells as well as human HSCs and effectively fuse with the cells. Furthermore, hydrodynamic titers were highly predictable for the transduction efficiency, confirming accuracy and validity of our new titration methodology for surface-engineered viral vectors.

[0392] VSV-G-Based Viral Vectors Can Be Targeted to CD90. In addition to measles- pseudotyped viral vectors, the VSV-G-based system was evaluated for surface engineering following the previously described strategy by Dobson et al. Dobson, et al., 2022, Nat Methods 19, 449-460 Native targeting of the VSV-G envelope for the LDL-receptor (LDL-R) were further knocked out to introduce specific point mutations as described by Nikolic et al (FIG. 15A) (Nikolic, et al., 2018, Nat Common 9, 1029). The CD90 scFv was introduced onto the capsid using a CD8 hinge and ICAM domain (FIG. 15B). Similar to the measles vectors, four VSV-G variants were generated with V1 (WT) recognizing LDL-R, V2 (KO) removing LDLR recognition, V3 (WT / CD90) recognizing LDL-R and CD90, and V4 (KO / CD90) only binding to CD90 (FIG. 15C).

[0393] The number of total viral as well as RNA-loaded particles was determined using the ZetaView® instrument (FIG. 15D). Regardless of the modifications and addition of the CD90 scFv, all four envelope variants equally produced viral particles. However, the loading efficiency of viral particles (frequency of RNA-loaded particles within total particles) decreased as more engineering was done with the lowest loading efficiency seen in V4.

[0394] To test functionality of the surface-engineered VSV-G vectors, K562 cells were exposed to increasing doses of all four viruses (FIG. 15E). Similar to the measles variants, the V2 (KO) variant failed to fuse with the cells, the V3 (WT / CD90) version demonstrated a gain of function relative to V1 (WT), and V4 (KO / CD90) regained the ability to fuse with cells comparable to V1 (WT). Transduction efficiency of K562 for variants V1 , V3, and V4 was dose-dependent, with no unwanted uptake seen for V2 even in the highest dose.

[0395] On-Target Efficiency of CD90-Targeted Viral Vectors. To test the on- and off-target activity of the CD90-targeted measles- as well as VSV-G-pseudotyped viral vectors, co-cultureexperiments were performed mixing CD90-lacking (off-target) and CD90-expressing (on-target) suspension cell lines. Raji cells, human B lymphoblastoid cells, do present very low levels of CD90 protein on the cell surface (off-target), whereas Jurkat cells, immortalized human T lymphocytes, express CD90 protein at high levels (on-target) (FIG. 16A). GFP-transduced Jurkat cells and GFP- Raji cells were mixed and exposed to measles and VSV-G variants 1 and 4 in a serum-free, transduction enhancer-free, suspension culture system, and mScarlet expression flow- cytometrically determined on day 3-5 post-transduction (FIG. 16B). When the variant H1 / V1 (WT) virus was used, mScarlet expression was seen for Raji as well as Jurkat cells by flow cytometry. However, when the cell mix was exposed to variant H4 / V4 (KO / CD90), a strong preference of mScarlet signal was seen in the CD90+Jurkat cells (on-target) and only minimal transduction in the CD90low / _Raji cells (off-target) (FIG. 16B). To quantify the on-target specificity, the ratio was calculated of mScarlet+GFP- cells within total GFP- Raji to mScarlet+GFP+cells within total GFP+Jurkat cells (FIG. 16C). For both virus types, the CD90-targeted variant 4 showed significantly higher on-target specificity in comparison to the variant 1 (WT) confirmed CD90-mediated target specificity.

[0396] Discussion. Here, the successful design of CD90-targeted measles- and VSV-G-based viral vectors equipping viral capsids was demonstrated with an anti-CD90 scFv. Surface engineering did not impact titers, binding, nor fusion of viral vectors to CD90-expressing cell lines and primary human HSCs. Most importantly, targeted vectors demonstrated enhanced on-target specificity in mixed cultures of cell lines as well as for human HSCs within bulk CD34+HSPCs ex vivo. NTA-based methods were further described to reliably validate surface engineered viral vectors measuring their size, loading-efficiency, and targeting functionality. Hydrodynamic titration of engineered vectors is highly reproducible and predictive of the transduction efficiency, providing a reliable surrogate to functional readouts on cell lines which are not applicable to surface engineered vectors.

[0397] The development of targeted gene therapy agents has been very successful for non- hematological diseases (Kedmi, et al., 2018, Nat Nanotechnol 13, 214-219; De, et al., 2018, Hum Gene Ther Methods 29, 146-155; van Haasteren, et al., 2018, Expert Opin Biol Ther 18, 959-972; Kleinlutzum, et al., 2017, Front Oncol 7, 127; and Wei, et al., 2020, Nat Commun 11 , 3232). Previous work has shown that by conjugating antibodies or scFvs to the surface of nanoparticles (NPs) and viruses, one can reliably target cells of interest (Benedict, et al., 1999, Hum Gene Ther 10, 545-557; Brendel, et al., 2015, Mol Ther 23, 63-70; Frecha, et al., 2008, Blood 1 12, 4843- 4852; Kedmi, et al., 2018, Nat Nanotechnol 13, 214-219; Kleinlutzum, et al., 2017, Front Oncol 7, 127; Niu, et al., 2018, Biomaterials 167, 132-142; Rosenblum, et al., 2020, Sci Adv 6; Zheng, etal., 2013, Journal of Controlled Release 172, 426-435; Kneissl, et al., 2012, PLoS One 7, e46667; Moffett, et al., 2017, Nat Common 8, 389; and Veiga, et al., 2018, Nat Common 9, 4493). Targeted delivery however depends on the optake of the vector after attaching to the cell / antigen, which coold not be achieved with CD34-targeted HSC gene therapy vectors (Benedict, et al., 1999, Hom Gene Ther 10, 545-557). In this example, CD90-targeted viral vectors were designed that soccessfolly fosed and transdoced CD90-expressing cell lines as well as CD34+CD90+HSCs with secondary colony-forming potential. Althoogh the exact mechanism of CD90-mediated cellular optake is not known, cytomegaloviros (CMV) and Homan Immonodeficiency Viros (HIV) ose CD90 as a part of their cellolar entry process (Rodnicka, et al., 2009, J Virol 83, 6234-6246; Li, et al., 2016, J Virol 90, 9766-9781 ; and Li, et al., 2015, PLoS Pathog 1 1 , e1004999). CMV entry was interropted when CD90 was either blocked with an antibody or downregolated osing an siRNA approach (Li, et aL, 2015, PLoS Pathog 11 , e1004999). In addition, liposomes conjogated with anti-CD90 antibodies have been shown to be capable of binding and internalizing through the CD90 antigen on liver cancer cells, demonstrating efficient uptake of CD90-targeted lipid nanoparticles (LNPs) also in vivo (Yang, et aL, 2016, Oncotarget 7, 35894-35916). Disclosed viral vectors have been genetically modified to lose their native binding (LDL-R for VSV-G; CD46 and CD150 for measles), but their ability to fuse with the target cell remains intact. The CD90 scFv is therefore likely acting as a binder, whereas cellular entry remains to be dependent on viral capsid. More in-depth studies can determine whether CD90 is actively involved in the uptake, which pathways are triggered, and whether this system can also be used completely independent from viral fusion proteins. Regardless of the exact pathways involved, CD90 protein was internalized on primary human HSCs without any obvious impact on the cells proliferation and differentiation potential.

[0398] A major bottleneck for the development of surface engineered viral vectors for HSC gene therapy is the inability to quickly and reliably verify the vectors before use. Genetically engineered viral vectors lose their native binding and functional titration methods on HEK293T or HT 1080 cell lines are not possible. In addition, validation of antigen / target recognition is needed to ensure that surface-engineered viral vectors are functional and targeting ligands have been successfully integrated into the capsid. To close this gap, a methodology was developed to titrate and validate viral vectors using nanoparticle tracking analysis (NTA). NTA allows for the efficient and reliable quantification of viral vector concentration, size, and mRNA loading. Most importantly, hydrodynamic viral titers correlated with the transduction efficiency on cell lines for wildtype variants, providing a surrogate method for the traditional titration on cell lines or other qPCR- based strategies. NTA was further used to demonstrate successful antigen recognition of targetedvectors. Custom-designed fluorochrome-conjugated CD90 protein was bound by the scFv- decorated VSV-G- and measles-pseudotyped viral vectors, resulting in a size increase measured by NTA and confirming fully functional antigen recognition when the targeting ligand was presented on the virus. The measurement of these parameters is nearly instantaneous and provides a new alternative to traditional titration methods of wildtype, surface-engineered viral vectors, as well as virus-like particles. As viral vectors often undergo freezing and thawing, quality control and quantification of viral particles can even be performed right before applying them to the cells or injecting them in vivo, providing an opportunity to adjust and carefully control viral titers in real-time.

[0399] A major advantage of the disclosed CD90-targeted viral vectors is the ability to incorporate them into already existing ex vivo gene therapy pipelines focusing on the genetic modification of CD34+HSPCs. Further purification of the CD90+subset for direct targeting and to enhance the transduction efficiency (Radtke, et al., 2020, Mol Ther Methods Clin Dev 18, 679-691) would potentially no longer be needed to transduce long-term engrafting HSCs and likely require even lower viral titers reducing the overall costs. Even the purification of CD34+cells from the leukapheresis products may no longer be necessary, providing a vector to realize closed-system vein-to-vein applications with the transduction of cells in bags and immediate infusion of the transduced cells back into the patient. Finally, direct intravenous or intraosseous injection of the HSC-targeted viral vectors would be warranted to further simplify HSC gene therapy, providing full portability, and consequently better accessibility.

[0400] The disclosed CD90-targeted vectors improve already existing ex vivo HSC gene therapies as well as overcome current hurdles to apply gene therapy in vivo. The development of targeted viral vectors increases the on-target efficiency, safety, and accessibility of HSC gene transfer for a variety of different diseases and will be a step in democratizing access to gene therapies.

[0401] Materials and Methods. mRNA extraction, cDNA synthesis, and heavy / light chain sequencing of the hybridoma cells. mRNA was extracted from the hybridoma cells using an RNAeasy kit (Qiagen USA, MD). Extracted mRNA was run on TapeStation (Agilent Technologies, Santa Clara, CA) for integrity. cDNA was generated from mRNA using an applied biosystem high capacity RNA-to-cDNA kit (Thermo Fisher Scientific, Waltham, MA). Primers for the variant regions of the antibody were acquired from the literature (Sun, et aL, 2012, World J Microbiol Biotechnol 28, 381-386) and ordered using IDT. CD90 ab internal reverse primer (TTC AGT CAC CAT GCT GTT GAC) (SEQ ID NO: 187) forward primer (CCA GCA GAA GCC AGG ATC) (SEQ ID NO: 188). Antibody variable regions were sequenced using on an Applied Biosystems 3730x1DNA Analyzers (Thermo Fisher Scientific, Waltham, MA). Raw sequencing data was analyzed using International ImMunoGeneTics V-QUEry and STandardization program to align and annotate the antibody sequence.

[0402] Modeling and visualization of proteins. The antibody structure was predicted with SabPred (Dunbar, et al., 2016, Nucleic Acids Res 44, W474-W478). Antibody-protein docking was predicted using Swissdock (Grosdidier, et al., 2011 , Nucleic Acids Res 39, W270-W277). Visualizations were generated with Pymol (The PyMOL Molecular Graphics System, Version 2.0 Schrodinger, LLC).

[0403] Cloning of virus plasmids. Measles plasmid were provided by Els Verhoeyen (Levy, et al., 2017, Blood Adv 1 , 2088-2104). Cloning was performed using restriction enzymes from New England Biolabs ([NEB] Ipswich, MA) and Gibson Assembly Master Mix (NEB). DNA tiles containing the desired sequences to be cloned into the plasmids were acquired from Codex DNA (Codex DNA, San Diego, CA). Cloned products were confirmed via sequencing on an Applied Biosystems 3730x1 DNA Analyzers (Thermo Fisher Scientific, Waltham, MA). The relevant sequences are in FIGS. 12A, 12C-12E, 15A, and 15B.

[0404] VSV-G Knockout (KO) and targeted binder plasmids were provided by the Baker Laboratory (Institute of Protein Design, University of Washington, Seattle, WA). Cloning of CD90 scFv into the targeted binder plasmid was performed by Eco721 -FastDigest (Thermo Fisher Scientific, cat # FD0364) and Eco1471 -FastDigest (Thermo Fisher Scientific, cat # FD0424) restriction enzyme digest of targeted binder plasmid followed by Gibson Assembly (NEB, cat # E261 1 S) with synthesized Gibson block sequence (Integrated Data Technologies, Coralville, IA) containing the CD90 scfv sequence (FIGS. 12D, 12E, and 16A-16C). Successful cloning was confirmed by EcoRI (NEB, cat # R3101 S) restriction enzyme digest, followed by sequencing on Applied Biosystems 373xl DNA Analyzers (Thermo Fisher Scientific, Waltham, MA).

[0405] Virus production. HEK293T cells were expanded on gelatin coated plates and transfected with plasmids mixed with polyethylenimine (PEI) (Polysciences, Warrington, PA) at a pg plasmid to ug PEI ratio. Measles virus plasmid ratios were 27 pg pLenti-EF1 a-mScarlet-l-WPRE transfer plasmid, 17 pg 2ndgeneration Gag / Pol Helper plasmid pCMVdelta8.74, 10 pg measles hemagglutinin Delta 24H, and 10 pg measles fusion Delta F per 15 cm cell plate. VSV-G virus plasmid ratios were 27 pg pLenti-EF1 a-mScarlet-l-WPRE transfer, 17 pg 2ndgeneration Gag / Pol Helper plasmid pCMVdelta8.74, 10 pg VSV-G fusion, and 10 pg of relevant targeting ligand plasmid per 15 cm cell plate. Twenty-four (24) hours after transfection, the media was changed to LV-MAX (Thermo Fisher Scientific) media with 20 mM HEPES (Thermo Fisher Scientific) and 1 X L-glutamine (Thermo Fisher Scientific). Virus production media was allowed to sit for a further24 hours, then the supernatant was collected, spun down at 800 g to remov...

Claims

CLAIMSWhat is claimed is:1 . An antibody or binding fragment thereof that binds CD90 comprising a variable heavy chain comprising a complementarity determining region (CDR) heavy (H) 1 , a CDRH2, and a CDRH3; and a variable light chain comprising a CDR light (L)1 , a CDRL2, and CDRL3, wherein: the CDRH1 comprises a sequence as set forth in SEQ ID NO: 41 , the CDRH2 comprises a sequence as set forth in SEQ ID NO: 42, the CDRH3 comprises a sequence as set forth in SEQ ID NO: 43, CDRL1 comprises a sequence as set forth in SEQ ID NO: 53, the CDRL2 comprises the sequence YTS, and the CDRL3 comprises a sequence as set forth in SEQ ID NO: 54, according to IMGT; the CDRH1 comprises a sequence as set forth in SEQ ID NO: 44, the CDRH2 comprises a sequence as set forth in SEQ ID NO: 45, the CDRH3 comprises a sequence as set forth in SEQ ID NO: 46, the CDRL1 comprises a sequence as set forth in SEQ ID NO: 55, the CDRL2 comprises the sequence as set forth in SEQ ID NO: 195, and the CDRL3 comprises a sequence as set forth in SEQ ID NO: 54, according to Kabat; the CDRH1 comprises a sequence as set forth in SEQ ID NO: 47, the CDRH2 comprises a sequence as set forth in SEQ ID NO: 48, the CDRH3 comprises a sequence as set forth in SEQ ID NO: 46, the CDRL1 comprises a sequence as set forth in SEQ ID NO: 55, the CDRL2 comprises the sequence as set forth in SEQ ID NO: 195, and the CDRL3 comprises a sequence as set forth in SEQ ID NO: 54, according to Chothia; the CDRH1 comprises a sequence as set forth in SEQ ID NO: 49, the CDRH2 comprises a sequence as set forth in SEQ ID NO: 50, the CDRH3 comprises a sequence as set forth in SEQ ID NO: 43, the CDRL1 comprises a sequence as set forth in SEQ ID NO: 55, the CDRL2 comprises the sequence as set forth in SEQ ID NO: 195, and the CDRL3 comprises a sequence as set forth in SEQ ID NO: 54, according to North; or the CDRH1 comprises a sequence as set forth in SEQ ID NO: 56, the CDRH2 comprises a sequence as set forth in SEQ ID NO: 57, the CDRH3 comprises a sequence as set forth in SEQ ID NO: 58, the CDRL1 comprises a sequence as set forth in SEQ ID NO: 67, the CDRL2 comprises the sequence as set forth in SEQ ID NO: 68, and the CDRL3 comprises a sequence as set forth in SEQ ID NO: 69, according to Contact.

2. The antibody or binding fragment thereof of claim 1 , wherein the variable heavy chain has at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 40 and the variable light chain has at least 90% sequence identity to the sequence as set forth in SEQ ID NO:

3. The antibody or binding fragment thereof of claim 1 , wherein the variable heavy chain has the sequence as set forth in SEQ ID NO: 40 and the variable light chain has the sequence as set forth in SEQ ID NO: 193.

4. The antibody or binding fragment thereof of claim 1 , wherein the variable heavy chain is encoded by a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 22 and the variable light chain is encoded by a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 190.

5. The antibody or binding fragment thereof of claim 1 , wherein the variable heavy chain is encoded by a sequence as set forth in SEQ ID NO: 20 and the variable light chain is encoded by a sequence as set forth in SEQ ID NO: 190.

6. The antibody or binding fragment thereof of claim 1 , wherein the antibody or binding fragment thereof further comprises a human constant region.

7. The antibody or binding fragment thereof of claim 6, wherein the human constant region comprises a human heavy chain constant region and / or a human light chain constant region.

8. The antibody or binding fragment thereof of claim 7, wherein the human heavy chain constant region comprises an IgG, IgM, IgD, IgA, or IgE heavy chain constant region.

9. The antibody or binding fragment thereof of claim 7, wherein the human heavy chain constant region comprises an IgG heavy chain constant region.

10. The antibody or binding fragment thereof of claim 9, wherein the IgG heavy chain constant region comprises an IgG 1 heavy chain constant region, an lgG2 heavy chain constant region, an lgG3 heavy chain constant region, and an lgG4 heavy chain constant region.11 . The antibody or binding fragment thereof of claim 9, wherein the IgG heavy chain constant region comprises an lgG1 heavy chain constant region.

12. The antibody or binding fragment thereof of claim 9, wherein the IgG heavy chain constant region comprises an lgG4 heavy chain constant region with an S228P mutation.

13. The antibody or binding fragment thereof of claim 9, wherein the IgG heavy chain constant region comprises the sequence as set forth in SEQ ID NOs: 15-19.

14. The antibody or binding fragment thereof of claim 7, wherein the human light chain constant region comprises a human IgK light chain constant region or a human IgA light chain constant region.

15. The antibody or binding fragment thereof of claim 14, wherein the human IgK light chain constant region comprises the sequence as set forth in SEQ ID NO: 13.

16. The antibody or binding fragment thereof of claim 14, wherein the human IgA light chainconstant region comprises the sequence as set forth in SEQ ID NO: 14.

17. The antibody or binding fragment thereof of claim 1 , wherein the antibody further comprises an Fc modification.

18. The antibody or binding fragment thereof of claim 1 , wherein the antibody or binding fragment thereof is humanized.

19. The antibody or binding fragment thereof of claim 1 , wherein the antibody or binding fragment thereof is PEGylated.

20. The antibody or binding fragment thereof of claim 1 , wherein the antibody or binding fragment thereof is conjugated to polyglutamic acid (PGA).

21. The antibody or binding fragment thereof of claim 20, wherein the PGA shields a positive charge of a polymeric nanoparticle.

22. The antibody or binding fragment thereof of claim 1 , comprising M428L / N434S mutations.

23. The antibody or binding fragment thereof of claim 1 , comprising G236A / S239D / A330L / I332E mutations.

24. The antibody or binding fragment thereof of claim 1 , wherein the antibody or binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises the sequence as set forth in SEQ ID NO: 3 and the light chain comprises the sequence as set forth in SEQ ID NO: 7, or the heavy chain comprises a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 3 and the light chain comprises a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 7.

25. The antibody or binding fragment thereof of claim 1 , wherein the antibody or binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises the sequence as set forth in SEQ ID NO: 5 and the light chain comprises the sequence as set forth in SEQ ID NO: 7, or the heavy chain comprises a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 5 and the light chain comprises a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 7.

26. The antibody or binding fragment thereof of claim 1 , wherein the antibody or binding fragment thereof comprises a signal peptide.

27. The antibody or binding fragment thereof of claim 26, wherein the signal peptide comprises a sequence selected from SEQ ID NO: 191 , SEQ ID NO: 192, SEQ ID NO: 8, or SEQ ID NO: 9.

28. A multi-domain binding molecule comprising at least two binding domains, wherein at leastone binding domain of the at least two binding domains comprises the antibody or binding fragment thereof of claim 1 .

29. The multi-domain binding molecule of claim 28, wherein the multi-domain binding molecule comprises a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

30. The multi-domain binding molecule of claim 28, wherein the multi-domain binding molecule comprises an immune cell engaging molecule.

31. The multi-domain binding molecule of claim 30, wherein the immune cell engaging molecule activates a T cell, natural killer (NK) cell, B cell, or macrophage.

32. The multi-domain binding molecule of claim 31 , wherein the T cell is a CD3 T cell, a CD4 T cell, a CD8 T cell, a central memory T cell, an effector memory T cell, and / or a naive T cell.

33. The multi-domain binding molecule of claim 30, wherein a binding domain of the immune cell engaging molecule binds CD3, CD28, CD8, NKG2D, CD8, CD16, KIR2DL4, KIR2DS1 , KIR2DS2, KIR3DS1 , NKG2C, NKG2E, NKG2D, NKp30, NKp44, NKp46, NKp80, DNAM-1 , CD11b, CD11c, CD64, CD68, CD119, CD163, CD206, CD209, F4 / 80, IFGR2, Toll-like receptors 1-9, IL-4Ra, or MARCO.

34. The multi-domain binding molecule of claim 30, wherein a binding domain of the immune cell engaging molecule binds CD3, CD28, 4-1 BB, or CD27.

35. The multi-domain binding molecule of claim 30, wherein a binding domain of the immune cell engaging molecule binds CD8, CD16, NKG2A, NKG2D, KIR2DL1 , KIR2DL2 / 3, KIR2DL4, KIR3DL1 , NKp44, or KLRG1.

36. The multi-domain binding molecule of claim 28, wherein the at least two binding domains comprise at least two copies of the antibody or binding fragment thereof of claim 1 .

37. The multi-domain binding molecule of claim 36, wherein the at least two copies are joined by a linker.

38. The multi-domain binding molecule of claim 37, wherein the linker is a Gly-Ser linker.

39. The multi-domain binding molecule of claim 38, wherein the Gly-Ser linker is (GlyxSery)nwherein x and y are independently an integer from 0 to 10 provided that x and y are not both 0 and wherein n is an integer of 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10.

40. The multi-domain binding molecule of claim 28, comprising 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the antibody or binding fragment thereof of claim 1 .41 . The multi-domain binding molecule of claim 28, wherein the multi-domain binding molecule is a dimer, trimer, tetramer, pentamer, hexamer, or heptamer.

42. The multi-domain binding molecule of claim 36, wherein the at least two copies are linked to an Fc region of an antibody.

43. The multi-domain binding molecule of claim 42, wherein the Fc region is an IgA Fc region or an IgM Fc region.

44. The multi-domain binding molecule of claim 42, wherein the Fc region is an IgA Fc region having the sequence as set forth in SEQ ID NOs: 198 and 199.

45. The multi-domain binding molecule of claim 42, wherein the Fc region is an IgM Fc region having the sequence as set forth in SEQ ID NOs: 202-211.

46. The multi-domain binding molecule of claim 42, wherein the Fc region comprises a multimerizing fragment of an IgA Fc region or a multimerizing fragment of an IgM Fc region.

47. The multi-domain binding molecule of claim 46, wherein the multimerizing fragment of the IgA Fc region comprises an IgA tailpiece.

48. The multi-domain binding molecule of claim 47, wherein the IgA tailpiece has a sequence of residues 331 -352 as set forth in SEQ ID NO: 198 or the sequence of residues 318-340 as set forth in SEQ ID NO: 199.

49. The multi-domain binding molecule of claim 46, wherein the multimerizing fragment of the IgA Fc region comprises an IgA CA3 domain and an IgA tailpiece.

50. The multi-domain binding molecule of claim 46, wherein the multimerizing fragment of the IgA Fc region comprises an IgA CA2 domain, an IgA CA3 domain, and an IgA tailpiece.51 . The multi-domain binding molecule of claim 46, wherein the multimerizing fragment of the IgA Fc region comprises an IgA CA1 domain, an IgA CA2 domain, an IgA CA3 domain, and an IgA tailpiece.

52. The multi-domain binding molecule of claim 46, wherein the multimerizing fragment of the IgM Fc region comprises an IgM tailpiece.

53. The multi-domain binding molecule of claim 46, wherein the multimerizing fragment of the IgM Fc region comprises a Cp4 domain and an IgM tailpiece.

54. The multi-domain binding molecule of claim 46, wherein the multimerizing fragment of the IgM Fc region comprises a Cp3 domain, a Cp4 domain, and an IgM tailpiece.

55. The multi-domain binding molecule of claim 46, wherein the multimerizing fragment of the IgM Fc region comprises a Cp2 domain, a Cp3 domain, a Cp4 domain, and an IgM tailpiece.

56. The multi-domain binding molecule of claim 46, wherein the multimerizing fragment of the IgM Fc region comprises a Cp1 domain, a Cp2 domain, a Cp3 domain, a Cp4 domain, and an IgM tailpiece.

57. The multi-domain binding molecule of claim 46, wherein the multimerizing fragment of the IgM Fc region has the sequence as set forth in SEQ ID NO: 204.

58. A single-chain variable fragment (scFv) comprising an antibody binding fragment of claim 1 .

59. The scFv of claim 58, further comprising a linker.

60. The scFv of claim 59, wherein the linker comprises a sequence as set forth in SEQ ID NO: 66.61 . The scFv of claim 59, wherein the linker comprises a Gly-Ser linker.

62. The scFv of claim 61 , wherein the Gly-Ser linker comprises a sequence as set forth in SEQ ID NO: 64, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81 , SEQ ID NO:

82. SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, or SEQ ID NO: 91 .

63. The scFv of claim 61 , wherein the Gly-Ser linker comprises a sequence as set forth in SEQ ID NO: 66.

64. The scFv of claim 58, having the sequence as set forth in SEQ ID NO: 194.

65. A conjugate comprising the antibody or binding fragment thereof of claim 1 linked to a toxin, a drug, a detectable label, a radioisotope, a particle, or a bead.

66. The conjugate of claim 65, wherein the toxin comprises a holotoxin or a hemitoxin.

67. The conjugate of claim 65, wherein the drug comprises actinomycin D, anthracycline, auristatin, calicheamicin, camptothecin, CC1065, colchicin, cytochalasin B, daunorubicin, 1 - dehydrotestosterone, dihydroxy anthracinedione, dolastatin, doxorubicin, duocarmycin, elinafide, emetine, ethidium bromide, etoposide, gramicidin D, glucocorticoids, lidocaine, maytansinoid, mithramycin, mitomycin, mitoxantrone, nemorubicin, PNU-159682, procaine, propranolol, puromycin, pyrrolobenzodiazepine, taxane, taxol, tenoposide, tetracaine, trichothecene, vinblastine, vinca alkaloid, or vincristine.

68. The conjugate of claim 65, wherein the detectable label comprises a fluorescent label, an enzyme label, chemiluminescent label, a spectral colorimetric label, or an affinity tag.

69. The conjugate of claim 68, wherein the fluorescent label comprises blue fluorescent protein, cyan fluorescent protein, green fluorescent proteins, luciferase, orange fluorescent protein, red fluorescent protein, far red fluorescent protein, or yellow fluorescent protein.

70. The conjugate of claim 68, wherein the enzyme label comprises horseradish peroxidase, hydrolase, or alkaline phosphatase.71 . The conjugate of claim 68, wherein the chemiluminescent label comprises lucigenin, luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, or oxalate ester.

72. The conjugate of claim 68, wherein the spectral colorimetric label comprises colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, and latex) beads.

73. The conjugate of claim 68, wherein the affinity tag comprises a sequence as set forth in SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO:28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 1 1 , SEQ ID NO: 167, SEQ ID NO: 168, SEO ID NO: 169, or SEQ ID NO: 170.

74. The conjugate of claim 65, wherein the radioisotope comprises actinium-225, iodine-131 , arsenic-21 1 , iodine-131 , indium-1 1 1 , yttrium-90, lutetium-177, astatine-21 1 , bismuth-212, or bismuth-213.

75. The conjugate of claim 65, wherein the radioisotope comprises228Ac,111Ag,124Am,74As,211At, 209At,194Au,128Ba,7Be,206Bi,245Bk,246Bk,76Br,11C,14C,47Ca,254Cf,242Cm,51Cr,67Cu,153Dy, 157Dy,159Dy,165Dy,166Dy,171Er,250Es,254Es,147Eu,157Eu,52Fe,59Fe,251Fm,252Fm,253Fm,66Ga, 72Ga,146Gd,153Gd,68Ge,3H,170Hf,171Hf,193Hg,193mHg,160mHo,130l,131l,135l,114mln,185lr,42K, 43K,76Kr,79Kr,81mKr,132La,262Lr,169Lu,174mLu,176mLu,257Md,260Md,28Mg,52Mn, "Mo,24Na, 95Nb,138Nd,57Ni,66Ni,234Np,15O,1820s,189mOs,191Os,32P,201Pb,101Pd,143Pr,191Pt,243Pu, 225Ra,81Rb,188Re,105Rh,211Rn,103Ru,35S,44Sc,72Se,153Sm,125Sn,91Sr,173Ta,154Tb,127Te, 234Th,45Ti,166Tm,230U,237U,240U,48V,178W,181W,188W,125Xe,127Xe,133Xe,133mXe,135Xe, 85mY,86Y,90Y,93Y,169Yb,175Yb,65Zn,71mZn,86Zr,95Zr, or97Zr.

76. The conjugate of claim 65, wherein the radioisotope does not emit daughter radionuclides.

77. The conjugate of claim 65, wherein the particle comprises a metal nanoparticle, liposome, a polymer nanoparticle, or a lipid nanoparticle.

78. The conjugate of claim 65, wherein the bead comprises a magnetic or a paramagnetic bead.

79. A recombinant protein comprising the antibody or binding fragment thereof of claim 1 linked to a viral structural protein.

80. The recombinant protein of claim 79, wherein the viral structural protein comprises measles virus hemagglutinin (H), measles virus fusion (F), vesicular stomatis virus G (VSV-G), influenza hemagglutinin, ebolavirus glycoprotein (GP)1 or GP2, coronavirus spike, lassa virus glycoprotein precursor (GP) or spike, nipah virus NiV-G or NiV-F, rabies virus glycoprotein (G), respiratory syncytial virus fusion protein (F) or attachment protein (G), human immunodeficiency virus (HIV) gp41 , HIV gp120, paramyxovirus F protein, filovirus GP2 protein, flavivirus E protein, alphavirus E1 protein, herpes simplex virus gH or gB protein, or arenavirus G2 protein.81 . The recombinant protein of claim 79, wherein the viral structural protein comprises measles virus hemagglutinin.

82. The recombinant protein of claim 79, wherein the viral structural protein comprises VSV-G.

83. The recombinant protein of claim 81 , wherein the measles virus hemagglutinin comprises a cytoplasmic tail truncation of up to 30 residues.

84. The recombinant protein of claim 81 , wherein the measles virus hemagglutinin comprisesmutations Y481 A, R533A, S548L, and F549S.

85. The recombinant protein of claim 79, wherein the antibody or binding fragment thereof comprises an scFv.

86. A pseudotyped viral vector comprising an antibody or binding fragment thereof of claim 1 linked to a viral structural protein.

87. The pseudotyped viral vector of claim 86, wherein the viral structural protein comprises measles virus hemagglutinin (H), measles virus fusion (F), vesicular stomatis virus G (VSV- G), influenza hemagglutinin, ebolavirus glycoprotein (GP)1 or GP2, coronavirus spike, lassa virus glycoprotein precursor (GP) or spike, nipah virus NiV-G or NiV-F, rabies virus glycoprotein (G), respiratory syncytial virus fusion protein (F) or attachment protein (G), human immunodeficiency virus (HIV) gp41 , HIV gp120, paramyxovirus F protein, filovirus GP2 protein, flavivirus E protein, alphavirus E1 protein, herpes simplex virus gH or gB protein, or arenavirus G2 protein.

88. The pseudotyped viral vector of claim 86, wherein the viral structural protein comprises a measles virus hemagglutinin.

89. The pseudotyped viral vector of claim 86, wherein the viral structural protein comprises VSV- G.

90. The pseudotyped viral vector of claim 86, wherein the pseudotyped viral vector is a retroviral vector, an adenoviral vector, or an adeno-associated viral (AAV) vector.91 . The pseudotyped viral vector of claim 90, wherein the retroviral vector comprises a lentiviral vector or a foamy virus vector.

92. The pseudotyped viral vector of claim 86, wherein the antibody or binding fragment thereof is linked to the viral structural protein with a linker.

93. The pseudotyped viral vector of claim 92, wherein the linker is a Gly-Ser linker.

94. A pseudotyped viral vector comprising a viral vector expressing the recombinant protein of claim 79.

95. The pseudotyped viral vector of claim 94, wherein the viral vector comprises a retroviral vector, an adenoviral vector, or adeno-associated viral (AAV) vector.

96. A recombinant receptor that, when expressed by a cell, comprises an extracellular component comprising the scFv of claim 58.

97. The recombinant receptor of claim 96, wherein the recombinant receptor comprises a chimeric antigen receptor (CAR) or engineered T cell receptor (eTCR).

98. The recombinant receptor of claim 97, wherein the CAR comprises an intracellular component linked to the extracellular component by a transmembrane domain.

99. The recombinant receptor of claim 98, wherein the intracellular component comprises an effector domain comprising: 4-1 BB (CD137), CD3Y, CD35, CD3c, CD3 , CD27, CD28, DAP10, ICOS, LAG3, NKG2D, NOTCH1 , 0X40, ROR2, SLAMF1 , TCRa, TCR , TRIM, Wnt, Zap70, or a combination thereof.

100. The recombinant receptor of claim 98, wherein the transmembrane domain comprises a transmembrane region of: an a, p or chain of a T-cell receptor; CD28; CD27; CD3; CD45; CD4; CD5; CD8; CD9; CD16; CD22; CD33; CD37; CD64; CD80; CD86; CD134; CD137; CD154; or a combination thereof.

101. The recombinant receptor of claim 97, wherein the CAR further comprises a spacer region.

102. The recombinant receptor of claim 97, wherein the eTCR comprises a constant alpha domain (Ca) and a constant beta domain (Cp).

103. The recombinant receptor of claim 102, wherein the scFv of claim 58 is linked to the Cadomain and / or the Cp domain.

104. A nucleic acid encoding the antibody or binding fragment thereof of claim 1 .

105. The nucleic acid of claim 104, wherein the nucleic acid is codon-optimized.

106. The nucleic acid of claim 104, wherein nucleic acid comprises a variable heavy chain coding sequence and a variable light chain coding sequence, wherein the variable heavy chain coding sequence comprises the sequence as set forth in SEQ ID NO: 22 and the variable light chain coding sequence comprises the sequence as set forth in SEQ ID NO: 190, or the variable heavy chain coding sequence comprises a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 22 and the variable light chain coding sequence comprises a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 190.

107. The nucleic acid of claim 104, wherein the nucleic acid comprises a heavy chain coding sequence and a light chain coding sequence, wherein the heavy chain coding sequence comprises the sequence as set forth in SEQ ID NO: 2 and the light chain coding sequence comprises the sequence as set forth in SEQ ID NO: 6, or the heavy chain coding sequence comprises a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 2 and the light chain coding sequence comprises a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 6.

108. The nucleic acid of claim 104, wherein the nucleic acid comprises a heavy chain coding sequence and a light chain coding sequence, wherein the heavy chain coding sequence comprises the sequence as set forth in SEQ ID NO: 4 and the light chain coding sequence comprises the sequence as set forth in SEQ ID NO: 6, or the heavy chain coding sequencecomprises a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 4 and the light chain coding sequence comprises a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 6.

109. A genetic construct comprising the nucleic acid of claim 104 operably linked to a sequence encoding a viral structural protein.

110. The genetic construct of claim 109, wherein the viral structural protein comprises measles virus hemagglutinin (H), measles virus fusion (F), vesicular stomatis virus G (VSV-G), influenza hemagglutinin, ebolavirus glycoprotein (GP)1 or GP2, coronavirus spike, lassa virus glycoprotein precursor (GP) or spike, nipah virus NiV-G or NiV-F, rabies virus glycoprotein (G), respiratory syncytial virus fusion protein (F) or attachment protein (G), human immunodeficiency virus (HIV) gp41 , HIV gp120, paramyxovirus F protein, filovirus GP2 protein, flavivirus E protein, alphavirus E1 protein, herpes simplex virus gH or gB protein, or arenavirus G2 protein..

111. The genetic construct of claim 109, wherein the viral structural protein comprises measles virus hemagglutinin.

112. The genetic construct of claim 109, wherein the viral structural protein comprises VSV-G.

113. The genetic construct of claim 109, wherein the nucleic acid is operably linked to the sequence encoding the viral structural protein by a sequence encoding linker.

114. The genetic construct of claim 1 13, wherein the sequence encoding the linker comprises the sequence as set forth in SEQ ID NO: 78, SEQ ID NO: 164, or SEQ ID NO: 165 or a sequence having at least 95% sequence identity to a sequence as set forth in SEQ ID NO: 78, SEQ ID NO: 164, or SEQ ID NO: 165.

115. The genetic construct of claim 1 13, wherein the linker comprises the sequence as set forth in SEQ ID NO: 64 or SEQ ID NO: 66 or a sequence having at least 95% sequence identity to a sequence as set forth in SEQ ID NO: 64 or SEQ ID NO: 66.

116. A cell genetically modified to express the antibody or binding fragment thereof of claim 1 , the pseudotyped viral vector of claims 86 or 94, or the recombinant receptor of claim 96.

117. The cell of claim 116, wherein the cell is an immune cell.

118. The cell of claim 1 17, wherein the immune cell is a T cell, B cell, natural killer cell, or macrophage.

119. A composition comprising the antibody or binding fragment thereof of claim 1 , the multidomain binding molecule of claim 28, the scFv of claim 58, the conjugate of claim 65, the pseudotyped viral vector of claims 86 or 94, the nucleic acid of claim 104, or the genetic construct of claim 109; and a pharmaceutically-acceptable carrier.

120. A formulation comprising the cell of claim 1 16 and a pharmaceutically-acceptable carrier.

121. A kit comprising the antibody or binding fragment thereof of claim 1 , the multi-domain binding molecule of claim 28, the scFv of claim 58, the conjugate of claim 65, the pseudotyped viral vector of claims 86 or 94, the nucleic acid of claim 104, the genetic construct of claim 109, or the cell of claim 116.

122. A method of delivering genetic engineering components to a CD90+ cell comprising administering the pseudotyped viral vector of claims 86 or 94 or the conjugate of claim 65.

123. The method of claim 122, wherein the conjugate comprises the antibody or binding fragment thereof linked to a particle.

124. The method of claim 122, wherein the genetic engineering components provide a therapeutic coding sequence to the CD90+ cell.

125. The method of claim 124, wherein the therapeutic coding sequence inserts or alters a coding sequence selected from ABCA3, ABCD1 , Akt, amyloid beta precursor protein (APP), angiopoietin 1 (Ang1 ), angiotensin-converting enzyme 2 (ACE2), antibodies to CD4, antibodies to CD5, antibodies to CD7, antibodies to CD52, etc., antibodies to IL1 , antibodies to IL2, antibodies to IL6, antibodies to TCR specifically present on autoreactive T cells, antibodies to TNF, arylsulfatase A, Bcl-2, brain derived neurotrophic factor (BDNF), cerebral dopamine neurotrophic factor (CDNF), C1q / tumor necrosis factor- related protein-3 (CTRP3), C9ORF72, C-C Motif Chemokine Receptor 1 (CCR1 ), CCR2, chemokine receptor 2 (CXCR2), CXCR4, CXCR7, ciliary neurotrophic factor (CNTF), CLN3, connexin 43 (Cx43), Csx / Nkx 2.5, CTLA, cystic fibrosis transmembrane conductance regulator (CFTR), Cytochrome b5 reductase 3 (CYB5R3), DRB1 *1501 / DQB1 *0602, Dyskerin Pseudouridine Synthase (DKC1 ), dystrophin, E2F4, endothelial nitric oxide synthase (eNOS), erythropoietin (EPO), extracellular regulating kinase 1 / 2, F8 (coagulation factor VIII), F9 (coagulation factor IX), Fanconi anaemia complementation group (FANC) family genes, Fas L, fibroblast growth factor-2 (FGF-2), fibroblast growth factor 4 (FGF4), Follistatin-like 1 , forkhead box protein (Foxa2), fused in sarcoma (FUS), GATA1 , GATA-4, glial cell line-derived neurotrophic factor (GDNF), globin family genes, granulocyte colony-stimulating factor (G-CSF), HBB, heme oxygenase-1 (HO-1 ), hepatocyte growth factor (HGF), hepatocyte nuclear factor 4a (HNF4a), hypoxia-inducible factor 1 a (HIF-1 a), insulin-like growth factor (IGF)-1 , Intercellular Adhesion Molecule (ICAM)-1 , interferon -beta (IFN-p), integrin a4, interleukin-1 receptor antagonist (IL- 1 Ra), interleukin 4 (IL4), IL10, IL12, IL13, IL-33, Islet-1 , Klotho gene, let-7d, leucine-rich repeat kinase 2 (LRRK2), lipocalin 2 (Len2), Mashl , microRNA-1 (miR-1 ), miR-16-5p, miR-21 , miR- 25, miR-30b-3p, miR-34, miR-101 -3p, miR-124, mlR-126, miR-133, miR-133b, mlR-181a,miR-199a, miR-199a-3p, miR-211 , miR-705, nerve growth factor (NGF), neuregulin 4 (Nrg4), neurotrophin-3 (NT3), NLX2.1 , Notch ligand Delta-like-4, Notchl receptor, nuclear factor erythroid-derived 2-like 2 (Nrf2), orphan receptor tyrosine kinase 2 (ROR2), P53, p130, Parkinson disease 2 (PARK2), PARK7, PARKIN, phox, platelet-derived growth factor (PDGF), presenilin 1 (PSEN1 ), PSEN2, protein tyrosine phosphatase, non-receptor type 22 (PTPN22), PTEN-induced kinase 1 (PINK1 ), pyruvate kinase, ribosomal protein genes, secreted Klotho protein (SKL), sirtuin 1 , soluble CD40, soluble interleukin 1 receptor II (sILI RII), sILI RI, soluble TNF alpha receptor I (sTNFRI), sTNFRII, somatostatin 2 (sST2), sonic hedgehog (Shh), superoxide dismutase 1 (SOD1 ), surfactant protein B (SFTPB), SFTPC, synuclein alpha (SNCA), TAR DNA-binding protein 43 (TDP43), telomerase reverse transcriptase (TERT), telomerase RNA component (TERO), TERF interacting nuclear factor 2 (TINF2), TNF-related apoptosis-inducing ligand (TRAIL), transforming growth factor (31 (TGF- 1), type 2 angiotensin II receptor (AT2R), tyrosine kinase receptor type 3 (TrkC), ubiquilin 2, vascular endothelial growth factor (VEGF), WASP Actin nucleation promoting factor (WAS), or Wnt / p- catenin.

126. The method of claim 122, wherein the CD90+ cells are hematopoietic stem cells or mesenchymal stem / stromal cells.

127. The method of claim 122, wherein the genetic engineering components comprise coding sequence, guide RNA, homology arms, a base editor, a clustered regularly interspaced short palindromic repeats (CRISPR) nuclease, a zinc finger (ZFN), a tai effector nuclease (TALEN), a meganucleases or a meganuclease-TALEN fusion (MegaTALE).

128. The method of claim 122, wherein the administering is in vivo or ex vivo.

129. The method of claim 122, wherein the administering comprises intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, intrathecal, intrauterine, intraplacental, intramuscular, intravesicular, and / or subcutaneous administration.

130. The method of claim 122, wherein the administering comprises pipetting.131 . The method of claim 122, further comprising isolating CD90+ cells.

132. A method of isolating CD90+ cells from a sample comprising contacting the sample with the conjugate of claim 65, thereby creating a conjugate-target complex; and separating the conjugate-target complex from the sample, thereby isolating CD90+ cells.

133. The method of claim 132, wherein the sample comprises bone marrow, peripheral blood, adipose tissue, amniotic fluid, amniotic membrane, dental tissue, placental and fetal membrane, sub-amniotic umbilical cord lining membrane, salivary gland, endometrium, limbbud, menstrual blood, skin, synovial fluid, or Wharton's jelly.

134. The method of claim 132, wherein the sample comprises hematopoietic stem cells or mesenchymal stem / stromal cells.

135. The method of claim 132, wherein the separating comprises affinity chromatography.

136. The method of claim 132, wherein the separating comprises magnetic separation.

137. The method of claim 132, wherein the separating comprises fluorescence activated cell sorting.

138. The method of claim 132, wherein the CD90+ cells are isolated with reduced or minimal manipulation.

139. The method of claim 138, wherein the CD90+ cells are isolated without electroporation, viral vectors, cell selection processes, or cell purification processes.

140. The method of claim 138 wherein the CD90+ cells are isolated solely by washing cells to remove platelets.

141. The method of claim 132, further comprising washing the sample.

142. The method of claim 132, further comprising washing the isolated cells.

143. The method of claim 132, further comprising expanding isolated cells.

144. The method of claim 132, further comprising culturing isolated cells.

145. The method of claim 132, further comprising genetically modifying isolated cells.

146. The method of claim 132, further comprising storing isolated cells.

147. The method of claim 132, further comprising cryopreserving isolated cells.

148. The method of claim 132, further comprising formulating isolated cells.

149. A method of treating a subject in need thereof comprising administering a therapeutically effective amount of the composition of claim 119 or the formulation of claim 120 to the subject thereby treating the subject in need thereof.

150. The method of claim 149, wherein the subject has a CD90-positive cancer.

151. The method of claim 149, wherein the administering comprises intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, intrathecal, intrauterine, intraplacental, intramuscular, intravesicular, and / or subcutaneous administration.

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